Characterization of Newcastle disease viruses isolated from migratory waterfowl in the Atlantic flyway. Four isolants of Newcastle disease virus (NDV) obtained from free-flying Canada geese in the Atlantic flyway were characterized and compared with the B1, LaSota, Roakin, and Texas-GB strains of NDV. The 4 isolants were identified as lentogenic strains on the basis of embryo mean death times of greater than 100 hours and relative lack of pathogenicity for 1-day-old and 3-week-old chickens. The hemagglutinin of 2 of the isolants was stable at 56 C for 15 minutes, 1 for 30 minutes, and the other for at least 2 hours. When held at 4 C for 24 hours, none of the 4 isolants could be eluted from chicken erythrocytes. All 4 of the isolants produced plaques in chicken embryo fibroblast cell cultures without additives, whereas the B1 and LaSota strains did not. The 4 lentogenic NDV isolants from Canada geese differed in many respects from currently used commerical vaccine strains (B1 and LaSota).


Viscerotropic velogenic Newcastle disease in turkeys: immune response following vaccination with either viable B1 strain or inactivated vaccine. When used as a vaccine, live letogenic B1 Newcastle disease virus (NDV) protects turkeys against challenge-exposure to viscerotropic velogenic NDV (VVNDV). Low-level passively-immune poults were vaccinated one, two, or three times at various intervals and their immunity challenged at various times from 1 to 10 months of age. Newcastle disease virus was isolated readily from either the blood, trachea, or vent of turkeys in all challenge groups (through 5 months of age) on the 3rd to 6th days postchallenge (PC) but after 14 days PC was isolated rarely. Virus was isolated from turkeys that had high titers of serum hemagglutination-inhibition antibodies at the time of challenge. The anamnestic antibody response appeared to be stronger in poults that had low antibody titers prior to challenge-exposure to VVNDV. In a small-scale study with an inactivated VVNDV vaccine, vaccinated poults were protected against challenge with the homologous viscerotropic virus. Parallel control studies on the infectivity of viscerotropic NDV for turkeys indicated that resistance to VVNDV increased with age.


Inclusion body hepatitis in Mexico.

Inclusion body hepatitis in Mexico. Inclusion body hepatitis is recorded for the first time in Mexico. Clinical signs, gross lesions, and histopathology in chickens are described, as well as chicken embryo lesions.


In vitro incorporation of 1-14C acetate by blood cells of normal ducks and ducks infected with Plasmodium lophurae. Several investigations have been made, on a qualitative basis, of in vitro incorporation of 1-14C acetate into the lipids of whole blood from normal ducks and ducks infected with Plasmodium lophurae (1,3,4). It was found generally that the percent labeling in blood cells and plasma was higher for infected ducks than for normal ducks. The present study was concerned with quantitative determination of the incorporation of 1-14 C acetate into the lipids of purified erythrocytes (RBC) and leukocytes (WBC) of normal and infected ducks.


Borrelia anserina in chickens previously exposed to Borrelia theileri. Exposure of 10-week-old White Leghorn chickens to sheep blood containing Borrelia theileri neither resulted in infection nor conferred protection against B. anserina.


Viscerotropic velogenic Newcastle disease in Turkeys: virus shedding and persistence of infection in susceptible and vaccinated poults. Susceptible turkeys and turkeys vaccinated with live lentogenic B1 strain Newcastle disease virus (NDV) were inoculated intracularly with viscerotropic velogenic (VV) Fontana strain NDV and studied for virus shedding and persistence of infection. Susceptible poults that survived infection (15%) continued to shed NDV from the intestinal tract up to 46 days postinoculation. Turkeys that were vaccinated with B1 strain NDV did not develop clinical signs when their immunity was challenged with VV Fontana strain virus. Virus was covered up to 53 days postchallenge (PC) from the cloaca of poults that were vaccinated once at 4 days of age and challenged at 1 month of age. Older turkeys that had been vaccinated one to three times did not generally shed virus after 4 days PC. Newcastle disease virus was recovered later in convalescence by the organ-culture method when swabs of trachea and cloaca were negative for virus. Persistent infection was detected as long as 88 days PC in organ cultures of cecal tonsil. Five of seven NDV isolants from organ cultures or from swabs caused fatal disease in chickens.


Viscerotropic velogenic Newcastle disease in turkeys: isolation of Newcastle disease virus from tracheal and cecal tonsil organ cultures. Tracheal and cecal-tonsil organ cultures were made from vaccinated turkeys that had survived challenge of immunity with viscerotropic velogenic strain of Newcastle disease virus (NDV). Culture fluids were tested to show that latent infections did exist in the vaccinated and challenged turkeys, thus indicating a possible carrier state. NDV was recovered from 6 of 159 turkeys examined. Preliminary tests indicate that 4 isolants are velogenic and 2 are lentogenic.


Viscerotropic velogenic Newcastle disease in turkeys: vaccination against loss of egg production. Live B1 Newcastle disease virus was administered to young turkeys either intraocularly or by driniking water, or by both methods. Protection against egg production loss was evaluated by challenge-exposure to viscerotropic velogenic Newcastle disease virus in drinking water. During 22 days postchallenge (PC), none of the vaccinated hens had morbidity, whereas 44% of the unvaccinated controls died 6-13 days PC. Percent egg production (PEP) of all groups 1-5 and 6-22 days PC were compared with their levels 1-5 days before challenge. For days 1-5 PC, changes were not significant. For days 6-22 PC, changes for all groups were siginficant lower. The controls had 0 production. Hens vaccinated only at 4 days or at 4 days and again at 4 weeks averaged one-third or less of prechallenge levels but were recovering. Those revaccinated at 4 months maintained 84-91% of their prechallenge levels and were considered satisfactory. Broodiness was a detracting factor in one group of hens vaccinated at 4 days, 4 weeks, and 51/2 months. They averaged two-thirds of prechallenge levels but were in decline.


Characterization of a new infectious bronchitis virus isolate. III. Cell-culture adaptation of Clark 333. The Clark 333 strain of infectious bronchitis virus (IBV) was substantially resistant to primary chicken cell-culture adaptation. More than 40 serial embryo passages were required before the virus would produce cytopathic alterations upon cell-culture inoculation. The cytopathic effect was characteristic of the effect of reported for IBV. Adaptation was not accomplished by alternating serial passages in embryo and cell-cultured systems. A careful monitoring of cell-culture fluid infectivity by embryo inoculation was found to be essential because absence of adaptation is accompanied by a loss in virus titer (3 to 5 serial passages). Helpful additional aids were immunofluorescence and cytopathology.


Viremic responses of genetically susceptible and resistant chickens to experimental infection with acute, mild, or both strains of Marek's disease herpesvirus. The development and persistence of viremia were followed in two lines of Single-Comb White Leghorns: one experimental line (WSU-VS) highly susceptible, and one commercial line (C-WL) relatively resistant to Marek's disease(MD). In the resistant C-WL chicken, viremia with a mild strain of MD herpesvirus (MDHV) persisted in all viremic birds through 8 weeks postinoculation (PI), while viremia with an acute strain of MDHV did not, resulting in a decrease in number of viremic birds after 2 weeks PI. In the susceptible WSU-VS chicken, viremia with acute MDHV persisted in all viremic birds whereas viremia with mild MDHV was detected in a decreasing number of birds after 6 weeks PI. The pattern of viremia observed in the dually infected groups simulated a combination of the responses of the two groups respectively inoculated with mild and acute MDHV. Whether inoculation was with acute MDHV alone, or together with mild MDHV, levels of viremia with acute MDHV were appreciably higher in the WSU-VS than in the C-WL chicken. In both lines, levels of viremia were higher with acute MDHV than with mild MDHV but viremia with acute MDHV could not be demonstrated in the C-WL bird at 6-8 weeks PI. Levels of viremia with mild MDHV were consistently and similarly low in both WSU-VS and C-WL chickens.


The neutralizing characteristics of strains of infectious bronchitis virus as measured by the constant-virus variable-serum method in chicken tracheal cultures. The constant-virus variable-serum neutralization test in chicken tracheal organ cultures was employed in cross-neutralization tests with the following infectious bronchitis viruses: Massachusetts 41, Connecticut, Iowa 97, Iowa 609, Holte, JMK, Clark 333, SE 17, Florida, and Arkansas 99. These viruses all proved serologically distinct by this method. Chickens immunized with the Connecticut strain and later challenged with Arkansas 99 were not protected against it.


Adenoviruses of chickens: serologic groups. By literature review and experimental studies, 57 adenovirus isolants from chickens were grouped into 10 distinct serotypes. In plaque-reduction tests, 20 antibody units were reacted with 32-320 plaque-forming units of virus. Viruses that were neutralized (80% or greater reduction in titer) by a given serum were considered to belong to that serotype. Used to distinguish among serotypes were their antisera. One-way neutralization occurred in only two instances; in both cases it was traced to contamination of the virus inocula used to induce specific antiserum. Recloning corrected the problem. Of the 10 serotypes described, all are known to exist in the United States, 7 were found in Northern Ireland, and at least 6 have been shown to occur in Japan.


Properties of the highly reactive SH groups of phosphorylase b. The reaction of rabbit muscle phosphorylase b with 5,5'-dithiobis(2-nitrobenzoic acid) (Nbs2) has been studied with stopped-flow spectrophotometry. Two highly reactive sulfhydryl groups per dimer reacted with Nbs2 within a few seconds, while the remaining SH groups needed several minutes and hours. Decomposition of the time curve revealed that the highly reactive SH groups can be divided into two subclasses: a fast type which reacted with a rate constant of 3 x 10-3 M-1 sec-1 and a more slowly reacting type disappearing with a rate constant of 0.3 x 10-3 M-1 sec-1. The reactivity of the slowly reacting type increased by a factor of about 2 in the presence of 1 mM AMP. Concurrently, the ratio between the fast reacting and the more slowly reacting subclasses decreased from 5.3 to 1.0. The AMP effect was greatly enhanced by glucose 1-phosphate. This enhancement was abolished in the presence of ATP. The finding that the ratio between the number of SH groups in the two subclasses of the highly reactive SH groups changed upon addition of ligand molecules indicates that the two subclasses reflect the different reactivities of the SH groups when the enzyme is present in different conformational states. It is suggested that the highly reactive SH group measured belong to the peptide: Gly-Cys-Arg-Asp.


Photosensitized inactivation of stem bromelain. Oxidation of histidine, methionine, and tryptophan residues. Pineapple stem bromelain was photooxidized in the presence of Methylene Blue used as a sensitizer. The essential sulfhydryl group of the enzyme protein rapidly became inaccessible to react with 5,5'-dithiobis(nitrobenzoic acid), but the reactivity was readily regained to the original level upon treatment with dithiothreitol. Even after such reduction, the photooxidized enzyme showed a markedly decreased hydrolytic activity on casein. Spectral examination revealed that the oxidized enzyme had tyrosine residues intact. Amino acid analysis showed significant decreases in histidine, ethionine, and tryptophan residues. Photoinactivation occurred in a similar manner also in the presence of tetrathionate which reversibly blocked the essential sulfhydryl group. It is concluded that the irreversible photoinactivation of stem bromelain must be related to the oxidation of histidine, methionine, and tryptophan residues. When the photooxidation was carried out a different pH values ranging from 4.0 to 8.3, the inactivation and the decrease in histidine content were found to be markedly pH dependent. Thus, the photooxidation experiment provided a method for directly measuring the apparent pKa of the ionization of the single histidine residue in stem bromelain. Apparent pKa values of 6.4 and 7.1 were obtained for the histidine imidazole in the absence and in presence of tetrathionate, respectively. In view of these normal pKa values for an imidazole, a mechanism of ionization of the active-site group in a plant thiol proteinase is proposed, in which the validity of mechanism involving a close electronic interaction between histidine and cysteine residues is seriously questioned.


Iron-donating properties of transferrin. The transferrin molecule has two specific metal-binding sites, each of which may provide iron for the biosynthesis of hemoglobin by reticulocytes. Diferric human transferrin was shown to be a better iron donor, per iron atom, for rabbit reticulocytes, than was monoferric transferrin obtained by isoelectric focusing. The difference in binding of 125I-labeled monoferric and differic transferrin to reticulocytes may be sufficient to account for the difference in iron uptake. In contrast, diferric and monoferric rabbit transferrin both donated iron to reticulocytes at the same rate, per iron atom. In an experiment using 55Fe/59Fe doubly labeled transferrin, one iron binding site of human transferrin was a better iron donor than the other. In rabbit transferrin, the two sites appeared to function equivalently. Care was taken in these experiments to demonstrate that labeled iron added to dilute solutions of transferrin was indeed specifically bound to the protein. A liquid scintillation counting procedure, simpler than existing methods, was developed to quantitate 55Fe and 59Fe in blood.


The alpha and beta subunits of Cyanidium caldarium phycocyanin: properties and amino acid sequences at the amino terminus. Phycocyanin was isolated and purified from the unicellular alga, Cyanidium caldarium. Subunits were prepared on a Bio-Rex-70 column developed stepwise with urea solutions (pH 1.9). The alpha subunit eluted in 8 M urea and the beta subunit eluted in 9 M urea. The alpha and beta subunits displayed absorption maxima at 660, 354, and 277 nm in 8 M and 9 M urea. The alpha:beta ratio of total absorbance under the 660-nm peak was 0.56 suggesting an alpha:beta phycocyanobilin chromophore ration of 1:2. On calibrated sodium dodecyl sulfate gels, the alphs subunit had an estimated molecular weight of 15,500 plus or minus 1100 and the beta subunit has an estimated molecular weight of 18,300 plus or minus 300. Minimum molecular weights based on one histidine residue per subunit were 16,300 for the alpha subunit and 18,750 for the beta subunit. Phycocyanin displayed a single visible absorption maximum at 625 nm and two positive circular dichroic bands at 632 and 610 nm. The alpha and beta subunits displayed single visible absorption maxima at 618 and 600 nm and single positive circular dichroic peaks at 620 and 585 nm, respectively. Two-dimensional maps of tryptic digests of the alpha and beta subunits revealed distinct patterns of peptides each of which was consistent with the lysine and arginine composition of these polypeptides. Maps of tryptic digests of phycocyanin contained 25 major peptides (a total of 27 lysine and arginine residues). Automated sequence analysis of separated subunits revealed a 70% homology within the first 27 residues at the amino terminus of the alpha and beta subunits of C. caldarium phycocyanin.


Negative cooperativity in the binding of thyroxine to human serum prealbumin. Preparation of tritium-labeled 8-anilino-1-naphthalenesulfonic acid. The binding of thyroxine (T4) and 8-anilino-1-naphthalenesulfonic acid (ANS) to human serum prealbumin was measured by equilibrium dialysis at pH 7.4 in 0.05 M phosphate-0.10 M NaCl at 25 degrees. The data were analyzed for the binding constants based on equations for (1) two independent sites and (2) two identical sites with negative interaction. Evaluation by the independent site model gave the following association constants: for T4 binding, KT1 = 1.0 x 10-8 M-1, KT2 = 9.5 x 10-5 M-1; for ANS binding, KA1 = 9.5 x 10-5 M-1, KA2 = 2.1 x 10-5 M-1. The interactive model gave constants kT = 5.5 x 10-7 M-1 and kA = 5.5 x 10-5 M-1. Interaction factors, alpha, defined such that -RT in alpha is the energy of interaction, were: alpha T = 0.041 AND ALPHA A = 0.62 for T4 and ANS, respectively. The "best fit" values for the number of sites were 2.0 and 1.6 for T4 and ANS, respectively. The binding of T4 to human prealbumin was competitive with ANS, and the binding constants evaluated from competition experiments were in agreement with those found for each ligand when studied separately. On the basis of analysis of X-ray data of human prealbumin (Blake et al.) there appear to be two identical T4 sites. It is therefore evident that the binding of T4 represents a case of negative cooperativity which is presumably due to interaction between ligands.


DNA-dependent RNA polymerase from Pseudomonas BAL-31. I. Purification and properties of the enzyme. The DNA-dependent RNA polymerase from Pseudomonas BAL-31, the host for bacteriophage PM2, has been purified 154-fold using differential centrifugation, chromatography on DEAE-cellulose, ammonium sulfate precipitation, and sucrose gradient centrifugations at low and high ionic strength. The resulting enzyme is free of enzyme activities which could interfere with transcription studies and is greater than 85% pure as judged by polyacrylamide gel electrophoresis. Like other bacterial RNA polymerases, its subunit structure is beta'beta sigma alpha2. From gel electrophoresis the beta', beta, and alpha subunits have approximately the same molecular weights as those from Escherichia coli, whereas the sigma subunit is 5% larger (89,000 vs. 85,000). A summation of the subunits yields a molecular weight of 485,000 for the holoenzyme. Like other bacterial RNA polymerases, it sediments as a monomer (15 S) at low ionic strength (0.065) and as a dimer (22 S) at high ionic strength (0.75). Its activity is stimulated three-fold by monovalent cations (K+,NH4+, NA+) with additional stimulation provided by divalent cations (Mg2plus, Mn2plus). The transcription of phage PM2 form I (supercoiled) DNA has an ionic strength optimum of 0.26 for continuous long-term synthesis, and over an ionic strength range of 0.09-0.46 "plateau-type" kinetics are not observed. The sigma subunit is required for optimal PM2 transcription. The enzyme is sensitive to the same inhibitors of transcription as the RNA polymerase from E. coli, it has a temperature optimum of 28 degrees, and it is 50% inactivated by heating 10 min at 41 degrees. It has template preference similar to E. coli polymerase and shows little preference for homologous templates. With various DNAs the order of template activities is T7 greater than PM2 I congruent to T4 greater than PM2 II (relaxed circular form) greater than lambda-c greater than calf thymus greater than BAL-31 DNA. Phage PM2 form I DNA is transcribed at a twofold greater rate than PM2 form II DNA by this enzyme.


DNA-dependent RNA polymerase from Pseudomonas BAL-31. II. Transcription of the allomorphic forms of bacteriophage PM2 DNA. Transcription of the supercoiled form (I) and the relaxed circular form (II) of bacteriophage PM2 DNA was studied utilizing the DNA-dependent RNA polymerase from its host, Pseudomonas BAL-31. Transcription of both templates is continuous for up to 2 hr, but proceeds at a two-fold higher rate on I than on II. This difference is mainly due to a 2.2-fold higher rate of chain initiation on I. When rifampicin (Rif) is added ater 10 min of synthesis, (1) transcription of II ceases by 30 min with a maximum product length of 7000 nucleotides (number average) being produced; (2) transcription of I continues with little rate reduction and with the product reaching 16,000 nucleotides (number average) by 2 hr. Sucrose gradient analysis shows that the product of II achieves maximum size 20 min after Rif addition and sediments in three peaks of 24, 33, and 39 S (approximately one-third, two-thirds, and one genome lengths). The product of I has a heterogeneous distribution and grows continuously with a large fraction reacting greater than 3 genome lengths by 90 min. The same differences in synthesis kinetics, Rif inhibition, and product size distribution are observed when I and II are transcribed by Escherichia coli RNA polymerase. These experiments show that (i) PM2 form I DNA is transcribed mainly by a process of continuous chain elongation, with little chain termination occurring; (ii) PM2 form II is transcribed by a process of continuous chain initiation, elongation, and termination of yield discrete products. Thus, the tertiary structure of circular DNA influences chain termination by RNA polymerase.


DNA binding proteins in the cytoplasm and in a nuclear membrane complex isolated from uninfected and adenovirus 2 infected cells. The DNA binding proteins in a nuclear membrane fraction that can synthesize DNA in vitro (referred to as "nuclear membrane complex") and in the cytoplasm of adenovirus infected and uninfected cells were isolated and characterized. Suspension cultures of human KB cells infected with human adenovirus 2 were treated with 25 mu-g/ml of arabinosylcytosine starting at 2 hr to block the synthesis of viral structural proteins, and then labeled with (3H)leucine from 6 to 24 hr after infection. Uninfected cells were treated similarly and labeled with (14C)leucine. The 3H-labeled proteins (infected cells) and 14C-labeled proteins (uninfected cells) isolated from the cytoplasm were mixed, as were the corresponding proteins isolated from the membrane complex, and each mixture was fractionated by stepwise elution from single-stranded DNA-cellulose columns. From 50 to 60% of the labeled protein in the membrane complex from infected cells and 40 to 50% of that from uninfected cells bound to DNA-cellulose in 0.05 M NaCl. Much less protein from the cytoplasm was bound to DNA cellulose, 20% from infected cells and 11% from uninfected cells. Gel electrophoresis of the mixture of 3H- and 14C-labeled proteins eluted from DNA-cellulose by different concentrations of NaCl revealed the following. (1) The 0.15 and 0.40 M NaCl eluates from the membrane complex of infected and uninfected cells contained a heterogenous mixture of similar polypeptides. (2) The 0.6 M NaCl eluate from the membrane complex derived from infected cells contained two major DNA binding proteins with molecular weights of 75,000 and 45,000 that were absent from uninfected cells. Large quantities of these two proteins were present in highly purified form in the 0.6 M NaCl eluate from the cytoplasm of infected cells. The DNA binding proteins of molecular weight 75,000 and 45,000 that are present in the cytoplasm are identical with those present in the membrane complex, as established by coelectrophoresis. (3) Two major cell-specific proteins of molecular weight 40,000 and 15,000-17,000 were present in the 2 M NaCl eluate of the membrane complex from uninfected and infected cells. A major cell-specific protein of molecular weight 33,000 was present in the 0.15 and 0.4 M NaCl eluates of the uninfected and infected cell cytoplasmic fractions. Aanalysis of cells labeled at 2-6 hr after infection in the absence of arabinosyl cytosine indicated that the synthesis of the DNA binding proteins of molecular weight 75,000 and 45,000 begins early after infection prior to the onset of viral DNA replication.


Isolation, characterization, and stability of the 30S ribosomal RNA complex from HeLa cells. The HeLa 30S rRNA molecule (historically designated 28S rRNA) can be dissociated into two components, a 7S rRNA and a large rRNA component which we call 29S rRNA. To evaluate conformational differences between the 30S rRNA complex and the isolated 29S rRNA component of the complex, viscosity, sedimentation velocity, circular dichroism, and ultraviolet absorption measurements with the two species were performed. Sedimentation equilibrium studies were also carried out with the 30S rRNA complex. In addition, the kinetics of the reaction which dissociates the 30S rRNA complex were characterized. The removal of glycogen-like molecules by cetyltrimethylammonium bromide prescipitation of the rRNA and the preequilibration of rRNA with solvent by Sephadex column chromatography were found to be essential for reproducibility. The s20,2o values for the 30S rRNA complex and the isolated 29S rRNA were determined from the experimental data obtained at various rRNA concentrations as 29.89 plus or minus 0.40 and 29.09 plus or minus 0.14, respectively. The corresponding intrinsic viscosity values were 74 plus or minus 5 and 67 plus or minus 5 cm3/g, respectively. The optical properties of the 30S rRNA and 29S rRNA were not significantly different. These results indicate that there is no significant conformational difference between 30S rRNA and 29S rRNA under the conditions studied. We conclude from the sedimentation equilibrium data that the molecular weight of 30S rRNA is 2.1 x 10-6. From the kinetic data, the 30S rRNA dissociation appears to be an irreversible, cooperative, and ionic strength dependent reaction which at an ionic strength of 0.051 has an activation enthalpy of 123.5 kcal/mol and an activation entropy of 0.21 kcal/(mol deg).


The binding of deoxycholate, Triton X-100, sodium dodecyl sulfate, and phosphatidylcholine vesicles to cytochrome b5. Cytochrome b5 is composed of two domains that can be isolated after tryptic cleavage as two polypeptide fragments. One fragment is globular and hydrophilic and contains the heme; the other fragment is rich in hydrophobic amino acids and is essential for recombination of cytochrome b5 with microsomal membranes (Ito, A., and Sato, R. (1968), J. Biol. Chem. 243, 4922; Spatz, L., and Strittmatter, P. (1971), Proc. Nat. Acad. Sci. U.S. 68, 1042). Equilibrium dialysis and sedimentation equilibrium measurements of the binding of deoxycholate, Triton X-100 and dodecyl sulfate show that neither intact cytochrome b5 nor its proteolytic fragments possess high affinity binding sites for any of these amphiphiles. However, each detergent binds to the protein in a highly cooperative manner at concentrations near the critical micelle concentration. Binding measurements using the separated tryptic fragments show that deoxycholate and Triton X-100 (both nondenaturing detergents) bind to the hydrophobic fragment to the same extent as to intact cytochrome b5, and not at all to the polar fragment. Sodium dodecyl sulfate (a denaturing detergent) is bound to both tryptic fragments, but 70% of the detergent is bound to the hydrophobic fragment although it comprises only 30% of the protein mass. Less detailed measurements were made with synthetic and natural phosphatidylcholines, and show that the intact protein is quantitatively incorporated into phosphatidylcholine vesicles, but that no interaction with the polar fragment occurs. These results are interpreted in terms of the hydrophobic domain of cytochrome b5 having a diffuse hydrophobic surface that can act as a nonspecific nucleus for the formation of a micelle with a variety of amphiphilic substances. This domain of the molecule will insert into any available hydrophobic environment, whether it be detergent micelles, synthetic phospholipid vesicles, or the microsomal membrane. The incorporation of cytochrome b5 into the microsomal membrane is only a specialized case of the general property.


Comparison of the resonance Raman spectra of carbon monoxy and oxy hemoglobin and myoglobin: similarities and differences in heme electron distribution. With 441.6-nm excitation, which is near the Soret band, we observe that the resonance Raman spectra of hemoproteins contain not only the bands between 650 and 1700 cm-1 which arise from vibrations of the conjugated macrocycle, but also bands below 650 cm-1, some of which involve vibrations of the iron pyrrole-nitrogen bonds. The spectra of the oxygen and carbon monoxide complexes of both myoglobin and hemoglobin are sufficiently similar to those of low spin met derivatives, that the electronic distribution on the heme for both ligands can be interpreted as that of a low spin ferriheme. This agrees with an earlier interpretation, by others, of comparative optical absorption spectra and, as pointed out previously, would imply that in the complex the ligands are bound as O2- and CO-. However, band frequencies and relative intensities differ somewhat between the carbon monoxide and oxygen complexes of the same protein, which indicates differences between the details of the pi-electron distributions in the corresponding complexes.


Studies of individual amino acid residues of the decapeptide tyrocidine A by proton double-resonance difference spectroscopy in the correlation mode. The cyclic decapeptide antibiotic tyrocidine A was studied by two relatively new methods, viz., correlation proton magnetic resonance (pmr) spectroscopy and double-resonance difference pmr spectroscopy. The correlation method of spectral accumulation provided pmr spectra of good resolution, and in addition the signal-to-noise ratio achieved per unit time of accumulation was much higher than that achieved by use of the conventional continuous wave (cw) method. Furthermore, when protonated solvents are used, the correlation mode of accumulation has a distinct advantage over pulse and fast Fourier transform (fft) methods currently in use. Double-resonance difference (drd) spectra of individual amino acid residues in tyrocidine A were obtained by the correlation method when the decoupling frequency was maintained at the center frequency of the appropriate C-alpha proton multiplet and at a level of power that totally decoupled vicinal C-alpha and C-beta protons; the resolution of these spectra was good, and the signal-to-noise ratio was high. The distinct patterns and spectral positions of the drd spectra were characteristic of the particular type of amino acid residue and, therefore, could be used as the basis for making assignments. Furthermore, the drd spectra revealed the spectral positions of individual C-alpha and C-beta proton transitions and therefore, upon spectral analysis, could provide the chemical shifts and coupling constants of these protons. Positions of transitions were revealed even though they were hidden by overlap in the corresponding conventional single- or double-resonance spectra.


Properties of hydroxylase systems in the human fetal liver active on free and sulfoconjugated steroids. The substrate specificity of the steroid sulfate-hydroxylating activity in microsomes from human fetal liver has been investigated. Twelve different C18, C19, C21, and C27 steroid sulfates and the corresponding free steroids were used as substrates. The introduction of a sulfate group on the steroid substrate was found to have two principal effects. (1) The hydrophilic sulfate group directs the steroid molecule so that it only interacts with the active site of cytochrome P-450 with its non-sulfurylated, hydrophobic end. (2) The sulfate group interacts with the enzyme surface resulting in exposure of a slightly different part of the hydrophobic end of the substrate to the active site of cytochrome P-450 than when the same end of the free steroid is exposed to the active site of the enzyme. As a consequence of these two effects of the sulfate group, the "steroid sulfate pathway" of steroid hydroxylations generally differs considerably from the "free steroid pathway," both from a qualitative and a quantitative aspect. This difference was found to be most pronounced with estrogens: whereas estradiol was not hydroxylated by human fetal liver microsomal preparations, estradiol 3-sulfate was both 15-alpha- and 16-alpha-hydroxylated. Thus, for certain steroids, sulfurylation is a prerequisite for further metabolism by microsomal hydroxylase systems. These results indicate the presence in human fetal liver microsomes of a multipotent, highly unspecific, hydrophobic "bulk" of cytochrome P-450. The existence of this hydroxylase system which efficiently hydroxylates steroid sulfates is probably of great physiological importance as a detoxifying mechansim in the human fetus.


Cholesterol in aqueous solution: hydrophobicity and self-association. Free energies of transfer of cholesterol monomer from water to organic solvents show that the hydrophibicity of this sterol molecule is significantly less than predicted from hydrophobic surface area considerations. It is suggested that this phenomenon may arise from unusual orientation of water molecules at the surface of the solute. From the direct measruemtn of the hydrophobic free energy of transfer and comparison with thermodynamic data on micelle formation reported previously we calcuate specific attractive interactions between cholesterol monomers in the micelle of 2-4 kcal/mol, which suggests the possiblity of self-association (phase separation) in mixed micellar systems such as sterol-lipid complexes.


The role of the state governments in educating the public about health. Fifty years ago health care was a private matter between a physician and his patient. Doctors practiced medicine; people could and did pay for their own health care; and legislators did not need to concern themselves much with health problems. In the meantime, instigated by a massive scientific knowledge explosion, we have spent billions on monstrous complexes to house ultramodern equipment and technology to fight acute, dreaded diseases. Doctors, who once treated human body as an entity, are so specialized that none seems to know any more that the head bone is still indirectly connected to the great toe. People, who incidentally are plagued with essentially the same chronic maladies that visited their predecessors five decades ago, are in real trouble if their complaint is simply a boil on the backside. First they must determine who specializes in this particular area. Next they are lucky if they can obtain an appointment before the boil bursts. Finally, they probably cannot afford to pay for the services rendered. The lawmaker steps in and pours more money into erecting more buildings and providing more specialists. We will not solve the dissonance between the mutually dependent group if we isolate ourselves, forgetting to listen, forgetting to educate, forgetting to communicate. Let me close with Sir William Osler's metaphor: How common the experience to enter a cold cheerless room in which the fire in the grate has died down, not from lack of coal, not because the coal was not alight, but the bits, large and small, falling away from each other have gradually become dark and cold. Break them with a poker, get them together, and what a change in a few minutes.


The Dr. Fox effect: a study of lecturer effectiveness and ratings of instruction. Students viewed one of six lectures which varied only in substantive teaching points (content) covered and seductiveness. These 207 students then rated the effectiveness of the presentation (satisfaction ratings) and completed a 26-item achievement test. Students who viewed high seduction lectures performed better on the achievement test than did students who viewed low seduction lectures. Similarly, students who viewed lectures high in content performed better on the cognitive test than did students who viewed low-content lectures. The relationship between staisfaction ratings and student achievement was not perfect. Students gave higher ratings to seductive lectures. However, ratings reflected differences in content-coverage only under low seduction conditions. The ratings were not sensitive to variations in content-coverage when lectures were highly seductive. The "Doctor Fox Effect" appears to be more than an illusion. Seductiveness affects both student ratings of instruction and achievement.


The development of views of specialties during four years of medical school. A total of 141 medical students at one medical school participated in a longitudinal study of medical specialty choice. Students indicated their choices in the freshman, sophomore, and senior years. They ranked specialties with respect to status and completed an adjective checklist for self and practitioners of five specialties. Measures of social attractiveness and similarity to self were applied for each specialty. Increasing numbers of students chose internal medicine over the four years. Pediatrics and psychiatry lost students, while family practice and surgery changed little between the first and fourth years. Surgery and medicine were ranked highest and family practice and psychiatry lowest as to status. Family practice was ranked highest and surgery lowest as to social attractiveness. In examining similarity to self, medical students regardless of specialty interest rated themselves as having traits similar to those they assigned to the family practitioner.


Foreign medical graduates who return home after U.S. residency training: the Peruvian case. With few exceptions, reports on foreign medical graduates (FMGs) are concerned with their academic, clinical, and adaptational problems during their training at American hospitals and universities. This report, part of a study of FMGs from Peru who have completed postgraduate training in the United States in the past 10 years, is concerned with the psychosocial and professional stresses of adaptation experienced by a group of 21 Peruvian doctors who have returned home to practice. The authors discuss the "push" factors and "pull" factors that motivated these physicians to return to Peru to pursue careers in academic medicine, private practice, and government service in contrast to many of their colleagues who have remained in the United States.


The effects of an information specialist on patient care and medical education. A medical librarian joined the pulmonary medicine health care team at Cook County Hospital in order to provide a quick response to information needs regarding patient care, graduate medical education, continuing education, and research. Regular attendance at rounds and conferences enabled the librarian to initiate immediately literature searches in response to both clinical problems and educational requirements. A basic reference supplied to the medical librarian/information specialist by a physician frequently expedited literature research. It was found that patient care and education were enhanced by the rapid access to recent information and that team members used the library more. Close cooperation between the information specialist and physicians significantly facilitated the searches and saved time for health care team members.


Medical student change during a psychiatry clerkship: evaluation of a program. The psychiatry clerkship at the University of British Columbia (U.B.C.) emphasizes the teaching model of the student as primary therapist. In the adult program this takes the form of a ward in which clinical clerks are the primary medical agents. In the child program each clerk assesses and treats a new family referral. While the student-ward model has been described previously, this is the first report which attempts to evaluate some aspects of this innovative approach to teaching clinical psychiatry. Student knowledge, attitudes, personality attributes, and expectations were assessed at the beginning and end of the eight-week rotation, and the changes are described. Principal findings included significant differences in the following: psychiatric knowledge, attitudes about psychiatric illness, ratings of the students by the psychiatrist-supervisor, and some student personality characteristics. Most students had rotation experiences which went beyond prior expectations. The changes which occurred were thought to be important, although modest. The implications of the changes are discussed. The level of the patient care in the programs described compared favorably with similar situations in which residents are the primary therapists.


Relationship between the mutagenic and base-stacking properties of halogenated uracil derivatives. The crystal structures of 5-chloro- and 5-bromouracil. Three-dimensional X-ray diffraction data were used to determine the crystal structures of 5-chlorouracil and 5-bromouracil, two mutagenic pyrimidine analogs that can substitute for thymine in DNA. Crystals of the two compounds are nearly isostructural. The space group is P21/c, with a equals 8.450(6), b equals 6.842(3), c equals 11.072(16) angstrom, beta equals 123.53(19) degrees for 5-chlorouracil, and a equals 8.598(3), b equals 6.886(1), c equals 11.417(5) angstrom, beta equals 123.93(3) degrees for 5-bromouracil. Intensity data were collected with an automated diffractometer. The structures were refined by full-matrix least-squares to R equals 0.058 for 5-chlorouracil and R equals 0.027 for 5-bromouracil. The analogs from planar, hydrogen-bonded ribbons that are nearly  identical to those found in the crystal structure of thymine monohydrate. As in many other structures of 5-halogenated uracil derivatives, the bases assume a stacking pattern that permits intimate contacts between the halogen substituents and the pyrimidine rings of adjacent bases. This stacking pattern involves halogen contacts that are significantly shorter than normal van der Waals interactions. The crystallographic results provide additional evidence that halogen substituents influence the stacking patterns of uracil derivatives, while exerting little direct effect on the hydrogen-bonding properties. The observed stacking patterns are consistent with the hypothesis that altered stacking interactions may account for the mis-pairing between 5-halogenated uracil bases and guanine residues within double-helical nucleic acids.


Free and membrane-bound ribosomes. II. Two-dimensional gel electrophoresis of proteins from free and membrane-bound rabbit reticulocyte ribosomes. Free and membrane-bound polysomes were isolated from rabbit reticulocytes. The membrane-bound polysomes were liberated form the membrane with deoxycholate. Monosomes were prepared from the two types of polysomes by incubation with puromycin. The ribosomal proteins were extracted and analyzed by two-dimensional gel electrophoresis. Two proteins of the large subunit, L11 and L17 present in the free monosomes were not found in the membrane-bound monosomes. On the other hand, four additional spots were found in the protein pattern of the membrane-bound monosomes.


Factors affecting incorporation of (14C) leucine into albumin and transferrin by the liver in the postnatal rat. The mechanisms responsible for the increase in incorporation of radioactive amino acid into albumin, transferrin and total soluble liver protein which occurs in the immediate postnatal period in the rat was investigated in rats delivered surgically in the last 2 days of gestation. The in vivo incorporation of [14C]leucine into the proteins in the liver was low at birth, but increased rapidly during the first half hour after delivery and then more gradually during the subsequent 4.5 h. Neonatal adrenalectomy had no effect on this pattern of results. Intraperitoneal administration of an amino acid supplement had little effect on [14C]leucine incorporation immediately after birth but increased incorporation at 0.5 h and eliminated the second phase of rising incorporation values between 0.5 and 5 h. The in vitro incorporation of 14C into albumin, transferrin and total protein by slices of the liver from animals immediately after delivery was as great as with slices from animals 5 h after delivery. It is concluded that the initial increase in synthesis of proteins in the liver in the first 0.5 h after delivery is probably due to an increase in the supply of metabolic energy due to improved oxygenation of the rats and that the slower increase in protein synthesis between 0.5-5.0 h results from an improved supply of amino acids to liver cells. It is unlikely that changes in the secretion of adrenal hormones in involved.


Protein synthesis in chloroplasts. IV. Polypeptides of the chloroplast envelope. Envelope membranes were isolated from washed chloroplasts of pea seedlings. As judged by the protein-to-chlorophyll ratio, average preparations contain less than 8 percent contamination with internal lamellar membranes. Electrophoresis on sodium dodecylsulphate polyacrylamide gels shows that the envelope membranes contain at least 25 polypeptides. The molecular weight distribution of the envelope polypeptides is different from that of the lamellar polypeptides, there being more polypeptides of molecular weights above 50 000 in the envelopes. Two envelope polypeptides become labelled when isolated intact chloroplasts are incubated in the light with (35S) methionine. One of these is similar in molecular weight to the main polypeptide labelled in lamellae, but the other is unique to the envelope fraction. Incorporation of label into both polypeptides is totally light-dependent and is inhibited by chloramphenicol. When (35S) methionine is fed to detached pea shoots with and without cycloheximide, the labelling of other envelope polypeptides is inhibited. We conclude that two polypeptides of the chloroplast envelop are synthesised by chloroplast ribosomes.


Globin mRNA from rabbit reticulocyte membrane-bound ribosomes. Messenger RNA has been isolated from both free and membrane-bound rabbit reticulocyte ribosomes. Membrane-bound ribosomes, which constitute less than 10% of total cellular ribosomes, are released from the membrane by deoxycholate but remain associated with the membrane fraction following treatment with 0.5 M KC1. The major messenger RNA species isolated from either free or bound ribosomes is globin messenger. Both alpha- and beta-globin chains are synthesized when the isolated messenger RNA from either free or membrane-bound reticulocyte ribosomes is added to a Krebs ascites cell-free system.


Neutral lipids from the skin of the rhino mutant mouse. The neutral lipids of the skin of the Rhino mutant mouse consist mainly of fatty acid esters of sterols, fatty alcohols and 1,2-alkane diols, with strikingly low amounts of triacylglycerols. Fatty acids of wax and sterol esters were predominantly even chain monounsaturates (63 per cent) between C16 and C36 with a surprisingly high proportion of long chain lengths: the principal peaks corresponded to C32, C34, C18, C30, and C22 monoenes. The fatty alcohols showed a somewhat similar pattern, but with an even greater preponderance of long chain lengths and only small proportions shorter than C24. sterols included cholesterol, as expected, but only to the extent of about 28 per cent; the larger fraction was shown to be lathosterol (5alpha-cholest-7-en-3beta-ol). The largest single fraction (35.6 per cent) of cutaneous lipids consisted of fatty acid esters of 1.2-alkane diols. The 1,2-alkane diols were completely saturated and included odd and even chain lengths, both straight and branched, in the C16-C24 range: predominant peaks were C20, C22(iso), C16, and C22. Fatty acids of diol esters ranged between C14-C36 with major concentrations of C18, C22, C32, and C34 monounsaturates and C20 and C16 saturates.


Changes in the activities of de novo fatty acid synthesis and palmitoyl-CoA synthetase in relation to myelination in rabbit brain. 1. Age-related changes in the activities of fatty acid synthetase and palmitoyl-CoA synthetase (EC 6.2.1.3) have been determined in rabbit brain from the foetal stage through to maturity. 2. Fatty acid synthetase was most active in the soluble fraction of brain homogenates at 5 days of age, prior to the active phase of myelination. Palmitic acid was the major fatty acid synthesised throughout development. 3. Palmitoyl-CoA synthetase had a constant specific activity in the full homogenate, but in the microsomal fraction reached a maximum specific activity at 15-20 days of age. The specific activity was higher than in the mitochondrial fraction which declined from birth. Most of the palmitoyl-CoA synthetase activity was present in the fraction containing cell membranes plus nuclei. 4. From a comparison of the total activities of the enzymes involved in the metabolism of fatty acids in the brain, de novo fatty acid synthesis may be rate limiting compared with esterification of synthesised fatty acids, but not in the further transformations of the synthesised fatty acids.


Fatty acid metabolism in the microsomal fraction of developing rabbit brain. 1. Incorporation of [14C]malonyl-CoA into fatty acid and lipid by the microsomal fraction from developing rabbit brain has been determined. 2. The specific activity for malonyl-CoA incorporation into preformed fatty acid reached a maximum at 15-20 days of age and the increase in enzyme activity paralleled that of palmitoyl-CoA synthesis in the microsomal fraction. 3. Most of the preformed fatty acid was derived from microsomal membrane lipid, and added acyl-CoA only slightly increased the incorporation. Inhibition occurred at concentrations in excess of 2 muM acyl-CoA. Endogenous acyl-CoA may be formed by acyl-group turnover through the action of phospholipase A and acyl-CoA synthetase. 4. Added 14C-labelled acyl-CoA was elongated to a longer chain-length product than endogenous fatty acid elongated with [14C]malonyl-CoA. 5. Both ATP and acyl-CoA influence the incorporation of elongated fatty acids into complex lipids, possibly through their effect on acyl-CoA synthetase.


Effect of halofenate and clofibrate on lipid synthesis in rat adipocytes. The free acids of the plasma lipid-lowering agents, halofenate and clofibrate inhibited the incorporation of radioactive glucose and pyruvate into fatty acids of isolated adipocytes prepared from rat epididymal fat pads. The concentration which inhibited fatty acid synthesis was dependent on the bovine serum albumin concentration in the incubation. The 50 per cent inhibitory concentration of the free acid of halofenate in 1 per cent, 2 percent and 4 per cent albumin was 0.9 mM, 2.3 MM and 4.4 mM, respectively. The potency of clofibrate was also lowered by increasing the albumin concentration. These compounds inhibited the uptake of both [14C]glucose and [14C]pyruvate to the same degree as the incorporation of these substrates into fatty acids. However, the drugs either had no effect on , or stimulated the uptake of palmitate by the cells. Leucine accumulation by the adipocytes was unaffected by halofenate (free acid) and inhibited by clofibrate (free acid). A comparison of these agents with (minus)-hydroxycitrate, kynurenate and cerulenin (inhibitors of ATP-citrate lyase, acetyl CoA carboxylase and fatty acid synthetase, respectively) on the oxidation of pyruvate suggested that they inhibited pyruvate metabolism at or near the enzyme, pyruvate dehydrogenase.


Substrate-selectivity of rat liver microsomal 1,2-diacylglycerol: CDP-choline(ethanolamine) choline(ethanolamine)phosphotransferase in utilizing endogenous substrates. Rat liver microsomes containing 1,2-diacylglycerols formed from the endogenous phosphatidycholines by the action of 1,2-diacylglycerol: CDPcholine cholinephosphotransferase (E.C. 2.7.8.2.) were used as a source of enzymes and substrates. A marked selectivity of 1,2-diacylglycerol: CDPethanolamine ethanolaminephosphotransferase (E.C. 2.7.8.1.) was revealed for utilizing endogenous hexaenoic diaglycerol, while cholinephosphotransferase utilized without marked selectivity the endogenous 1,2-diacylglycerol species differing in the degree of unsaturation. By using microsomes prepared after injecting the animals with 1-acyl lysophosphatidylcholines labeled with radioactive myristic, palmitic, and stearic acids, the selectivity of the transferases towards the saturated fatty acids in the 1-position of the endogenous substrates was studied. In incubation of the labeled microsomes with CMP, cholinephosphotransferase appeared to utilize 1-myristyl phosphatidylcholine most rapidly and, in decreasing order, the 1-palmityl and 1-stearyl species. Ethanolaminephosphotransferase utilized the endogenous 1-stearyl diacylglycerol in preference to 1-palmityl type.


Biosynthesis of galactosyl diglycerides by non-green fractions from chloroplasts. When either mitochondria, chloroplast stroma lamellae, or osmotically shocked chloroplasts were centrifuged through sucrose gradients, zones were always obtained at the 0.6 M-0.9 M boundary which were highly active in galactosyltransferase. These activities did not coincide with maxima for chlorophyll or cytochrome c oxidase activity. A second chlorophyll-free fraction was obtained at lower density, showing high galactosyltransferase activity when incubated after isolation. The results indicate that the highly active fractions originate from chloroplast envelopes.


Distribution of fatty acids incorporated into triacylglycerols by microsome/cytosol preparations from adipose tissue. Microsome/cytosol preparations from adipose tissue of the mouse, pig, rat and chicken and from pig liver synthesize triacylglycerols containing a fatty acid distribution consistent with that in their respective fats. Since the incorporation of fatty acids depends on the presence of glycerol phospahte and no loss of tritium occurs during the incorporation of sn-[2-3 H]glycerol 3-phosphate into triacylglycerols, it would appear that the specific distribution is a property of the transacylases of the glycerophosphate pathway. The apparent Michaelis constant for sn-glycerol 3-phosphate, measured either by sn-glycerol 3-phosphate or palmitate incorporation, averaged 1.4-10-4 M both the mouse and pig adipose tissue enzyme systems.


Electron diffraction study of the hexagonal polymorphic form of some choline-containing phosphpolipids. Since the kinematical diffraction assumption has been found to be applicable to the analysis of electron diffraction patterns from thin organic crystals, single crystal electron diffraction patterns were obtained from thin anhydrous microcrystals of a lecithin, a lysolecithin and a beef brain sphingomyelin. Crystal structure analysis using the hk0 intensity data affirms the correctness of a free methylene rotor model for this projection of the aliphatic chain packing. Because the aliphatic chains are affixed to the polar moiety of a phospholipid molecule, it is proposed that there is helical twisting along the polymethylene chains, rather than the rigid body chain rotation proposed for the hexagonal packing of long chain paraffins. Lack of upper layer diffraction from the lecithin crystals which would be indicative of repeats along the chains leads to a speculation of translational disorders along the chain axes.


Biosynthesis of retinol in bovine corpus luteum tissue. The bovine corpus luteum was found to contain retinol, as analyzed by fluorometry and the formation of anhydroretinol. After the incubation of beta-[15,15 foot-3H] carotene with slices of corpus luteum tissue the biosynthesis of radioactive retinol was demonstrated. Alumina column chromatography, thin layer chromatography, and high speed liquid chromatography were used to isolate and identify radioactive vitamin A. Retinol was also found to form a polar complex, from which could be released by acid hydrolysis.


Effect of trans-1,4-bis(2-chlorobenzylaminomethyl) cyclohexane dihydrochloride and carbon monoxide on hepatic cholesterol biosynthesis from 4,4,-dimethyl sterols in vitro. The drug trans-1,4-bis(2-chlorobenzylaminomethyl)cyclohexane dihydrochloride (AY-9944) almost completely inhibited the conversion of [2-14C] mevalonic acid, dihydro[14C]lanosterol, 4,4-dimethyl-5alpha-[2-3H2]cholesta-8,14-dien-3beta-ol and 4,4-dimethyl-5alpha-[2-3H2]cholest-8(14)-en-3beta-ol to 5alpha-cholest-7-en-3beta-ol and cholesterol by cell-free systems of rat liver. With the first three precursors, the inhibition was accompanied by an accumulation of radioactive 5alpha-cholesta-8,14-dien-3beta-ol, but this material could not be detected during inhibition of cholesterol biosynthesis from 4,4-dimethyl-5alpha-[2-3H2] cholest-8(14)-en-3beta-ol. Regardless of the nature of the precursor, trans-1,4-bis(2-chlorobenzylaminomethyl)cyclohexane dihydrochloride did not result in the accumulation of any delta5,7 sterols. Non-radioactive 5alpha-cholest-8(14)-en-3beta-ol inhibited the conversion of dihydro[14C]lanosterol to 4,4-dimethyl-5alpha-cholesta-8,14-dien-3beta-ol. Carbon monoxide resulted in a decrease in the rate of conversion of dihydro[14C]lanosterol to 4,4-dimethyl-5alpha-cholesta-8,14-dien-3beta-ol but had no effect on the rate of conversion of 4,4-dimethyl-5alpha-[2-3H2]cholesta-8,14-dien-3beta-ol to 5alpha-cholest-7-en-3beta-ol and cholesterol suggesting that cytochrome P-450 is involved neither in the oxidative removal of the 4-methyl groups nor in the oxidative introduction of the delta5 bond during cholesterol biosynthesis. In addition, the process of cholesterol and 5alpha-cholest-7-en-3beta-ol biosynthesis from 4,4-dimethyl-5alpha-[2-3H2]cholest-8(14)-en-3beta-ol was inhibited by carbon monoxide at a stage after the formation of 5alpha-cholest-8(14)-en-3beta-ol.


Identification of alpha-, beta-, gamma-, and delta-tocopherols and their contents in human milk. Human milk was found to contain naturally occuring beta-, gamma- and delta-tocopherol and gamma-tocotrienol in addition to alpha-tocopherol on thin-layer chromatography. Some of the tocopherols were also identified by gas-liquid chromatography and mass spectrometry. The mean content of alpha-, beta-, gamma- and delta-tocopherol was 319.3, 7.6, 23.9 and 4.9 mug/g lipid in twelve human colostra 52.5, 1.8, 10.2 and 2.3 mug/g lipid in three transitional milks and 61.7, 2.0, 9.2 and 1.1 mug/g lipid in three normal milks, respectively. A markedly high concentration of alpha-tocopherol was found in colostrum compared with transitional and normal human milk. Gamma-Tocotrienol was detected in colostrum as only one tocotrienol derivative on thin-layer chromatography. The importance of colostrum as the source of vitamin E for the newborn is suggested.


3-Ketosphingolipids: application to the determination of sphingolipids which contain 4-sphingenine. 1. Ceramides and cerebrosides, both with 2-hydroxy or non-hydroxy fatty acids, and sphingomyelins were converted quantitatively to the corresponding 3-keto derivatives with the oxidative reagent 2,3-dichloro-5,6-dicyanobenzoquinone. Sulfatides were converted to 3-ketocerebrosides. 2. The conversion of these sphingolipids to the alpha,beta-unsaturated ketone permits three methods of quantitation: in Method I, the absorption of the reaction product is measured at 230 nm; Method II utilizes high performance liquid chromatography and an ultraviolet light detector; in Method III, the product is reduced with NaB3H4 and the radioactivity of the reduced products is determined. All three methods can be used to measure sphingolipids in nanomole quantities. 3. Methods I and II have been applied to the determination of cerebroside and sulfatide content of normal and metachromatic leukodystrophy brains. In the white matter of the pathological brains, there was a greater accumulation of sulfatides containing non-hydroxy fatty acids than of those containing 2-hydroxy fatty acids. The ceramide levels of the cerebellum in a Farber's disease patient and a control were determined by Method II. Method III was used to determine the content of free ceramides in human sera.


9-Hydroxyprostaglandin dehydrogenase activity in the adult rat kidney. Regional distribution and sub-fractionation. 1. Catabolism of prostaglandin F2alpha in the adult rat kidney takes place by the following sequence of enzymatic steps: (1) 15-hydroxyprostaglandin dehydrogenase; (2) prostaglandin delta13-reductase; and (3) 9-hydroxyprostaglandin dehydrogenase. 2. 9-Hydroxyprostaglandin dehydrogenase activity was highest in the cortex with lesser amounts in the medulla and negligible activity detected in the papilla. A similar distribution was observed for 15-hydroxyprostaglandin dehydrogenase and prostaglandin delta13-reductase. 3. Most of the 9-hydroxyprostaglandin dehydrogenase activity in the homogenate was found in the high-speed supernatant as also observed for 15-hydroxyprostaglandin dehydrogenase and prostaglandin delta13-reductase. 4. These observations indicate that the rat kidney contains an abundance of prostaglandin-catabolising enzymes which favour formation of metabolites of the E-type.


Photoreactions of phosphorothioate and cysteamine-S-phosphate. Photosubstitution and photophosphoryl transfer. The photoreactions of phosphorothioate and cysteamine-S-phosphate were investigated. On irradiation of phosphorothioate a marked change in absorption spectrum was observed. The product migrated in high voltage electrophoresis, with different mobility from that of phosphorothioate and its dimer, or inorganic orthophosphate. It contained phosphate and sulfur in a ratio of 2: 1, without reducing properties. Therefore it was suggested that the product is either pyrothiophosphate, or a cyclic compound, with similar composition. On irradiation of phosphorothioate in the presence of potential phosphoryl group acceptor, such as glucose or galactose, 25-40% of the phosphoryl group was transferred. The formation of glucose 6-phosphate, or galactose 6-phosphate was observed. The photolysis of cysteamine-S-phosphate gave cysteamine, inorganic orthophospate and taurine. Under the same conditions of irradiation, inorganic orthophosphate or aminoethanol-O-phosphate were found to be stable.


Biosynthesis of phenylalanine, tyrosine, 3-(3-carbocyphenyl) alanine and 3-(3-carbocy-4-hydroxyphenyl) alanine in higher plants. Examples of the transformation possibilities for chorismic acid. 14C-labelled shikimic acid and double labelled shikimic acid tritiated stereospicifically at C-6 are incorporated into 3-(3-carboxyphenyl) alanine, 3-(3-carboxy-4-hydroxyphenyl) alanine, phenylalanine, and tyrosine in Reseda lutea L., Reseda odorata L., Iris x Hollandica cv. Prof. Blauw, and Iris x hollandica cv. Wedgwood. The experiments with 14C-labelled shikimic acid confirm that the aromatic carboxyl groups and rings in 3-(3-carboxyphenyl) alanine and 3-(3-carbocy-4-hydroxyphenyl) alanine derive from the carbocyl group and ring in shikimic acid whereas the experiments with double labelled shikimic acid demonstrate that the pro-6S-hydrogen atom is retained and the pro-6R-hydrogen atom lost in the biosynthesis of 3-(3-carboxyphenyl) alanine, phenylalanine, and tyrosine in the plants used. 3H was located in the ortho-position in the aromatic rings of phenylalanine and tyrosine but in a position para to the alanine side chain of 3- (3-carboxyphenly) alanine. No 3H was found in 3- (3-carboxy-4-hydroxyphenyl) alanine. This supports a derivation of the last two compounds from chorismic acid via isochorismic acid, isoprephenic acid, and 3'-carboxyphenylpyruvic acid and 3'-carbocy-4'-hydroxyphenylphruvic acid. The 3H/14C ratio in 3-(3-carboxyphenyl) alanine was found higher than in the precursor used. This isotope effect must operate by competition between the pathways from isoprephenic acid to 3'-carbocyphenylpyruvic acid and to 3'-carbocy-4'- hydroxyphenylpyruvic acic. The proposed biosynthetic pathways for the two carboxy-substituted amino acids are in agreement with their distribution patterns in the plant kingdom and suggest that they may derive from minor changes of enzymes involved in the general pathways of aromatic biosynthesis.


Intermediates in the metabolism of m-carboxy-substituted aromatic amino acids in plants. Phenylpyruvic acids, mandelic acids, and phenylglyoxylic acids. Tracer experiments with 14C-labelled precursors in Iris times hollandica cv. Wedgwood, Reseda Iutea L. And Keseda Odorata L. have demonstrated that 3-(3-carboxyphenyl) alanine and 3-(3-carboxy-4-hydroxyphenyl) alanine can be derived from the corresponding pyruvic acids, presumably by unspecific transaminations, and that (3-carboxyphenyl) glycine and (3-carboxy-4-hydroxyphenyl) glycine can be derived from the corresponding phenylglyoxylic acids. The glycine derivatives are derived from the alanine derivatives, and the corresponding mandelic acids are intermediates in these transformations. The corresponding phenylacetic acids are incorporated only slightly into the glycine derivatives, indicating that oxidation at the benzylic position in the C6-C3 compounds takes place early in the transformation. The corresponding cinamic acids are not metabolized at all in the plants.


Biosynthesis of p-aminophenylalanine: part of a general scheme for the biosynthesis of chorisimic acid derivatives. p-Aminophenylalanine is biosynthesized in Vigna vexillata (L.) A. Rich. from shikimic acid through a pathway different from that giving phenylalanine and tyrosine. Experiments with 1,6-14C-labelled shikimic acid demonstrate that the C3-side chain in p-aminophenylalanine is attached to the original C-1 in shikimic acid. The biosynthesis of p-aminophenylalanine in Vigna vexillata probably follows the same pathway as the biosynthesis of this amino acid in Streptomyces species where it is known to be an intermediate in the biosynthesis of chloramphenicol. It is proposed that the biosynthesis takes place through chorismic acid, 4-amino-3-enolpyruvylcyclohexa-1,5-dienecarboxylic acid, 3-(4-amino-1-carboxycyclohexa-2,5-dienyl) pyruvic acid, and 4'-aminophenyl-yruvic acid. It is proposed that chorismic acid can gave rise to 4-amino-3-enolpyruvylcyclohexa-1,5-dienecarboxylic acid, 2-amino-3-enolpyruvylcyclohexa-4,6-dienecarboxylic acid, and isochorismic acid, and that these three compounds and chorismic acid itself by simple rearrangements and elimination reactions can give rise to most known chorismic acid derivatives, i. e.p-hydroxybenzoic acid, 3,4-dihydroxy-3,4-dihydrobenzoic acid, phenylalanine, tyrosine, p-aminobenzoic acid, p-aminophenylalanine, anthranilic acid, 2-amino-3-hydroxy-2,3-dihydrobenzoic acid, 3-(3-carobxyphenyl) alanine, 3-(3-carbocy-4-hydrocyphenyl) alanine, salicylic acid, and 2,3-dihydroxy2,3-dihydrobenzoic acid.


Isolation and partial characterization of two minor glycoproteins from human erythrocyte membranes. Two minor glycoproteins GP-II and GP-III, were isolated from human erythrocyte membranes and characterized chemically and immunologically. The chemical composition of GP-II and GP-III was similar: GP-II consisted of 81% protein and 19% carbohydrate of which 4.9% was hexose, 5.4% hexosamine and 7.8% sialic acid. GP-III consisted of 76% protein and 24% carbohydrate of which 7.6% was hexose, 7.2% hexosamine and 8.1% sialic acid. The amino acid composition of GP-II and GP-III was also similar. GP-II and GP-III, however, differed in chemical composition from the MN glycoprotein. GP-II and GP-III were associated with the blood group activities Ss, I and A, but not with the MN antigens. GP-III had higher blood group activities per mug of protein than did GP-II. The specific activities for the Ss blood group antigens were increased 3-10-fold by purificantion of GP-III from the aqueous phase of chloroform methanol extracts.


Species variability in the modification of erythrocyte surface proteins by enzymatic probes. Bovine and equine erythrocytes have been studied by three different surface modification techniques to investigate the accessibility of the surface components to the external medium. Lactoperoxidase labeling of equine erythrocytes results in a significant labeling of only one membrane component, a 100 000-mol.wt polypeptide corresponding to the membrane-spanning Component III of human erythrocytes. The major sialoglycoprotein of the equine erythrocyte is not labeled. This is in contradistinction to the situation for human and bovine cells, where both components are labeled. The equine membrane sialoglycoprotein is also not markedly affected by pronase, chymotrypsin or trypsin treatment of whole cells under the treatment conditions used, although it can be cleaved by pronase in isolated membranes. Experiments with the isolated glycoprotein show that its cleavage by trypsin is quite selective, whereas cleavage by pronase and chymotrypsin is much more extensive. Labelling of bovine red cells by galactose oxidase treatment followed by reduction with 3H-labeled borohydride yields radioactivity in only one major peak, that corresponding increase in labeling. Equine erythrocytes don not show significant labeling by this technique unless a neuraminidase pretreatment has been performed. Then only the major glycoprotein is labeled. Thus the equine glycoprotein is apparently inaccessible to the cell surface by standard surface modification methods, although it is clearly a surface component. These experiments point out some of the limitations of surface labeling and proteolysis methods in probing the accessibility of membrane components. The results suggest that apparent inaccessibility of the equine glycoprotein is due partially to its structure and partially to its localization in the membrane.


Active secretion of calcium, sodium and chloride by adult rat duodenum in vitro. Active secretion of Ca2+ is observed from the serosal to the mucosal surface across adult rat duodenum in vitro when absorptive Ca2+ flux is saturated by a high [Ca2+]. Sodium and chloride are spontaneously secreted by this tissue with Cl secretion apparently accounting for about one-third of the short-circuit current when there is no absorptive co-transport of Na+.


Galactose transport across the serosal border of rabbit ileum and its role in intracellular accumulation. Unidirectional fluxes of D-galactose across the brush and serosal border of rabbit ileum were determined using the method described previously (Naftalin, R. J. and Curran, P.F. (1974) J. Membrane Biol. 16, 257-278). With ringer [Na] equals 75 meguiv., the Km for galactose influx across the brush-border is 5mM, with 0.1 mM ouabain present K-m equals 50 mM, the V (2.0 munol - CM-2-H-1) remains unaltered. The Michaelis parameters for galactose influx across the serosal border are K-m equals 59 plus or minus 9 mM and V equals 4.7 plus or minus 0.24 mumol-cm-2-h-1 and for efflux K-m equals 85 plus or minus 10 mM and V equals 6.8 plus or minus 0.7 mumol-CM-2-H-1. 2. 2-Deoxy-D-glucose and methyl beta-D-glucopyranoside inhibit galactose entry exclusively at the serosal and mucosal borders respectively, while 3-O-methyl-D-glucose inhibits galactose influx at both borders. 0.1 mM ouabain increases the K1 of 3-O-methylglucose for the serosal transport system (100 mM) is unaffected by ouabain. Inhibition of mucosal galactose transport by ouabain or by competition with other sugars results in a reciprocal increase in exit permeability and decrease in entry permeability. Inhibition of serosal galactose transport results in inhibition of both the entry and exit permeability, entry is more affected. 3. There is a small degree of permeability asymetry at the serosal border to galactose which is reduced by ouabain or removel of Na+ from the Ringer. Uptake of 14C-labelled galactose from the serosal solution into the tissue is also inhibited by addition of ouabain or Na+ removal. It is therefore considered that there is a weak active transport system for galactose at the serosal border. 4. Net transepithelial galactose flux is sufficiently high and serosal permeability to galactose sufficiently low to be consistent with the view that galactose is concentrated within the tissue fluid, after conviction (Naftalin, R.J. and Holman, G.D. (1974) Biochim. Biophys. Acta., 373, 453-470) across the mucosal border because it is reflected at the serosal boundary.


The effect of diamide and glutathione on the uptake of alpha-methyl-D-glucoside by slices of rat kidney cortex. The uptake of alpha-methyl-D-glucoside was stimulated in slices of rat kidney cortex by pretreatment with reduced glutathione. Diamide, an oxidizind agent with high specificity for GSH, caused an inhibition of alpha-methyl-D-glucoside uptake. These effects appeared to be related specifically to GSH, since dithiothreitol and mercaptoethanol did not increase alpha-methyl-D-glucoside uptake, and were not as effective as GSH in reversing the effects of diamide. GSH and diamide had no effect on the uptake of another sugar analog, 3-O-methylglucose, which is not actively transported. Kinetic studies indicated that GSH increased the apparent V without affecting K-m. The results are discussed in terms of the possible role of GSH in the process of sugar transport.


A comparison of a spin-label and a fluorescent cell membrane probe using pure and mixed monomolecular films. Monocular films studies of 12-nitroxide stearic acid and 12-(9-anthroyl) stearic acid reveal that deviations from the behavior of the parent molecule (stearic acid) are as much dictated by the polar, or nonpolar, nature of the probe group as by its size. In mixed films under membrane-like conditions, the spin label probe, 12-nitroxide stearic acid, exhibits positive deviations from ideality and should read too high a fluidity. The picture is, however, complicated by a tendency of this probe molecule to adopt a bent conformation, a tendency apparently enhanced by specific interactions with the lecithin zwitterion. 12-(9-anthroyl) stearic acid, in contrast, shows only negative deviations from ideality in mixed dipalmitoyl lecithin films and should read too low a fluidity.


Demonstration of a class of proteins loosely associated with secretory granule membranes. It is shown, in this study, that rat secretory granule membrane preparations, as prepared by the method of Amsterdam et al. [(1971) J. Cell Biol. 50, 187-200], contain a protein fraction which is removed by washing in isotonic medium. This fraction contains unusually high levels of Pro, Gly and Glx, and appears to label rapidly if the rats are pulsed with [14-c] amino acids prior to removal of the glands. The fraction, which may represent specifically adsorbed secretory protein(s) or peripheral membrane protein, is significant to investigators using this model system to study secretory phenomena.


RNA - DNA hybridization on membrane filters with fragmented mammalian DNA. The possibilities of using fragmented mammalian DNA for hybridization on membrane filters were investigated. The adsorption and release of fragmented DNA were studied as influenced by various factors of the hybridization procedure. It was found that working with preparations sufficiently homogeneous in molecular weight with fragment size 4.8-6.5 S, dissolved in 6 times SSC at neutral pH, the adsorption on filters was almost 100%. After incubation of filters for 18 h in 2 times SSC at 65 degrees C about 50% of the fragmented DNA and 20% of the high molecular weight DNA were released. The degree of release differed for the different families of repeated DNA sequences. Lowest release was obtained with the highly repeated DNA (20%) and highest with the unique DNA (63%), i.e. the release was inversely proportional to the renaturation rate of DNA. In the course of release of fragmented total DNA the material remaining on the filters became enriched in highly repeated sequences, due to selective release of the slowly reassociating fractions. As a result, the percentage of fragmented DNA which hybridized with heterogeneous nuclear RNA was higher than that of high molecular weight DNA. The thermal stabilities of the hybrids with fragmented and high molecular weight DNA were identical. The conditions are defined which permit application of the membrane filter hybridization technique to fragmented mammalian DNA.


Molecular weights of maize mitochondrial and cytoplasmic ribosomal RNAs under denaturing conditions. The molecular weights of maize cytoplasmic and mitochondrial rRNAs were determined by gel acrylamide electrophoresis under non-denaturing and denaturing conditions. The molecular weights of mitochondrial rRNAs (0.76-10-6 and 1.25-10-6) exceeded those of cytoplasmic rRNAs (0.67-10-6 and 1.19-10-6) when electrophoresed in 8 M urea at 60 degrees C. Electrophoresis in 1.1 M formaldehyde resulted in similar values except for heavy mitochondrial rRNA, which exhibited a higher molecular weight than observed in 8 M urea. The observed values for cytoplasmic rRNAs, especially the heavy component, represent a decrease from estimates obtained under non-denaturing conditions. This is the first report of the electrophoretic examination of higher plant rRNAs under denaturing conditions.


On the interaction of ribonuclease U-2 and substrate analogues. 1. The interaction of ribonuclease U-2 (RNAase U-2) with its substrate analogues has been investigated by a gel filtration method. At pH 4.5 and 30 degrees C, the apparent binding strength of the substrate analogues was in the following order; adenylate greater than guanylate greater than inosylate greater than cytidylate among 2'-nucleotides and 2'- greater than 3'- greater than 5'- among adenylate isomers. The formation of an equimolar complex of RNAase U-2 and 2'-nucleotide was indicated from the Scatchard plot. 2. The interaction of RNAase U-2 with 2'-adenylate or 2'-guanylate was observed spectrophotometrically. The complex of RNAase U-2 and 2'-adenylate yielded not only an absorption difference spectrum having a broad positive peak at 280 to 285 nm and a negative trough at 256 nm but also a circular dichroic difference spectrum having a positive peak at around 250 nm and a negative trough at around 290 nm. The complex of RNAase U-2 and 2'-guanylate gave a similar difference spectrum to that of the RNAase T-1 - 3'-guanylate complex, in absorption as well as in circular dichroism.


The cell-free translation of Rauscher leukemia virus RNA into high molecular weight polypeptides. Rauscher leukemia virus (RLV) 65-S RNA, 35-S mengovirus RNA and reticulocyte A-rich RNA each stimulated cell-free protein synthesis in a JLS-V5 cell derived S-30 system. rRNA, however, was not stimulatory in this system. Of the stimulated protein products only those synthesized in response to added RLV RNA were immune-precipitable with anti-RLV rabbit serum. Furthermore, cell-free incubations with pactamycin at a concentration which specifically inhibits initiation and not elongation prevented the stimulation of amino acid incorporation in response to added RLV RNA. Analysis of the polypeptides synthesized by the cell-free system in response to reticulocyte A-rich RNA, showed them to be globin-like and, therefore, also mRNA specific. The RLV RNA-directed product included at least two classes of polypeptides (mol. wts of 140 000-185 000 and 50 000-75 000) both of which were larger than the group specific polypeptides of mature virions. None of the internal structural polypeptides of mature virions were synthesized in response to RLV RNA. The large molecular weight, viral-specific polypeptides are candidate precursor polyproteins which may represent the translational products of a polycistronic mRNA with a single initiation site.


Mechanism of inhbition of protein systhesis initiation by diacetoxyscirpenol and fusarenon X in the reticulocyte lysate system. The mechanism of inhibition of peptide chain initiation by diacetoxyscirpenol and fusarenon X, (trichotecene mycotoxins), was studied in the reticulocyte lysate protein-synthesizing system. The mycotoxins did not inhibit formation of the complex between Met-tRNA-f and the 40S ribosomal subunit nor the initiation codon AUG-promoted combination of the complex with  60S ribosomal subunit. Formyl-methionyl-valine accumulated when the system was incubated with the mycotoxins and fMet-tRNA-f. This suggests that the inhibition occurs after the formation of the first peptide bond.


Schizophrenia and organic brain syndrome with trisomy 8 (group-C trisomy 8 [47, XX, 8+]). A supernumerary autosomal syndrome in the C group of submetacentric autosomes has not yet been delineated phenotypically, although cases of an extra C-group autosome have been reported. Recent technical advances (fluorescence and Giemsa banding) now permit positive identification of each autosome. The present case reports the presence of schizophrenia and organic brain syndrome in association with trisomy 8. It is the sixth case of trisomy 8 reported to date. A stress-diathesis model is used to explain the development of the psychiatric disturbances. The patient's autosomal aberration is regarded as diathetic, and the multiple congenital anomalies, surgical interventions, infections, familial, and other environmental responses as stress. This report provides further data for the phenotypic delineation of a trisomy 8 syndrome.


Dopamine antagonism by thioridazine in schizophrenia. One of the leading current theories of the etiology of schizophrenia is excessive activity of some brain dopaminergic tracts. One of the major objections to the theory is that thioridazine is clinically as effective a treatment of schizophrenia as other neuroleptic drugs but appears to have much less dopamine-blocking properties than these agents in man and laboratory animals. Serum prolactin levels are increased by dopamine receptor-blocking drugs. We have found that thioridazine is as effective as chlorpromazine, trifluperazine, and prolixin enanthate in increasing serum prolactin levels in unmediated schizophrenic patients, indicating it is an effective dopamine-blocking agent.


The origin of land plants: a matter of mycotrophism. It is hypothesized that terrestrial plants are the product of an ancient and continuing symbiosis of a semi-aquatic ancestral green alga and an aquatic fungus-an oomycete. The Siluro-Devonian "explosive" colonization of land, and indeed the very evolution of plants, was possible only through such mutualistic partnerships-partnerships that were equipped to cope with the problems of desiccation and starvation associated with terrestrial existence.


Sodium dodecyl sulfate in protein chemistry. This review summarizes in a brief manner the main aspects of the application of sodium dodecyl sulfate (SDS) to protein chemistry. The principal problems of SDS-polyacrylamide gel electrophoresis are described, as well as the anomalous behavior of protein-SDS complexes and the inactivation of enzymes due to variable binding of SDS to the polypeptides studied. The particular value of SDS in elucidating the protein composition of biological membranes and in membrane-reconstitution experiments is discussed.


[The effect of methyluracil on protein and amino acid concentration in HEp-2 cells]. Methyluracil doses from 1 to 50 mg/ml induced an increase of total nucleinic acids in the nuclei and the protein in the cytoplasm of the HEp-2 cells. 20 mg/ml of methyluracil gave a progressive increase in the nucleinic acid concentration in the cells--from 122 to 197 %. A relatively stable increase of protein at all the time intervals was induced by a dose of 30 mg/ml (132--145%). This is in favour of an increase in cell metabolic processes under the influence of methyluracil.


Peroxidase-H2O2-halide system: Cytotoxic effect on mammalian tumor cells. Myeloperoxidase, H2O2, and a halide constitute a potent antimicrobial system. A cytotoxic effect of this system on a line of mouse ascitic lymphoma cells (LSTRA) is demonstrated here using four different assay systems: 51Cr release, trypan blue exclusion, inhibition of glucose C-1 oxidation, and loss of oncogenicity for mice. Deletion of each component of the system, preheating the peroxidase, or addition of azide, cyanide, or catalase abolished the cytotoxicity. Myeloperoxidase was effective with either chloride or iodide as the halide, while lastoperoxidase was effective with iodide but not chloride. The iodinated thyroid hormones, triiodothyronine and thyroxine, could substitute for the halide, and H2O2 could be replaced by a peroxide-generating enzyme system such as glucose and glucose oxidase or by H2O2 producing bacteria such as pneumococci or streptococci. The possibility is raised that the peroxidases of inflammatory cells and certain biologic fluids may affect tumor initiation or growth in vivo.


Arsenic intoxication as a cause of megaloblastic anemia. We have described a case of chronic arsenic intoxication associated with pancytopenia and megaloblastic erythropoiesis. The patient had the typical laboratory manifestations of effective erythorpoiesis due to a megaloblastic process, including macroovalocytes, mild pancytopenia, low reticulocyte index, increased marrow cellularity with erythroid hyperplasia, and morphologic evidence of megaloblastic maturation in the marrow. The patient's serum folate and vitamin B12 were normal, and the anemia regressed without therapy. Our case suggests that the combination of megaloblastosis with normoblastic or megaloblastic karyorrhexis,should raise the suspicion of arsenic intoxication in the mind of the observer. In addition, arsenic should be added to the list of agents causing a reversible megaloblastic anemia.


Red cell life span in sickle cell-hemoglobin C disease with a note about sickle cell-hemoglobin O ARAB. Red cell survival was measured in ten subjects with S-C disease and one with S-O Arab (alpha 2 beta 2-121 glu yields lys) disease using both DF32p and 51Cr as tags. Red cell volume was slightly reduced in most patients (87% plus or minus 20% of predicted normal). In nine SC patients, mean red cell life (DF32p) was 28.9 plus or minus 4.0 days. For one SC subject it was significantly longer (47.9 days), as it was for the one with S-O Arab. The S-O Arab subject had irreversibly sickled cells in the peripheral blood, shereas those with SC had few (less than 1/1000 red cells) or none. The S-O Arab hemolysate gelled at a hemmoglobin concentration (16.2 g/100ml) near that for sickle cell anemia hemolysates (15.9 plus or minus 1.0 g/100 ml; n equals 8) but significantly lower than that for SC hemolysates (21.6 plus or minus 1.9 g/100 ml; n equals 5). It seems likely that properties of S-C red cells other than their relative ease of sickling contribute significantly to their rate of hemolysis.


Measurement of vitamin B12-binding proteins of plasma. I. Technique. The unsaturated binding capacities (UBBC) of individual vitamin B12-binding proteins in plasma were measured by a two-step procedure. Transcobalamin II (TC II) was separated by precipitation with ammonium sulfate; the "R"-type binders remaining soluble were then divided into two components by bath separation with anion exchange on DEAE-cellulose. The two R components were designated alpha1-R (TC 1) and alpha2-R (third binder, fetal binder, PV binder, TC III). Ten normal sera were studied by this technique giving a separation into TC III and total plasma R identical to that obtained simultaneously by gel filtration. The mean UBBC of TC II was 969 plus or minus 204 pg of 57 Co B12 per ml of serum. The mean contamination of the precipitated TC III with plasma R was 3%. The UBBCs of alpha 2-R and alpha 1-R were 127 plus or minus 42 and 40 plus or minus 12 pg/ml, respectively. The mean contamination of the R fraction by TC II was 14% as evaluated by gel filtration. By isoelectric focusing it was found that the alpha1-R contained principally those components isoelectric at pH isoelectric at pH of 2.9-3.2, while alpha2-R was made up of those components isoelectric at pH of 3.6 or greater.


Some factors affecting fibrinogen precipitation by ristocetin: ultrastructure of precipitates. Fibrinogen in aqueous solution is precipitated by the antibiotic ristocetin. This reaction is inhibited by albumin and facilitated by low temperature. Resolubilized fibrinogen clots in the presence of thrombin. Ristocetin-precipitated fibrinogen takes the form of fibrils or clumps, composed of irregularly spaced, structure-less particles. The addition of ristocetin to washed platelets suspended in fibrinogen-containing media produces fibrinogen clumps in both the media and in the surface cannalicular system of the platelets. The changes in light transmission (aggregation curves) are due to both platelet aggregation and fibrinogen clumping. The role of the latter is confirmed by the observation that the addition of ristocetin to inert latex particles suspended in fibrinogen solution produces typical aggregation curves. This phenomenon is prevented by the addition of albumin to the media. We conclude that (1) if fibrinogen is present in any artificial system, albumin should be included in the media to prevent fibrinogen precipitation; and (2) statements about aggregation of any particulated materials by ristocetin should not be based solely on light-transmission changes, but should also include a description of the morphologic appearance.


Direct effects of thyroid hormones on bone marrow erythroid cells of rats. A stimulatory effect on bone marrow cellularity was observed in normal and nephrectomized rats continuously infused with T3 and T4. Results of bone marrow studies are expressed in absolute numbers of total nucleated erythroid cells per milligram of femoral marrow at the beginning and after 8 hr of continuous intravenous infusions. Administration of T3 and T4 to nephrectomized rats produced a marked and significant increase in total erythroid cells counted. After differential analyses of the nucleated erythroid elements, a significant increase in all erythroid cell types was also observed. Similar results were seen in a control group of rats in which both ureters have been previously ligated and in groups of nephrectomized rats receiving rabbit antiserum against erythropoietin before starting the intravenous infusions of T3 and T4. These results indicate that stimulation of marrow erythropoiesis produced by thyroid hormones in our system is not dependent on renal or extra-renal production of erythropoietin. The progressive introduction of T3 and T4 into the circulation of rats with bilateral nephrectomy or ureter-ligated normal rats, may overload the mechanism of transport of these hormones in plasma. As a consequence, a progressive increase in free active forms of T3 and T4 in plasma may occur. Our interpretation of the present findings is that thyroid hormones stimulate directly bone marrow erythropoiesis. This stimulation is clearly evident when high levels of free active forms of thyroid hormones are present in plasma.


Ferritin iron absorption in man.

Ferritin iron absorption in man. The iron absorption from ferritin and hemosiderin biosynthetically labeled with radioiron was studied in 108 subjects. The geometric mean absorption of ferritin iron in both normal and iron-deficient subjects was 1.9 percent. Its mean absorption ranged from 0.9 percent in normal subjects to 2.5 percent in subjects with moderate iron deficiency and 5.7 percent in subjects with marked iron deficiency. The administration of this iron compound with vegetals in a meal showed distinctly lower absorption values than the absorption from either maize, wheat, or soybean. Ferritin iron absorption was also different from that of ferric chloride when they were administered together as a drink or mixed with maize or liver. The iron absorption from ferritin was markedly increased when it was administered with either meat or liver, but it did not reach the absorption level of these foods. It is still to be elucidated whether the difference in iron absorption between ferritin and vegetable foods administered together reflect that this iron is incompletely miscible with a nonheme iron pool or that it really forms a third iron pool.


Pressure-volume characteristics of foot veins in normal cases and patients with venous insufficiency. In order to evaluate pressure-volume characteristics of foot veins in patients with venous insufficiency compared with a control group, the foot-volumetric method was utilized, combined with intravenous pressure measurements.  Calculations of compliance and elastance were preformed within a fixed pressure interval, where the veins were filled or almost filled.  The investigation demonstrates a lower compliance and a higher elastance in the varicose veins compared with the control cases.  This is contrary to most previous investigations, in which the veins have not been fully distended, as the measurements were preformed in the lying position with rather low venous pressure.  Changes of compliance and elastance were most marked in cases with advanced venous disease and skin changes of the ankle region.  The observed changes of a 'greater stiffness' of the foot-vascular system can be explained by fibrotic or phlebosclerotic changes.  In addition to the observations of slightly altered elasticity factors, we observed volume changes after exercise which seem to be related to capillary filtration.


Inhibition of in vivo Neural Vasoconstriction by exogenous catecholamines. Intaarterial infusions of norepinephrine and dopamine markedly attenuated the mesenteric vasoconstrictor response to periarterial nerve stimulation in anesthetized cats, but did not attenuate the response to intravenous norepinephrine.  The degree of attenuation was independent of stimulation voltage, but inversely related to frequency.  The results are compatible with previous in vitro evidence that NE and DA decrease adrenergic transmitter release during nerve stimulation.


Differentiation of calcium activation mechanisms in vascular smooth muscle by selective suppression with verapamil and D 600-1. Mechanical activity of the isolated portal vein and thoracic aorta of the guinea-pig was recorded and the effects of verapamil and D 600 (methoxy-verapamil) on the dose-response curves to noradrenaline were measured. Extracellular electrical activity in portal vein was also sometimes recorded. Two calcium activation mechanisms could be differentiated: a "spike activation mechanism" (SAM) inhibited by verapamil and D 600, and a "spike-free activation mechanism" (SFAM) resistant to these antagonists in their specific concentration range (up to 10-minus 5 mol/1). In portal vein, both mechanisms were similarly dependent on extracellular calcium, indicating a D 600-resistant system for transmembrane calcium fluxes. The response of portal vein to increased potassium concentration was also tested. Species differences and differences in the specificity of various calcium antagonistic drugs complicate the picture of calcium antagonism in vascular smooth muscle.


Vibration-induced inhibition of vascular smooth muscle contraction. Vascular smooth muscle is known to be exposed to an oscillating strain under physiological and patho-physiological conditions as well as in different occupational and environmental situations.  The effect of vibrations of smooth muscle seems to be largly unknown.  In the present experiments on isolated preparations of the rat portal vein and the rabbit thoracic aorta, imposed sinusoidal changes in length were found to cause prompt reduction in active force, the extent of which was dependent on amplitude (1-10% of tissue length, peak to peak, i.e. approximately plus or minus 50-500 mum) and frequency of  vibration (1-400 Hz) as well as on the prevailing level of active and passive forces.  Vibration caused only small and inconsistant reductions of passive force of vascular smooth muscle.  The results are in accordance with the hypothesis that vibrations exert a direct action on the contractile process by causing an increased rate of detachment of actin-myosin cross-links.  It is suggested that, in vivo, vibrations may affect the diameter of conduit arteries locally in the case of turbulent blood flow as seen in post-stenotic dilation and arterio-venous anastomosis.  Possibly, even the normal pulse pressure oscillations may sometimes tend to inhibit the smooth muscle activity in such arteries and thereby influence their diameters.


Ajamline-induced changes in mechanical and electrical activity of vascular smooth muscle. The effects of ajmaline on vascular smooth muscle were studied using helical aortic strips and portal veins of male rats. This report is based on the results of 104 mechanical experiments. In 30 additional experiments electrical activity was recorded simultaneously at different points of the preparation using extracellular methods (liquid paraffine or pressure electrodes technique). Ajmaline induces relaxation of aortic helical strips (activated by 2.0 mug/l norepinephrine) to 75 percent of initial tension in a dose of 0.6 mg/l, to 50 percent by 2.0 mg/l, and to 25 percent by 4-5 mg/l. The relaxation slope is shifted to the right by increasing the [Ca++]o from 2.0 to 4.0 mM, by increasing the initial norepinephrine concentration to 4.0-10 mug/l, or by KCl depolarization, [K+]o ranging from 15 to 60 mM. The relaxing effect of ajmaline on aortic strips can partly be attributed to a change in electrical activity with a dose-dependent conduction impairment or block and, at high concentrations, also a decrease in the frequency of pacemaker excitations. Experiments on aortic strips in K+ contracture show relaxation independent of changes in phasic electrical events. Ajmaline ranging from 0.2 to 80-100 mg/l causes on the portal vein a marked increase in amplitude and a small decrease in frequency of rhythmical contractions. Integrated isometric force reaches 300 percent of initial values. The increasing amplitude of contractions is related to a prolongation of excitation trains, while the frequency and amplitude of the individual spike are reduced. Our results suggest that the effects of ajmaline on the mechanical and electrical activity of vascular smooth muscle may be partly related to a reduction in Ca++ and probably Na+ conductance.


Hyperglucagonaemia in the surgical patient. Twenty-one patients had serial samples of blood taken before, during, and after operation for the measurement of plasma glucagon, plasma insulin, and blood glucose concentrations. A significant rise in plasma glucagon level was noted during the operation. In contrast the plasma insulin concentration fell during the operation and rose in the postoperative period despite hyperglycaemia during and after the operation. These findings show that hyperglucagonaemia is a physiological consequence of a surgical operation and that the relationship of plasms glucagon to plasma insulin is complex.


I alpha-hydroxycholecalciferol: a treatment of renal bone disease. Three patients with chronic renal failure on maintenance haemodialysis have been treated with 1 alpha-hydroxycholecalciferol (1 alpha-OHCC), a synthetic vitamin D analogue. A daily dose of 2 mug by mouth produced a significant increase in both calcium absorption from the gastrointestinal tract and calcium content of bone. Treatment with 1 alpha-OHCC appears to be effective in cases of metabolic bone disease associated with chronic renal failure.