Evaluation of the mutagenicity of aminoglycoside antibiotics in Salmonella typhimurium and Saccharomyces cerevisiae. The mutagenicity of aminoglycoside antibiotics (KM, AKM, DKB, RSM, AMK, GM, TOB) has been studied in cells of the bacteria Salmonella typhimurium and in the yeast Saccharomyces cerevisiae. The bacterial strains (Ames') monitor reverse mutation (point mutation) and the yeast strain D5 monitors mitotic crossing-over, mitotic gene conversion and point mutation. None of these antibiotics demonstrated any mutagenic activities in either the bacteria or the yeast.


Rapamycin (AY-22,989), a new antifungal antibiotic. IV. Mechanism of action. Rapamycin, an antifungal antibiotic produced by Streptomyces hygroscopicus showed a strong candicidal activity, which could not be reversed by sterols. It has no effect on efflux of K+, Pi ir U.V. absorbing materials and cell permeability of Candida albicans. Thus, in its action it differs from the polyenes. Mechanism of action of rapamycin appears to be different from many known antifungal agents. In C. albicans, rapamycin at the minimum growth inhibitory concentration inhibited: 1) phosphate incorporation into nucleic acids, 2) acetate incorporation into lipids and 3) substrate respiration of amino acids. The effect on amino acid metabolism was expressed as inhibition of oxidative deamination. At low concentrations rapamycin caused degradation of P32-labeled intracellular macromolecules. Inhibition of threonine incorporation into cell wall and leucine incorporation into cellular protein was observed at relatively higher concentrations of rapamycin. The antibiotic showed no effect on cell-free protein synthesizing systems of Escherichia coli, rat liver and C. albicans and in the mitochondrial enzyme systems. Whether the lethal action of rapamycin on C. albicans is primarily due to one of the above effects or is the result of combined effect on some of these biosynthetic parameters remains to be established.


Effects of relaxation training on pulmonary mechanics in children with asthma. An experiment, designed to overcome shortcomings in previous work, was conducted to investigate the potential symptomatic benefits of relaxation training in the treatment of asthma in children. Fourteen chronic, severely asthmatic children received three sessions in which they rested quietly, followed by five sessions of relaxation training, and finally three sessions of relaxing as trained previously. Pulmonary function was assessed, in a manner far more definitive than in previous studies, before and after each session, and three additional times at 30-minute intervals thereafter. Tension in the frontales muscles, heart and respiration rates, and skin temperature and conductance were also monitored. Heart rate and to some extent muscle tension results tended to confirm the attainment of relaxed states. However, the lung function results failed to substantiate the previous, preliminary findings of a clinically meaningful change in pulmonary function following relaxation. The status of relaxation in the treatment of asthma was discussed.


Relaxation treatment of pseudoinsomnia and idiopathic insomnia: an electroencephalographic evaluation. Twenty-nine insomniacs underwent four consecutive sleep laboratory evaluations before and after receiving tension-release relaxation training, no-tension-release relaxation training, or no-treatment. On the basis of the discrepancy between subjective and EEG-defined measures of latency to sleep onset, subjects were classified as pseudoinsomniacs or idiopathic insomniacs. As predicted, tension-release relaxation was significancy more effective than the other two conditions on subjective sleep measures, regardless of insomnia subtype and on objective sleep measures only for idiopathic insomniacs. Subjective improvement was maintained at 12-month followup. Numerous differences between the two subtypes emerged on pretherapy and during-therapy measures distinct from the latency measures, but changes on those variables were unrelated to outcome improvement.


A simple and rapid purification method for Escherichia coli DNA polymerase I. We report a simple, three-step method for the purification of Escherichia coli DNA polymerase I. Its advantages over other procedures are ease and rapidity, the absence of an autolysis or any high speed centrifugation step, and applicability to large quantities of material. In addition, RNA polymerase can be isolated as a by-product. We have applied this method to purify DNA polymerase both from wild type E. coli cells and from cells bearing a lambda prophage carrying the polA gene (Kelley, W.S., Chalmers, K., and Murray, N.E. (1977) Proc. Natl. Acad. Sci. U.S.A. 74, 5632-5636). This latter source amplifies the amount of DNA polymerase in the cells by at least 10-fold.


Differential inhibition of host and viral thymidylate synthetases by folylpolyglutamates. The ability of folate analogues to inhibit host and viral thymidylate synthetases was measured using the corresponding Escherichia coli and T2-phage-induced enzymes. In the absence of Mg2+, 6 x 10(-7) M pteroylhexaglutamate inhibited the T2-phage-induced synthetase by 50%, but at least 100-fold greater levels of this compound were necessary to inhibit the E. coli synthetase by this amount. At 2.5 x 10(-6) M pteroylhexaglutamate, at least 80% inhibition of the T2-phage synthetase could be obtained with little or no inhibition of the E. coli enzyme. The pteroylmonoglutamate was about 2 orders of magnitude less inhibitory towards the T2-phage enzyme than the pteroyltri- to -heptaglutamates. However, upon addition of Mg2+ to the assay mixture, the inhibition produced by pteroylhexaglutamate was essentially reversed, with the E. coli synthetase now increasingly inhibited by this compound and the T2-synthetase only minimally impaired. Methotrexate and N10-formyl-2-amino-4-hydroxyquinazoline, although inhibitory to both enzymes in the presence or absence of Mg2+, did not show this differential selectivity. These results suggest that certain folate analogues may be useful in distinguishing between a host and an infecting organism's thymidylate synthetase and could thus provide an additional means of screening for potential chemotherapeutic agents.


Location of the sugar-binding site of L-arabinose-binding protein. Sugar derivative syntheses, sugar binding specificity, and difference Fourier analyses. The sugar-binding site of the L-arabinose-binding protein, an essential component of the high affinity L-arabinose uptake system in Escherchia coli, is located deep in a cleft formed by the asymmetric contributions from both of the two similar domains. The site was unambiguously identified with the electron-rich substrate analog 6-bromo-6-deoxy-D-galactose in a difference Fourier analysis. The observation that the original native structure might have been solved with bound L-arabinose necessitated the synthesis of a heavy atom analog, its structure consistent with the known sugar-binding specificity of the protein. Difference Fourier maps (3.5 A) of crystals soaked in 46 mM analog showed a peak 3.5 times background, which is attributed to the -CH2Br moiety of the analog. Superposition of a difference map onto a 2.8-A native electron density map indicated that the difference peak is 6 to 7 A from the reactive single cysteine (Cys-64) and partially coincident with an "extraneous" density found in the native map. This "extraneous" peak was previously attributed to a bound L-arabinose molecule, and its presence accounts for the early failures of difference Fourier analyses of crystals soaked in or co-crystallized with L-arabinose to locate the sugar-binding site.


The properties of a bacteriophage T5 mutant unable to induce deoxyuridine 5'-triphosphate nucleotidohydrolase. Synthesis of uracil-containing T5 deoxyribonucleic acid. Bacteriophage T5 induces a deoxyuridine 5'-triphosphate nucleotidohydrolase (dUTPase) activity during infection of Escherichia coli. A T5 mutant (T5 dut) unable to induce this dUTPase activity has been isolated. Although this mutant is viable, the E. coli dUTPase activity is not sufficiently active to exclude uracil from the progeny DNA and about 3% of the thymine is replaced by uracil. When the mutant is grown in an E. coli dut host about 12% of the thymine in the progeny DNA is replaced by uracil. T5 phage containing 12% uracil can replicate in uracil-DNA glycosylase-deficient (ung) hosts with high efficiency, but fail to replicate in ung+ hosts. The amount of thymine replaced by uracil in the progeny produced in dut hosts is nearly independent of the ung genotype, indicating that the host uracil-DNA glycosylase-dependent repair pathway is not operating efficiently to remove uracil from T5 progeny DNA.


Stimulation of the glucose transport system in isolated mouse pancreatic acini by cholecystokinin and analogues. Cholecystokinin and analogues increased the uptake of 2-deoxy-D-glucose and 3-O-methylglucose into isolated mouse pancreatic acini. This uptake was mediated by a facilitated glucose transport system that was saturable, stereospecific, and was inhibited by both phloretin and cytochalasin B. In agreement with previous studies of acinar function, caerulein was more potent and pentagastrin less potent than cholecystokinin in increasing sugar transport. The cholinergic analogue carbachol mimicked the effect of caerulein; atropine completely abolished the effects of carbachol but was without influence on the effects of the polypeptide hormones. In contrast, secretion, as well as dibutyryl cyclic AMP and dibutyryl cyclic GMP, had no effect on 2-deoxy-D-glucose uptake. Two lines of evidence suggested that hormonal stimulation of this sugar transport system was related to mobilization of cellular Ca2+. First, depletion of cellular Ca2+ by incubation of acini with ethylene glycol bis(beta-aminoethyl ether) N,N,N',N'-tetraacetic acid (EGTA) reduced the effect of caerulein. Second, the Ca2+ ionophore A23187 mimicked the effects of caerulein on 2-deoxy-D-glucose uptake when Ca2+ was present in the medium.


Conformational and ligand binding properties of the isolated domains from the beta 2 subunit of Escherichia coli tryptophan synthetase investigated by the reactivity of their cysteines. A mild proteolytic treatment of the dimeric beta 2 subunit of Escherichia coli tryptophan synthetase (L-serine hydrolase (adding indole) EC 4.2.1.20) is known to nick each polypeptide chain into two complementary fragments, F1 and F2 (Högberg-Railbaud, A., and Goldberg, M.E. (1977) Proc. Natl. Acad. Sci. U.S.A. 74, 442-446). The reactivity of the cysteines in the isolated or associated fragments is studied and used to characterize the structural and functional properties of these fragments. It is shown that the total number of cysteines, their reactivity to dithiobisnitrobenzoate, and their protection by various ligands are the same in the nicked and intact enzyme, thus demonstrating the close structural analogy between these two proteins. In the isolated F1 fragments two cysteines are reactive and two are buried, thus confirming that this fragments has a compact, globular structure. Various ligands tested fail to produce any modification of the cysteines in the isolated fragments, thus suggesting that none of the fragments alone carries a binding site for the substrates and coenzyme.


The effect of template secondary structure on vaccinia DNA polymerase. Vaccinia virus DNA polymerase will utilize a substrate consisting of phi X174 DNA primed with a strand of a unique restriction fragment, but the reaction is inefficient. Examination of the reaction products by alkaline agarose gel electrophoresis revealed a few discrete fragments, each corresponding to an extended primer strand. This result implies that specific barriers exist on the phi X174 template which impede, but do not completely halt, the progress of the enzyme. Only a few per cent of the template molecules were completely copied. Similar findings were reported by Sherman and Gefter using Escherichia coli DNA polymerase II and fd DNA (J. Mol. Biol. (1976) 103, 61-76). Several observations suggest that the barriers are regions of template secondary structure. Some barriers are more effective than others, and they increase in both effectiveness and number as the temperature is decreased. The same barriers are observed with T4 DNA polymerase, but none are detected with E. coli DNA polymerase I. Finally, the major barriers are located in regions of the phi X174 sequence known to contain hairpin structures of relatively high stability. The exact stopping point of one of the major barriers is within the duplex stem of a hairpin structure. These results show that DNA polymerases are a useful probe of the secondary structure of a single-stranded DNA.


The role of glyoxylate in the regulation of biodegradative threonine dehydratase of Escherichia coli. The activity of biodegradative threonine dehydratase of Escherichia coli K12 was reversibly inhibited by glyoxylate in the presence of AMP. Kinetic analysis showed that the inhibition was mixed with respect to L-threonine and competitive in terms of AMP; the inhibitory effect of glyoxylate was less pronounced at high protein concentrations. Incubation of dehydratase with L-threonine shifted the absorption maximum of the enzyme-bound pyridoxal phosphate from 413 to 425 nm; addition of glyoxylate completely prevented the threonine-mediated spectral shift. In addition to the inhibitory effect, incubation of purified enzyme with glyoxylate resulted in a progressive, irreversible inactivation of the enzyme and formation of inactive protein aggregates. The rates of inactivation were decreased with increasing concentrations of protein and AMP. During inactivation by glyoxylate, the 413-nm absorption maximum of the native enzyme was replaced by a new peak at 385 nm. Experiments with [14C]glyoxylate showed a rapid binding of 1 mol of glyoxylate per 147,000 g followed by a slow binding of 3 additional mol of glyoxylate; the glyoxylate-protein linkage was stable to acid precipitation and protein denaturants. Competition binding experiments revealed that pyruvate (which also inactivated the E. coli enzyme, Feldman, D.A., and Datta, P. (1975) Biochemistry 14, 1760-1767) did not interfere with the binding of glyoxylate or vice versa, suggesting that the two keto acids may occupy separate sites on the enzyme molecule. Nevertheless, experiments on enzyme inactivation using glyoxylate plus pyruvate reveal mutual interactions between these ligands in terms of lack of additive effect, retardation in the spectral shift due to glyoxylate, and stabilization of the enzyme in the presence and absence of AMP. We conclude from these results that the control of biodegradative threonine dehydratase is governed by a complex set of regulatory events resulting from reversible and irreversible association of these effectors with the enzyme molecule.


Quantitative analysis of two-dimensional electrophoretograms. A method for quantitative analysis of complex film density distributions in autoradiograms is described. The method is intended particularly for measuring the distribution of radioactivity among the proteins resolved by two-dimensional gel electrophoresis but should, of course, be suited to analyzing other two dimensional separations. The film density distribution is first digitized by a high speed rotating drum scanner to generate the image data array that is stored on a magnetic disk. Subsequent analysis involves: 1) data averaging, 2) detection of contours and of their locations, 3) splitting of overlapping spots, 4) conversion of film density to radioactive intensity by means of calibration films, and 5) differentiation and integration to measure the total radioactivity contained in the protein which generates a spot in the autoradiogram. The product of the analysis is a numbered contour map and a table listing coordinates and radioactivity content of each resolved spot. Coordinate transformations for comparison and matching of autoradiograms are also described. A set of utility programs print and graph the data at intermediate stages of the analysis in order to facilitate the checking of procedures and programs.


Purification and properties of L-Aspartate-alpha-decarboxylase, an enzyme that catalyzes the formation of beta-alanine in Escherichia coli. L-Aspartate-alpha-decarboxylase, an enzyme that catalyzes the production of beta-alanine, has been purified to apparent homogeneity from Escherichia coli. The properties of the enzyme are: (a) pH optimum of 6.8 to 7.5, (b) temperature optimum of 55 degrees C, (c) Km for L-aspartate of 0.16 mM, and (d) molecular weight of 58,000. The activity of the enzyme is inhibited by reagents (hydroxylamine, phenylhydrazine, and sodium borohydride) that react with carbonyl groups, but no pyridoxal phosphate is present. The compound containing the carbonyl group has been identified as covalently bound pyruvate. Approximately 1 mol of pyruvate was found/mol of enzyme. That the enzyme has a biosynthetic function rather than a catabolic role is indicated by the observations that a mutant (designated as E. coli 99-2) which requires either beta-alanine or pantothenic acid for growth contains only trace amounts of enzyme activity, whereas it is present in substantial amounts in the parent strain (E. coli W) and in a spontaneous revertant of the mutant.


Isolation and characterization of recombinant clones containing the chicken adult beta-globin gene. We have isolated and characterized two independent clones containing the chicken adult beta-globin gene. Each clone contains a 6.2-kilobase-pair Eco RI restriction fragment of chicken erythrocyte DNA inserted into the vector, lambda gtWES . lambda B. The orientation of the inserted fragment is opposite in the two clones. Characterization of the clones by electron microscopic R-loop studies, by restriction enzyme mapping, and by filter hybridization shows that the adult beta-globin gene is interrupted by at least one small and one large intervening sequence. In addition to the complete adult beta-globin gene, at least part of a second beta-globin-like gene was identified about 2.7 kilobase pairs from the 3'-end of the adult gene. The two independent clones, while very similar, do differ at two Msp I restriction endonuclease sites in regions flanking the adult beta-globin gene.


Evidence for mitochondrial localization of the hormone-responsive pool of Ca2+ in isolated hepatocytes. Conditions are described that allow chlortetracycline, a fluorescent probe of membrane-associated Ca2+, to monitor the content of the major exchangeable pool of intracellular Ca2+ present in the isolated rat hepatocyte. Chlortetracycline fluorescence is decreased in cells whose Ca2+ content is diminished by treatment either with carbonylcyanide-m-chlorophenylhydrazone or with ionophore A23187. Norepinephrine releases Ca2+ from this exchangeable pool and decreases both the fluorescence signal and its subsequent response to A23187. Previous suggestions that chlortetracycline fluorescence is localized in the mitochondria of liver and other cells is supported by comparison of the fluorescence that follows the addition of chlortetracycline to intact hepatocytes and to isolated hepatic microsomes and mitochondria. Identification of the hormone-responsive pool of Ca2+ with the mitochondria is strengthened by comparison of the total calcium content of mitochondria isolated from control and hormone-treated animals. The uptake and release of Ca2+ in control and hormone-treated hepatocytes rendered permeable by treatment with digitonin is also consistent with this interpretation.


Regulation of glycogen synthesis and glucose utilization in Escherichia coli during maintenance of the energy charge. Quantitative correlation of changes in the rates of glycogen synthesis and glucose utilization with simultaneous changes in the cellular levels of both glucose 6-phosphate and fructose 1,6-diphosphate. Treatment of nitrogen-starved cultures of Escherichia coli W4597(K) with sodium azide results in simultaneous changes in both glucose 6-phosphate and fructose 1,6-diphosphate as well as in the rate of glycogen synthesis. Based on these observations, a comprehensive equation was developed which relates the cellular levels of both of these hexose phosphates with the rate of glycogen synthesis. This relationship apparently represents the interaction in vivo between the rate-limiting enzyme of bacterial glycogen synthesis, glucose 1-phosphate adenylyltransferase (adenosine diphosphoglucose synthetase, EC 2.7.7.27), and its substrate glucose 1-phosphate (reflected by glucose 6-phosphate) and its major allosteric activator fructose diphosphate. The form of the equation that describes this relationship was determined from studies presented here of the kinetic properties of the E. coli W4597(K) enzyme in the presence of physiological concentrations of its substrates and modulators. We show here and in subsequent reports of this series that the comprehensive relationship between glycogen synthesis and hexose phosphates can serve as a reference to evaluate the possible participation of new factors in the regulation of glycogen synthesis. Treatment with NaN3 did not change the cellular level of glucose 1-phosphate adenylyltransferase. The value of the adenylate energy charge, (ATP + 1/2 ADP)/(ATP + ADP + AMP), was maintained despite losses of up to 35% in cellular adenylates. The quantitative co-variance between hexose phosphates and the cellular rate of glucose utilization that we previously described for other metabolic conditions was also observed in the azide-treated cultures. We integrate the new information into the system of coordinated regulation of glycogen synthesis, glycolysis, and glucose utilization that we proposed previously.


Evidence for new factors in the coordinate regulation of energy metabolism in Escherichia coli. Effects of hypoxia, chloramphenicol succinate, and 2,4-dinitrophenol on glucose utilization, glycogen synthesis, adenylate energy charge, and hexose phosphates during the first two periods of nitrogen starvation. We studied the effects of decreased aeration, chloramphenicol succinate, and 2,4-dinitrophenol on the cellular rates of glycogen synthesis and glucose utilization and on the cellular concentrations of adenine nucleotides, glucose 6-phosphate, fructose 1,6-diphosphate, and phosphoenolpyruvate during the first two periods of nitrogen starvation of Escherichia coli W4597(K). A quantitative relationship between the changes in the rates and the accompanying changes in the hexose phosphates is demonstrated. However, the relationship for glycogen synthesis is different in different sets of metabolic conditions. We suggest that this difference reflects a change in the steady state level of a previously unknown effector of ADP-glucose synthetase (glucose 1-phosphate adenylyltransferase, EC 2.7.7.27) the rate-limiting enzyme of bacterial glycogen synthesis. We show that the properties of the hypothetical in vivo effector are consistent with the inhibitory effects of ppGpp (guanosine 3'-diphosphate 5'-diphosphate) and pppGpp (guanosine 3'-diphosphate 5'-triphosphate) on this enzyme in vitro. In addition, tetracycline, an inhibitor of the synthesis of these nucleotides, apparently prevents the change in the quantitative relationship. The relationship between glucose utilization and the hexose phosphates is altered at the transition to Period II of nitrogen starvation. We propose that this change reflects the alteration of the cellular steady state level of an unknown effector of the glucose phosphotransferase system. In contrast to the ATP-hexose phosphate system of shared regulatory effects, the specific effects of the unknown effectors allow the rates of glucose utilization and glycogen synthesis to be altered independently of each other and independently of changes in the rate of glycolysis. This independence allows a greater latitude of response for the individual pathways in more severe metabolic stress or in accommodating the metabolic changes necessary for long term survival.


Biosynthesis of yeast mannoproteins. Synthesis of mannan outer chain and of dolichol derivatives. The Saccharomyces cerevisiae mutants affected in the structure of mannan outer chain were found to synthesize dolichol diphosphate-linked oligosaccharides identical in size to those of the wild type strain. The mannosyl transferases involved in the synthesis of the outer chain had an absolute requirement for manganese ions and were activated when enzymatic preparations were stored at 2 degrees C, whereas the transferases responsible for the formation of dolichol monophosphate mannose and dolichol diphosphate oligosaccharides were drastically inactivated from the onset of storage and required magnesium or manganese ions, the former being more effective than the latter. Both sets of enzymes could be separated by ion exchange chromatography. In vitro conditions that enhanced the synthesis of dolichol monophosphate mannose did not stimulate the incorporation of mannose residues into the outer chain. It is concluded that dolichol monophosphate mannose is not an intermediate in the synthesis of the outer chain and that this part of mannan and the dolichol diphosphate oligosaccharides are synthesized by different mannosyltransferases.


The primary structure of calf chymosin.

The primary structure of calf chymosin. The complete amino acid sequence of calf chymosin (rennin) (EC 3.4.23.4) has been determined. The sequence consists of a single peptide chain of 323 amino acid residues. The primary structure of the precursor part of calf prochymosin was published previously (Pedersen, V.B., and Foltmann, B. (1975) Eur. J. Biochem. 55, 95-103), thus we are now able to account for the total 365 amino acid residues of calf prochymosin. Comparison of the sequence of calf prochymosin with that of pig pepsinogen A (EC 3.4.23.1) shows extensive homology. In the precursor part of the sequence, 15 residues are located at identical positions, as compared to 189 identical residues in the respective enzymes. Furthermore comparison to Penicillium janthinellum acid proteinase (penicillopepsin) (EC 3.4.23.7) shows that 76 residues are common to this enzyme and to the two gastric proteinases. These homologies in sequence further suggest that the folding of the peptide chain in chymosin is very similar to that of other acid proteinases.


Purification and molecular characterization of two inhibitors of yeast proteinase B. A rapid purification procedure for large scale preparations of yeast proteinase B inhibitors 1 and 2 (IB1 and IB2) is described. By disc gel electrophoresis, amino acid analysis, and end-group determinations, each of the inhibitors is homogeneous. Both inhibitors are polypeptides with molecular weights of 8,500, containing 74 residues. No components other than amino acids could be detected. There is no significant difference in the amino acid compositions of the two inhibitors as analyzed after acid hydrolysis. Both polypeptides are characterized by the total absence of arginine, tryptophan, and sulfur-containing amino acid residues. The proteinase B inhibitors of yeast, therefore, differ fundamentally from proteinase inhibitors of many other organisms, which generally contain a large number of disulfide bridges. Both proteinase B inhibitors have threonine as the NH2-terminal residue and -Val-His-Thr-Asn-COO- as the COOH-terminal sequence. Comparison of peptide maps after tryptic digestion reveals that the two inhibitors differ definitely in only a few tryptic peptides. The inhibitors are rapidly inactivated by digestion with carboxypeptidase A from bovine pancreas at pH 8.5. Inactivation occurs stoichiometrically with the release of threonine, the penultimate residue at the COOH-terminal end of both inhibitors.


A pleiotropic defect of membrane synthesis in a thermosensitive mutant tsC42 of Escherichia coli. Synthesis of membrane proteins in a thermosensitive mutant of Escherichia coli K12, tsC42, that has a defect in a mechanism of cell cycle-dependent duplication of membrane enzymes was examined by sodium dodecyl sulfate polyacrylamide gel electrophoresis. The cells were labeled differentially with [14C]- and [3H]arginine and the membrane proteins synthesized at nonpermissive and permissive temperatures were compared. The results showed that at the nonpermissive temperature, the syntheses of cytoplasmic membrane proteins and outer membrane proteins were reduced more than 70% and 50%, respectively. No significant accumulation of precursor molecules of membrane proteins at the nonpermissive temperature was detected in pulse-chase experiments. It is therefore assumed that the mutant has a defect in a gene that regulates the biosynthesis of many membrane proteins.


Fumarate reductase of Escherichia coli. Elucidation of the covalent-flavin component. Fumarate reductase is a membrane-bound terminal oxidase which is induced when Escherichia coli is grown anaerobically. The purified enzyme is composed of two polypeptide chains of 69,000 and 24,000 daltons and contains 1 mol of covalently bound flavin adenine dinucleotide per mol of enzyme. Fluorescence scanning of SDS-polyacrylamide gels of the protein shows that the flavin is attached to the large subunit. The hypsochromic shift of the 372 nm band of riboflavin to 350 nm in both native fumarate reductase and a flavin peptide released by proteolytic digestion indicates that the flavin is attached via position 8 alpha of riboflavin. Based on the spectral properties and pH-fluorescence dependence we have identified the linkage as 8 alpha-[N(3)-histidyl]FAD.


Gene amplification causes overproduction of the first three enzymes of UMP synthesis in N-(phosphonacetyl)-L-aspartate-resistant hamster cells. Mutant Syrian hamster cells resistant to N-(phosphonacetyl)-L-aspartate (PALA), a transition state analog inhibitor of aspartate transcarbamylase, overproduce CAD, a multifunctional protein which catalyzes the first three reactions of de novo UMP biosynthesis. Increased levels of a single mRNA cause the overproduction of CAD in all PALA-resistant mutants examined thus far. A recombinant plasmid containing a 2,3-kilobase insert complementary to the 3'-proximal region of this 7.9-kilobase mRNA has been prepared and used to show that the CAD gene is amplified in each of the 10 PALA-resistant mutants examined. Rates of association of CAD sequences in DNA isolated from PALA-sensitive and PALA-resistant cells with labeled plasmid DNA indicated that the degree of amplification is approximately equal to the degree of overproduction of protein and mRNA in each mutant. The patterns of digestion of these DNAs with restriction enzymes confirmed this result and showed that the lower limit for the size of the amplified unit is 19 kilobases, much larger than the mRNA. A comparison of restriction endonuclease digests of the cloned cDNA with digests of genomic DNA indicated that part of this difference is attributable to intervening sequences in the CAD gene. A 10.2-kilobase RNA which contains CAD sequences is found in cytoplasmic fractions from some PALA-resistant mutants but not in wild type cells. Restriction patterns were analyzed by a new method in which fragments of DNA are transferred from agarose gels to diazo paper with a high efficiency which is independent of size.


Potentiation of the chemotherapeutic action of 5-fluorouracil by combination with cytidine or guanosine on HRS-sarcoma. The chemotherapeutic action of 5-fluorouracil monotherapy on HRS-Sarcoma in mice was compared with those of 5-fluorouracil nucleoside combinations (thymidine, cytidine or guanosine). The curative action of 5-fluorouracil was potentiated without increasing its toxicity, when cytidine or guanosine were applied at definite intervals before or after 5-fluorouracil.


Early mitochondrial damage in the induction of haemorrhagic necrosis in the Crocker sarcoma (S 180) by endotoxin. Disturbances in the functional properties of tumor mitochondria have been studied during the course of induction of haemorrhage brought about by endotoxin in the murine Crocker sarcoma (S 180). Extensive impairment of function was already present in mitochondria isolated from control tumors, as shown by low respiratory control ratios. The existing mitochondrial damage intensified promptly in response to injection of endotoxin long before the onset of haemorrhage at 4 h. The nature of the additional damage took two forms, depending on the duration of exposure to endotoxin; first, at 30 min, a true uncoupling of oxidative phosphorylation was seen, largely reversible in vitro by pre-treatment of the isolated organelles with bovine serum albumin (BSA). Second, at 1 h and later, oxygen utilisation in the presence of succinate, ADP and inorganic phosphate (Pi) was depressed. The pre-addition of BSA consistently lowered respiration rates with succinate and Pi in all preparations. The extent of endogenous inhibition of the adenine nucleotide translocase appeared unaltered by endotoxin in vivo.


Oesophageal cancer in Greenland: selected epidemiological and clinical aspects. During 1955--1974, forty cases of oesophageal cancer were diagnosed among indigenous Greenlanders. The annual incidence rates per 100,000, age adjusted to the "world" population, were 16.3 for males and 11.8 for females in 1955--1964. The corresponding rates in 1965--1974 were 15.9 for males and 6.7 for females with a male:feamle ratio of 2.4:1. These rates rank among the moderately high rates found in India, Puerto Rico and France and in the black population in the US. Age distribution, anatomical location and prognosis followed the normal pattern for oesophageal cancer disease. No particular occupational trend was apparent and there was no difference between towns and settlements. However, a statistically significant geographical gradient of frequency was found with higher rates in the southernmost 3 districts. The traditional Greenland diet may contain concentrations of precursors sufficient to create carcinogenic levels of nitrosamines. Further studies are needed in particular of environmental factors such as foodstuffs, homebrewed beer, drinking water and cigarette smoking. Attention should be focused on the special ecological conditions in southern Greenland.


Immunofluorescent and histochemical localization of AMP deaminase in skeletal muscle. Fluorescent antibody staining experiments with both isolated myofibrils and muscle fibers grown in culture show that AMP deaminase is bound to the myofibril in the A band. The strongest staining occurs at each end of the A band. The approximate width of the fluorescent stripes and their relation to the A band remains constant as a function of sarcomere length. Removal of enzyme from the myofibrils leads to loss of staining, and readdition of purified enzyme restores the original staining pattern. A histoenzymatic method for the detection of AMP deaminase activity in cultured fibers gives comparable localization. The results are consistent with the previous observation (Ashby, B. and C. Frieden. 1977.J. Biol. Chem. 252:1869--1872) that AMP deaminase forms a tight complex in solution with subfragment-2 (S-2) of myosin or with heavy meromyosin (HMM).


Transmembrane communication in cells chronically infected with measles virus. The transmembrane association of the measles virus hemagglutinin and hemolysin surface proteins with intracellular viral antigens was studied. Rabbit antisera monospecific for measles virus matrix and nucleocapsid proteins and a human antiserum containing specificities for both the hemagglutinin and hemolysin proteins were used to study the co-capping of these proteins in human Lu 106 cell-line, chronically infected with measles virus. Capping of the surface-associated envelope components was accompanied by co-capping of the matrix and nucleocapsid proteins, the latter being localized mainly within the inclusions. This demonstrated transmembrane communication between surface-associated envelope components and the intracellular measles virus matrix and nucleocapsid proteins. The results demonstrated the existence of a linkage between viral inclusions and viral proteins associated with cell membranes. In the presence of cytochalasin B (1--2 micrograms/ml), co-capping of the matrix protein was unchanged or slightly enhanced, whereas co-capping of the nucleocapsid protein decreased, indicating that actin filaments may mediate the communication between viral nucleocapsids and the cell membrane.


Tuftsin-macrophage interaction: specific binding and augmentation of phagocytosis. The binding of [3H]tuftsin to normal and in vivo stimulated mouse peritoneal macrophage populations was studied at 22 degrees C. The [3H]tuftsin binding to thioglycollate-stimulated macrophages was shown to be rapid and saturable, with an equilibrium dissociation constant (K(D)) (calculated from a Scatchard plot) of 5.3 X 10(-8) M. The calculated number of binding sites per macrophage amounts to approximately 72,000. Binding competition studies with unlabelled tuftsin yielded a K(D) of 5.0 X 10(-8) M. [3H] [N-Acetyl-Thr1]tuftsin, an inactive analog of tuftsin, failed to bind specifically to thioglycollate-stimulated macrophages. [N-Acetyl-Thr1]tuftsin and the tripeptide [Des-Arg4]tuftsin failed to compete for tuftsin binding sites, while [D-Arg4]tuftsin, an analog with small tuftsin-like activity, exhibited a low degree of inhibition of [3H]tuftsin binding. Thus a rather high degree of specificity is involved in the binding of the tetrapeptide. Normal as well as six different macrophage populations induced by stimulation with thioglycollate, concanavalin-A, starch, mineral oil, glucan and Bacillus Calmette Guerrin (BCG), exhibited a similar degree of binding of [3H]tuftsin. Corynebacterium parvum (CP)-stimulated macrophages, on the other hand, showed a 6- to 10-fold-lower capacity for tuftsin binding. Under similar experimental conditions, mouse fibroblast and lymphocyte preparations revealed no detectable specific binding. Tuftsin augmented the phagocytic response of normal and stimulated macrophages assessed both for phagocytosis mediated via the Fc-receptor and via non-specific receptors. CP-stimulated macrophages did not exhibit an increased phagocytic response upon treatment with tuftsin.


Pancreatic alpha- and beta-cell function in pheochromocytoma. Arginine infusion tests were carried out in seven patients with pheochromocytoma before and after extirpation of the tumors in order to evaluate pancreatic islet alpha- and beta-cell function during the state of endogenous catecholamine excess. Six of the patients had glucose intolerance; one did not. Preoperatively, the pancreatic glucagon response was suppressed, while the insulin response was comparable to that in normal control subjects. Plasma glucose levels decreased rapidly after the beginning of arginine infusion in all patients. Theses changes during the infusion were evident in the one patient without glucose intolerance. Postoperatively, the glucagon response and plasma glucose changes were normalized. In addition to the obvious suppression of pancreatic alpha-cell function in our patients with pheochromocytoma, it seems likely that pancreatic beta-cell function also was suppressed; there was no enhancement of the insulin response to arginine during the period of chronic hyperglycemia, a situation in which a synergistic effect between glucose and arginine might be expected.


Pancreatic alpha-cell function in diabetic hemochromatotic subjects. To clarify further the etiology of the carbohydrate intolerance in idiopathic hemochromatosis, we investigated the glucose, insulin, C-peptide, and glucagon responses to arginine (0.5 g/kg) infused during 30 min in lean normal subjects; in insulin-requiring subjects with hemochromatosis, genetic diabetes, and total pancreatectomy; and in nondiabetic cirrhotic subjects without portosystemic shunting. Serum insulin, C-peptide, and glucagon responses (30K antibody) were determined by RIA, and glucose level was determined by a glucose oxidase technique. Hemochromatotic and genetic diabetic subjects had similar basal glucose (157 +/- 25 vs. 168 +/- 40 mg/dl) and C-peptide (0.73 +/- 0.42 vs. 0.65 +/- 0.22 ng/ml) values, with subnormal C-peptide peak responses to stimulation (1.05 +/- 0.38 and 1.40 +/- 0.83 vs. 3.95 +/- 0.4 ng/ml in normals; P less than 0.05). No glucagon or C-peptide response to arginine was seen in any pancreatectomized subject. Similar but excessive glucagon levels were present in hemochromatosis, diabetes, and cirrhosis under basal conditions (166 +/- 24, 232 +/- 111, and 263 +/- 116 vs. 76 +/- 15 pg/ml; P less than 0.05) and after arginine stimulation (782 +/- 80, 834 +/- 123, and 902 +/- 275 vs. 489 +/- 81 pg/ml; P less than 0.05) when compared with normals. The excessive glucagon levels found in hemochromatosis, diabetes mellitus, and cirrhosis contrast to the absent response in pancreatectomized subjects and indicate that generalized islet cell destruction is not the major factor in diabetic hemochromatotic subjects.


Indirect immunofluorescent localization of prolactin to the cytoplasm of decidua and trophoblast cells in human placental membranes at term. An indirect immunofluorescent technique was used to determine the localization of cytoplasmic human PRL (hPRL) in fresh and incubated human placental membranes at term. In both fresh and 8-h incubated samples of amnion, amniochorion decidua, or chorion decidua obtained from three placentas, we found specific reproducible localization of hPRL to the cytoplasm of decidua and trophoblast cells. The decidua cells appeared to be the most intensely fluorescent. No specific hPRL immunofluorescence was noted in the amniotic epithelium of fresh or incubated samples of amnion and amniochorion decidua. These data suggest that the trophoblast decidua cell layer is the site of PRL localization and possibly synthesis in placental membranes at term and may be the origin of amniotic fluid PRL in humans.


Reduced gonadotropin secretion in postmenopausal women during treatment with a stimulatory LRH analogue. The potent and long-acting LRH agonist D-Ser(TBU)6-EA10-LRH was administered in a daily subcutaneous dose of 5 microgram to 5 postmenopausal women for a period of 10 days. The LRH analogue produced a significant decrease in both the basal FSH and LH levels and the gonadotropin responses to the agonist. The estrogen levels in serum remained unchanged during the study period. The results suggest that D-Ser(TBU)6-EA10-LRH has a direct inhibitory effect at the pituitary level.


Glucose memory of pancreatic B and A2 cells: evidence for common time-dependent actions of glucose on insulin and glucagon secretion in the perfused rat pancreas. The influence of previous exposure to glucose on the subsequent B- and A(2)-cell secretory responses to arginine was investigated in the perfused pancreas of the rat. Arginine (8 mM) was administered in two brief (9 min) pulses separated by a period of 66 min. In pancreata from 18-h-fasted animals the two pulses of arginine elicited biphasic glucagon secretory responses, while stimulation of insulin release was barely detectable. When 27.7 mM glucose was administered for 30 min during the intervening period up to 20 min before the second pulse of arginine, the glucagon response to arginine was diminished by 55% while the insulin release was markedly increased in comparison with the first pulse. 8.3 mM glucose, when administered before the second pulse of arginine, exerted effects that were smaller but otherwise similar to those of 27.7 mM glucose.The inclusion of 3.9 mM glucose during the stimulation periods with arginine decreased the glucagon and greatly increased the insulin secretory response. Under these conditions, previous exposure to 27.7 mM glucose inhibited the glucagon and enhanced the insulin response to the second stimulatory pulse of arginine to the same relative degree as when arginine was administered alone. Diazoxide (2 mM), when administered together with 27.7 mM glucose, almost completely inhibited insulin release induced by the presence of glucose, yet did not influence the modulation exerted by glucose on the subsequent insulin and glucagon secretory response to arginine. Conversely, these effects of the glucose pulse could not be reproduced by 1 mug/ml of porcine insulin. Previous exposure to glyceraldehyde (10 mM) mimicked the glucose effects.Also, in pancreata from fed rats, previous exposure to 27.7 mM glucose markedly inhibited subsequent arginine-induced glucagon secretion while the concomittant insulin response was enhanced.IT IS CONCLUDED THAT: (a) both A(2)- and B-cell responsiveness is modulated by a previous exposure to glucose which produces opposite effects in the two cell types, (b) this action of glucose does not depend on its insulin-releasing capacity, and (c) instead, a "memory" of glucose is induced as a consequence of the metabolism of the sugar in the A(2) and B cells.


Bacteria causing symptomatic urinary tract infection or asymptomatic bacteriuria. One thousand bacterial isolates from consecutive suprapublic aspirations of urine in two groups of patients with symptomatic urinary tract infection and two groups with asymptomatic bacteriuria were studied. Staphylococci accounted for 13 and 27% of the isolates from symptomatic patients but for only 3 and 4% of strains from asymptomatic bacteriuria. Conversely, 18 and 30% of Escherichia coli isolates from asymptomatic bacteriuria were auto-agglutinable when tested with antisera to the common urinary O-serogroups, whereas such strains accounted for only 5 and 7% of the E. coli from symptomatic patients. The high prevalence of auto-agglutinable E. coli in asymptomatic bacteriuria was accompanied by a reduction in the frequency of the common uninary O-serogroups rather than other smooth strains.


Identification of Pseudomonas pseudomallei in the clinical laboratory. Ninety-one strains of Pseudomonas pseudomallei were tested in the API 20E system and in equivalent conventional tests. Except for utilisation of citrate there was good correlation between API and conventional tests. Seven-digit profiles were constructed from each strain after 48 hours' incubation, and numerical codes 2 006 727, 2 206 706, 2 206 707, and 2 206 727 accounted for 77% of strains tested. API 20 E, in combination with tests for heat stability of alkaline phosphatase, resistance to colistin and gentamicin, oxidative attack only of glucose, and acid from maltose, was found to provide a simple method for positive identification of all strains of this organism in two days.


Anti-i cold agglutinins in choriocarcinomatosis: trophoblastic i antigen. The fetal antigen i has been demonstrated by indirect immunofluorescence on cord erythrocytes and placental trophoblast. A patient with disseminated choriocarcinoma developed high-tire anti-i cold agglutinins, and minor elevation of anti-i titres was seen in four out of six further patients with treated choriocarcinoma. In normal pregnant women, 6% showed similar increases in anti-i titres.


A comparison of three tests for rubella antibody screening. Sera from 1000 women attending antenatal clinics were screened for the presence of rubella antibody by haemagglutination inhibition (HI), single radial haemolysis (SRH), and enzyme-linked immunosorbent assay (ELISA). With 925 sera concordant results were obtained with all three tests. There were 46 false-negative HI tests due to the necessity of allowing for residual non-specific inhibitors. With SRH there was one false positive and three that were negative by SRH but HI- and ELISA-positive. By ELISA 14 sera gave a false-positive result (OD 405 nm greater than or equal to 0.2). Ten of these could be eliminated by taking an OD 405 nm of 0.5 as the threshold but then another 10 sera became false negatives.


Malakoplakia of the prostate: a report of two cases and a review of the literature. Two cases of malakoplakia of the prostate are documented, bringing the total described in the world literature to 15. This literature is reviewed, and typical features of the condition are established. The majority of the cases have had proven previous episodes of Escherichia coli infection of the urinary tract, but bacilliform organisms have yet to be demonstrated in the diseased prostatic tissue. Five of the reported cases have also had important additional disease, including three instances of malignant lung tumour. Finally, it is emphasised that recognition of prostatic malakoplakia as an entity will prevent its confusion with prostatic carcinoma.


An articulation disorder in 17th-century Germany. Apical trill was regarded as the correct pronunciation of R in 17th-century German, but malarticulations of this difficult sound were widespread. Two of the most common substitutions were the uvular trill and L. The first was satirically described in a novel by Christian Weise (1673), while Rosinus Lentilius (1698), a physician, dealt with the L-substitution in a more scholarly but no less humorous manner. Annotated translations of these texts are presented here along with an introduction. The study suggests there may be more to the earlier history of speech disorders than is generally supposed.


A proposed network to improve access to high-quality health care for Medicaid-eligible families. There is today both a need and an opportunity to develop and test a variety of models--organizational and financial--for improving the delivery of health care services. This article describes the structure and functioning of one such model and highlights the organizational problems expected to arise during its implementation. The proposed health plan is intended to facilitate the access of Medicaid-eligible, inner-city families to already available health services. The central hypothesis is that in low-income urban areas the elementary schools offer an organizational focus for the development of a health plan. As a prepaid, community-based model, this plan is designed to address the issues of accessibility, equity, accountability, continuity of care, and consumer participation, primarily through the development of a coordinating agency, the health plan office (HPO), which assures the linking of consumers and providers of health care. Adapted from the Kaiser-Permanente model, the HPO also assumes responsibility for marketing, enrollment, coordination of services, consumer advocacy, and quality surveillance.


Biosynthesis and secretion of milk proteins: a review. Recent years have seen a great increase in the knowledge and understanding of milk proteins. Arising from several origins including the blood stream and various cellular sources, many of the proteins found in milk are products of the secretory cells directly involved in the synthesis and secretion processes of various milk components. The lactation-specific proteins present in major amounts are synthesized in the rough endoplasmic reticulum (RER) under genetic control and undergo further post-translational modifications in their secretory route from the RER through the Golgi apparatus and secretory vesicles before ejection into the lumen with other milk components. Various molecular aspects of these mechanisms and their control are now understood, but many remain to be described.


Hemodiafiltration using readily available equipment. Hemodiafiltration as a blood purification method is a combination of hemodialysis and hemofiltration. With this procedure low molecular substances are predominantly cleared by diffusion while middle molecules are cleared mainly by convection. This report shows how hemodiafiltration can be done using readily available equipment: a regular dialysis machine, an "open membrane" and the COTRAL device. Treatment could be reduced to about 2/3 of dialysis time. With a TMP of 600 mmHg a filtration rate of 60 - 70 ml/min at a blood flow of 200 ml/min and a dialysate flow rate of 500 ml/min the following. Clearance values were obtained: Urea 140 ml/min, creatinine 128 ml/min and inulin 76 ml/min. Increasing the blood flow to 300 ml/min and dialysate flow to 1000 ml/min clearances were: urea 192 ml/min, creatinine 161 ml/min and inulin 91 ml/min. During a 3.5 hour treatment period max. 92.4 urea and 6.3 g creatinine and 36 mg B2 microglobulin were extracted. In our opinion this in our hand advantageous procedure deserves further study.


Hemodynamics during hemodialysis, sequential ultrafiltration and hemofiltration. Circulatory parameters were determined by cardiac catheterization in patients on maintenance hemodialysis. They were studied in three groups during conventional hemodialysis, sequential ultrafiltration and hemofiltration. All three groups revealed significant reduction of cardiac output, stroke volume, pulmonary artery pressure and plasma volume. In the hemodialysis group hemodynamic parameters were unstable; specifically, hypotension, increased heart rate, and only minimal increase of peripheral resistance were observed. The other two groups showed only minor changes in circulatory parameters despite high ultrafiltration rates. Blood pressure and heart rate remained stable. On the other hand, peripheral vascular resistance increased remarkably. There is compelling evidence that during hemofiltration and sequential ultrafiltration, the patient's ability for vasoconstrictive counterregulation is better maintained than during conventional hemodialysis.


Modification of etching patterns in bovine dental enamel. It is presumed that the etching pattern is controlled by the residual organic content of dental enamel. Pretreatment with 1.ON NaOH sould remove the organic material and modify the etching pattern. SEM studies and other tests for physical and chemical properties show that the predicted modification of the etching pattern, when the tooth surface is pretreated with NaOH solution, occurs apparently without other changes or properties.


Overview of alcoholism.

Overview of alcoholism. Dental practitioners should gain an understanding of the effects of alcohol in both nonalcoholic patients (about a third of whom can be expected to show minor alcohol-related problems) and in the 10% to 25% of their patients who have such severe alcohol problems that they could be labeled alcoholic. Although alcoholism can be a loosely applied term, it is possible to use fairly rigorous criteria to identify the primary alcoholic and, with resulting insight, to outline the probable future course of problems. Alcoholism can be viewed as a disease with a natural history that is somewhat predictable. Its causes probably consist of a variety of social and psychological factors; evidence for a genetic contribution has been fairly established. Once the alcoholic is identified, dental problems should be treated, but referral to an agency for treatment should also be seriously considered. Such rehabilitation programs offer general support and helping mechanisms to aid the alcoholic to achieve improved functioning in life.


Review of pharmacologic and toxicologic effects of alcohol. Oral neglect and dental diseases are common; consumption of alcohol is common. To appreciate the frequency of alcoholism, the dental specialist must recognize that no persons will seek attention for alcoholism. Suspicion of alcohol related dental disease is the key. Heavy smoking, malnutrition, bleeding gingiva, cancer of the head and neck, and enlargement of the parotid glands should all raise a question of alcoholism. Alcohol affects most organ systems, but toxicity is extremely variable. Single organ systems may be severely damaged whereas others are spared. Current evidence suggests that variations on the total amount of alcohol consumed and genetic variations in specific pathways may contribute to the multifaceted clinical presentations of alcoholism. Because alcohol is the single most common drug ingested by patients, common interactions of drugs that have dental significance should be suspect. Most commonly, these interactions involve anesthetic agents and sedative hypnotic drugs. Dental specialists may encounter alcoholic persons in intoxicated, withdrawing, or rehabilitating states. In any of these phases, appropriate recognition and judicious support of the dental problems will contribute to the overall well-being of these persons.


Induction of hyporesponsiveness to particulate antigen by feeding: the sequence of immunologic response to fed antigen. The sequence of immunologic response to feeding with particulate antigen (sheep red blood cell, SRVC) was studied in inbred Dark Agouti (DA) rats. Feeding of SRBC to rats led to systemic sensitization as demonstrated by the presence of agglutinating and hemolytic antibodies to SRBC in sera. These humoral responses as well as the number of specific plaque-forming cells (PFCs) to SRBC in the spleen and mesenteric lymph nodes of these animals could be boostered by the intravenous administration of SRBC during the early stages of feeding. With prolongation of feeding both hemolytic and agglutinating antibodies in sera dropped below presensitization levels. In addition, upon parenteral challenge with SRBC the number of PFCs to SRBC and the humoral antibody responses elicited in SRBC-fed rats were found to be significantly lower than those of controls. Antigen-reactive cells, however, were not completely abolished even after 10 wk of feeding. The results demonstrate that a diminished responsiveness to particulate antigen achieved by prolonged feeding is preceded by a state of immunity and is not absolute. The mechanism underlying the phenomenon remains to be elucidated.


Appropriateness of vitamin and mineral prescription orders for residents of health related facilities. A study was made of the medical charts of 433 elderly patients admitted to four Health Related Facilities in upper New York State, to determine whether the respective prescription orders for vitamins and minerals were appropriate to the given diagnoses and, if not, identify the variables correlated with inappropriateness. For 11.1 percent of the patients, vitamins or minerals were prescribed when there was no specific diagnostic indication, or they were not prescribed when the medical diagnosis indicated that they should have been. Women and medicaid patients were more likely than men and self-paying patients to be treated inappropriately with respect to vitamins and minerals, but the differences were not satistically significant.


An in vivo and in vitro study of an aging-related neuron cytoplasmic-binding antibody in male Fischer rats. Fischer 344 male rats showed a low average level (less than ++) of in vivo as well as in vitro neuron binding antibody at 3, 15, and 26 months. However, a significant increase with age in average intensity of binding was observed with both the in vivo and the in vitro conditions. If a criterion of ++ is used as an antibody binding level likely to have biological consequences, the percent of in vivo cases showing foci of ++ or greater intensity also rises with age from 20 to 80%; a criterion of +++ gives an in vivo rise with age of only from 10 to 20%. The in vitro incidence with the ++ criterion is already 80% at three months and rises to 90%; with the +++ criterion the incidence with age rises from 20 to 80%. No evidence was obtained to support the concept that there is a weakening of the blood-brain-barrier in 26 month rats either by the systemic injection of trypan blue or by a comparison of intensity of antibody binding between extra- and intra-barrier neurones.


Deep fasciitis of the biceps region.

Deep fasciitis of the biceps region. An infection of the biceps fascia, later myositis, and spreading to the pectoralis is described in six patients. The majority were synergistic infections with an anaerobic Peptococcus, Eikenella, or Bacteroides and an aerobic Streptococcus. Five of the patients were drug abusers. Excision of the biceps and portions of the pectoralis major was necessary in four patients.


[The present state of hormonal investigation in amenorrhoea (author's transl)]. The value or plasma radio-immune assays of steroid and pituitary hormones in amenorrhoea is discussed. The accent is placed on the need for repeated plasma sampling. Dynamic tests aimed at investigating the hypothalamo-hypophysial-ovarian axes are analysed. A proposal is put forward for a scheme of investigation that will make better use of these tests.


[Combined hydramnios with fetal ascites due to intra-uterine meconium peritonitis (authors' transl)]. It is possible with echotomography to make out fetal ascites in the last months of pregnancy. One of the causes of combined fetal ascites and hydramnios among several other possible ones is primary meconium peritonitis, which diagnosis can be confirmed after birth.


Dominant lethal gene test of 5-thio-D-glucose in male mice. Genetic effects of 5-thio-D-glucose (5-thio) were tested in male mice by the dominant lethal gene test. Males were treated with phosphate buffered saline (PBS) or 5-thio dissolved in PBS by oral intubation daily at 40 or 60 mg/kg for 7 or 35 successive days. Average implantations per pregnant female and the intrauterine postimplantation losses in females mated with 5-thio trated males did not show any significant differences when compared statistically with parallel controls. Furthermore, the compound had no effect on the appearance and mating behavior of the male mice. This is the first report on the genetic effects of 5-thio to show that the compound in the treatment regimens studied were not mutagenic in terms of the induction of dominant lethal mutation in male mice.


Quantitative relationships of the harmful effect of ionizing radiation on natural resistance of the organism to various infectious agents. Review of literature. On the basis of an analysis of data from the literature and our own experimental results, the conclusion can be drawn that there is inverse linear dependence between the dose of irradiation and natural resistance of the organism to infection with various infectious agents. With increasing doses of irradiation, the irradiated organism is exposed to greatest risk from the part of the agents of intestinal infections and representatives of normal microflora. These are followed by agents of various diseases of microbial nature. The least decrease can be observed in resistance to viruses.


Comparison of titration results of diphtheric antitoxic antibodies obtained by means of Jensen's method and the methods of tissue cultures and haemagglutination. In 33 human sera the determination of diphtheric antitoxic antibodies was performed in a double blind test using Jensen's method, the method of tissue cultures and the haemagglutination method. In the method of tissue cultures the antibody levels in the sera were determinated in the first and second experiment with the precision of +/- half dilution of the geometrical progression. In Jensen's method, the difference between the first and second measurements slightly exceeded +/- 1 dilution. In the haemagglutination method the error considerably exceeded the binary step dilution. In most cases, the determination fluctuated up to seven times the actual value. Differences among the mean values of examination results obtained by Jensen's method and the method of the tissue cultures are statistically insignificant. The differences between the haemagglutination method and both the other methods are statistically significant.


Rapid diagnosis of tick - borne encephalitis by immunofluorescence assay of specific serum IgM antibodies. The indirect IF technique, using suspensions of TBE virus infected and uninfected PS cells as antigen-containing substrate, furnishes a rapid and practical test making possible the detection of specific IgM class serum antibodies in the initial stage of clinically manifest TBE. It enables early confirmation of diagnosis already in the acute phase of the disease and thus it can be instrumental in differential diagnosis and rational therapy, e.g., the administration of specific hyperimmune gamma-globulin.


Characterization of a spontaneous murine B cell leukemia (BCL1). II. Tumor cell proliferation and IgM secretion after stimulation by LPS. A spontaneous BALB/c B lymphocyte leukemia could be stimulated in vitro by the polyclonal B cell activator lipopolysaccharide (LPS) and the conditions for activation were studied. Spleen cells or peripheral blood lymphocytes from tumor-bearing animals responded by increased DNA synthesis and the peak of activation occurred earlier than with normal mouse spleen cells. Tumor cells harvested from the spleen, but not from the peripheral blood, could be induced by LPS to secrete IgM. Direct demonstration that the response was due to tumor cell activation and not that of contaminating normal B lymphocytes was provided by karyotype analysis and by immunoprecipitation, which showed the restriction of light chains on secreted IgM molecules to the lambda isotype.


Macromolecular insoluble cold globulin (MICG): a novel protein from mouse lymphocytes. IV. Evidence for the plasma membrane distribution of MICG. Macromolecular insoluble cold globulin is a glycoprotein synthesized predominantly by T lymphocytes in the mouse. The present report details experiments demonstrating the plasma membrane distribution of MICG on T lymphocytes. By utilizing immunofluorescent techniques it was shown that MICG was located in the external cell surface of 98% of thymic lymphocytes and 60% of splenic lymphocytes. Furthermore, in spleen cells, it was demonstrated that T cells and not B cells were surface MICG positive. Antibody to MICG was able to cap all of the immunofluorescent-positive (60%) spleen cells. In contrast, anti-MICG antibody did not induce cap formation on thymus cells. Only when dilute solutions of antibody were used did MICG cap on the thymus cells. Employing limited proteolysis of thymus and spleen cells MICG was shown to be regenerated on the surface of T cells with a half-life of 3.5 hr. The distribution and cell surface characteristics of MICG are discussed in terms of a "receptor-like" function for this protein.


Demonstration of phosphorylcholine-specific IgE B cells in CBA/N mice. CBA/N mice, which did not make anti-PC IgM or IgG antibody against PC-conjugated T-dependent or T-independent antigens, produced IgE antibody to PC-determinant when they were immunized with PC-KLH. PC-specificity of IgE antibody produced in CBA/N mice was determined by inhibition of PCA reaction with free PC-hapten or C-polysaccharide or by absorption of reaginic activity in the serum with C-polysaccharide. The presence of T15 idiotype on anti-PC IgE antibody produced in CBA/N x BALB/c F1 males also showed that anti-PC IgE antibody in defective mice was PC-specific. The results suggest that PC-specific B epsilon cells may belong to a subpopulation distinct from PC-specific precursors for IgM and IgG responses.


Structural similarities between C6 and C7 of human complement. A new method for the isolation of C6 and C7 by affinity chromatography of human serum with anti-C6 and anti-C7 coupled to Sepharose is described. C6 and C7 prepared by this method are hemolytically fully active, homogeneous proteins obtained in 25% yield. A comparison of the properties of isolated C6 and C7 gave the following results: The amino acid composition of the two proteins is very similar. The m.w. calculated from the amino acid content is 124,800 for C6 and 120,800 for C7. Both components are single chain glycoproteins migrating upon electrophoresis at pH 8.6 as beta 2-globulins, Both proteins are polymorphic as detected by isoelectrofocusing in polyacrylamide gels and range in their isoelectric points from pH 6.15 to 6.7. The UV spectra reveal only minor differences; the extinction coefficients are: EC6 = 1.71 cm2 X mg-1 and EC7 = 1.92 cm2 X mg-1. CD-spectra show 8% alpha-helix and 10% beta-structure for C6 and 10% alpha-helix and 14% beta-structure for C7. The structural similarities of C6 and C7 suggest their evolution from a common ancestral gene.


C3 deposition in cholesterol-induced atherosclerosis in rabbits: a possible etiologic role for complement in atherogenesis. Hypercholesterolemia was induced in rabbits by feeding Purina Chow supplemented with cholesterol (5 g/kg body weight/day). The serum cholesterol levels of these rabbits increased progressively and after 3 to 5 months were 4 to 9-fold greater than those of the control animals. Decrease in total hemolytic complement was not apparent during the feeding regimen. Morphologic examination of aortae of these hypercholesterolemic rabbits showed typical atherosclerotic intimal plaques. Immunofluorescent microscopy with fluorescein (F)-labeled anti-rabbit C3 showed deposition of C3 in the intimal and inner medial layers as early as 3 months on high cholesterol diet. C3 deposits were also observed in the renal glomeruli and in the walls of coronary arteries. However, fluorescent studies failed to demonstrate the presence of IgG, IgM, and C4 at these sites. Tissues from control animals fed normal diets were negative for immunoglobulins, C3, and C4. These results suggest that the complement system may be implicated in the pathogenesis of cholesterol-induced atherosclerosis in rabbits.


Role of lipids in the immune response. II. An enhancer of antibody production (APS) that is a lipid closely associated with lymphocyte mitogenic factor (LMF). Lymphocyte mitogenic factor (LMF) is a lymphokine produced by dividing T cells. An activity coeluting with LMF from a Sephadex resin is extractable from LMF with chloroform-methanol. It enhances but does not initiate antibody formation measured by generation of plaque-forming cells (PFC). It does not cause proliferation of B cells. This activity elutes from a silicic acid column and produces a pattern on thin layer chromatography that suggests that the compound(s) responsible for the activity is a polar lipid.


The pathology and homing of a transplantable murine B cell leukemia (BCL1). The pathology and homing characteristics of a murine B cell leukemia are described. Experiments utilizing autoradiography to determine the early homing pattern of the leukemic cells revealed a pronounced localization of the labeled cells to the spleen. The cells that were seen in the white pulp showed preferential localization to the follicles or B cell domains. Tissue section immunofluorescence with antibodies to kappa- and lambda-light chains was used to study the initial mouse with this disease as well as to study the mice that were injected with in vivo passaged cells. These mice also showed predominant involvement of the spleen. Although the initial mouse with this disease had 200,000 lambda-bearing B lymphocytes per mm3 in the peripheral blood and closely resembled a human chronic lymphocytic leukemia patient, the studies described suggest that this murine B cell neoplasm is a lymphoma with a striking predilection for splenic involvement. The other organs including the bone marrow as well as the peripheral blood appeared to be involved secondarily. This unusual spontaneously occurring murine B cell disease provides a useful model for the investigation of certain commonly occurring human lymphomas and leukemias.


Markers of human T cell subsets identified by alloantisera. Analysis by the indirect fluorescence test followed by fluorescence-activated cell sorter (FACS) analysis has shown that antisera recognizing subsets of human T lymphocytes can be produced by planned immunizations involving HLA-A and HLA-B compatible donors. The reactivity of these antisera against some individuals of a population but not others shows that they recognize a polymorphic cell surface component. The reactive subpopulation largely overlaps with the JRA+ subset, which was previously shown to possess regulatory properties in functional assays. The specificity of the antisera for a T cell subset and the unrelatedness of the anti-B cell activity of the same antisera has been confirmed by two-color fluorescence tests.


Immunity to the T1699 murine mammary tumor. I. Thymic influence and long-term effect of irradiation on the humoral response. The thymus dependency of humoral immunity to syngeneic tumor antigens was investigated in T cell-deficient DBA/2 mice. ATXBM animals bearing the T1699 mammary tumor in the subcutaneous abdominal area displayed normal immediate but not delayed hypersensitivity reactions (DHR) to 3 M KCl-extracted T1699 antigens injected into the footpad. Sera from ATXBM tumor-bearers passively transferred immediate hypersensitivity but failed to support tumor-specific macrophage-mediated ADCC reactions. The synthesis of macrophage-mediated ADCC antibody was greatly reduced in the CXBM animals when compared to nonirradiated tumor-bearers. The CXBM mice, however, showed normal T cell function as measured by allograft rejection, antibody response to sheep erythrocytes, and DHR to T1699 antigens. Of all antibody classes and subclasses tested by indirect membrane fluorescence, only IgG2b was found to be produced at normal levels by either ATXBM or CXBM tumor-bearers. The results show that IgG2b antibody production in response to T1699 syngeneic tumor antigens is thymus independent and suggest that this antibody is the mediator of immediate hypersensitivity. The synthesis of macrophage-mediated ADCC antibody (IgG2a) was found to be not only thymus dependent but also sensitive to the long-term effects of irradiation and bone marrow repopulation.


A simple technique for harvesting lymphocytes cultured in Terasaki plates. A simple method has been developed for harvesting lymphocytes from Terasaki microplates based on a culture technique where the plates are inverted and the cells grown on the hanging meniscus of the medium in each well. Sixty individual filter discs were prepared in wells in a polycarbonate harvesting plate. A dry Terasaki plate was used to cut these filters with the harvester acting as a die. The inverted culture plate was then lowered onto the harvester to allow the cells on the meniscus from each well to be absorbed on the appropriate filter disc. The culture plate was then discarded and the cells on the filters washed in the harvester. The wash solutions were removed by suction through holes below each filter. The time taken to harvest the cultures from 60 wells of a plate was 3--5 min.


Antilymphocyte serum produced by immunization with purified mouse thymocyte plasma membrane. A predominantly plasma membrane fraction was isolated from mouse thymocytes and characterised. Immunization of rabbits with this material yielded highly potent antilymphocyte sera as measured by prolongation of skin allograft survival. Potency was maintained after repeated boosting of the rabbits, yet the generation of irrelevant antibodies was contained. Binding of antilymphocyte sera to lymphocytes was studied by immunofluorescence using the analytical facility of a fluorescence activated cell sorter (FACS-1). The results demonstrated that these anti-membrane sera recognise antigens common to thymocytes and spleen lymphocytes, bind more to T-cells and thymocytes than to B-cells and bind more avidly than antisera raised with intact cells. It was concluded that this thymocyte plasma membrane preparation is highly effective in raising powerful antilymphocyte serum.


Preparation of sera for subtyping of influenza A viruses by immunofluorescence. The conditions for preparation of type-specific and subtype-specific influenza A virus reagents to be used in the immunofluorescence technique have been evaluated. Type A-specific antibodies were prepared by passing an antivirion hyperimmune serum through an immunoadsorbent column containing antigens from disrupted virions of a different influenza A virus subtype. The type-specific antibodies were recovered from the immunoadsorbent by desorption with 3 M NaI. For subtype determination, antisera against the various hemagglutinins were used. Such sera could be prepared by removal of irrelevant influenza A virus antibodies from sera directed against purified virions and isolated peplomers, respectively. This was performed by passing the antisera through immunoadsorbent columns containing antigens from disrupted virions of appropriate strains. However, attempts to obtain a hemagglutin-specific antiserum from a serum directed against allantoic fluid virus suspension failed with this procedure. Antisera obtained after immunization with purified hemagglutinin were also elaborated. These sera were shown to be superior for subtyping of influenza A virus infections by immunofluorescence, but could not a priori be regarded as subtype-specific. The usefulness of subtype-specific sera has been demonstrated on clinical specimens for rapid virus diagnosis.


IgA contamination of IgG prepared on a protein A column. When protein A is used for the isolation of IgG from pooled human serum further purification steps may be necessary since the IgG is contaminated with approximately 30% of the IgA originally present in the starting material. No preference for binding of an IgA subclass to protein A was found. In addition part of the IgG3 subclass binds to protein A.


Novel enzyme immunoassay for 17 beta-estradiol. A novel approach to the enzyme immunoassay of analytes is presented and illustrated by demonstration of an estradiol assay. The assay depends on the competitive binding of estradiol and estradiol-DNP conjugate to immobilized anti-estradiol. The amount of estradiol-DNP bound is inversely proportional to the amount of free estradiol, and is measured through the use of peroxidase-labeled anti-DNP antibodies. The assay gives a dose-response curve from 10 pg/tube to 450 pg/tube and shows satisfactory correlation with radioimmunoassay.


Detection of antibodies specific to sodium dodecyl sulfate-treated proteins. Heavy meromyosin (HMM) denatured by sodium dodecyl sulfate (SDS) was injected into guinea pigs, either in the presence of 1 mg SDS/mg protein or after chromatography on Sephadex G-10 to remove detergent excess. Antigen-antibody interactions were analyzed by the microcomplement fixation technique. When HMM was injected in the presence of excess of SDS, the microcomplement fixation curves exhibited two maxima; one was specific to the random coil configuration of heavy meromyosin or myosin, and the other was common to several SDS-protein complexes. The latter peak disappeared when the excess SDS was removed from the immunogen by chromatography. Results showed the presence of antibodies directed either against SDS or against the non-specific SDS protein link.


Delineation of IgM-receptor bearing human T and B lymphocytes using a direct plaque forming cell (PFC) assay. Based on the observation that binding of IgM cytophilic antibodies to lymphocytes is temperature dependent, a direct plaque forming cell (PFC) assay was developed to detect IgM-receptor bearing human peripheral blood T and B lymphocytes. Lymphocytes were passively sensitized with IgM anti-SRBC molecules at 4 degrees C, added to SRBC monolayers then incubated at 37 degrees C with guinea pig complement to develop the plaques. The PFC assay has methodological advantages over rosetting methods which demonstrate IgM receptors, and under certain conditions is more sensitive than these rosette techniques. A mean of 17% of freshly isolated uncultured lymphocytes, enriched for B cells, formed direct plaques while a mean of 3% of T-enriched preparations formed direct plaques. However, if the lymphocytes were preincubated with vibrio cholerae neuraminidase (VCN) these figures increased to 46% and 35% respectively. The specificity of plaque formation by VCN-treated lymphocytes was established. SRBC sensitized with a F(ab')2 preparation of an IgG anti-SRBC reagent failed to bind to VCN-treated lymphocytes, inclusion of IgM, but not other Ig molecules in the test medium, inhibited plaque formation, and, most important, plaque formation by T and B cells was inhibited by F(c)5 mu but not by Fab mu fragments. These results indicate that T and B lymphocytes express IgM-class specific membrane receptors, that these receptors may be hidden on normal lymphocytes but are revealed by treatment with VCN and that the IgM receptor on VCN-treated lymphocytes is F(c)mu specific. These findings are discussed briefly with regard to other and partly contradictory data obtained after overnight in vitro lymphocyte culture. As demonstrated by direct PFC assay, the B cell IgM receptor is trypsin sensitive.


6-Thioguanine resistant peripheral blood lymphocytes in humans following psoralen, long-wave ultraviolet light (PUVA) therapy. A recently described method that enumerates variant 6-thioguanine resistant peripheral blood lymphocytes present in vivo in man as a potential marker of somatic cell mutations occurring in vivo was used to study 18 psoriatic patients receiving PUVA therapy, 16 conventinally treated psoriatic patients, 10 vitiligo patients receiving PUVA therapy and 7 untreated individuals with vitiligo. Variant lymphocyte frequencies determined for these individuals were compared with those determined for groups of 10 concurrent and 63 cumulative healthy control individuals. Variant frequencies were elevated in psoriatic and vitiligo patients receiving PUVA therapy and in conventionally-treated psoriatic patients. They were not elevated over control values in untreated vitiligo patients.


Amoeboid movement in human leucocytes: basic mechanisms, cytobiological and clinical significance. The present paper is an analytical review of the information available on amoeboid movement in human leucocytes. The reported evidence suggests that leucocyte locomotion is due to pressure developed in the cell cortex in the middle and posterior parts of the moving cell, that 4 nm fibrils may provide at least part of the ultrastructural basis of locomotion, that actin-like and myosin-like proteins may be involved in the mechanism of movement and that ATP may serve as an energy source. Leucocyte motility appears to be governed mainly by factors produced in the external medium. Neutrophil chemotaxis is the most antitubulin-susceptible cell mechanism known; from this observation an essential role of microtubule redistribution in chemotaxis is inferred. In contrast, the random movement of neutrophils is not appreciably affected by antimitotic concentrations of antitubulins. Amoeboid movement seems to be an important mechanism in the short-distance locomotion and immunological functions of leucocytes.


Incorporation of the Ca2+ -binding component (g2) into heavy meromyosin and subfragment-1 of pig cardiac myosin. A new protein component was found in heavy meromyosin and in subfragment-1 (S-1) prepared by chymotrypsin digestion of pig cardiac myosin in the presence of Ca2+. The molecular weight of this protein was estimated as 15,000 dalton. It was able to bind Ca2+ and showed a similar UV absorption spectrum to that of the g2 light chain. Heavy meromyosin and subfragment-1 which contained the 15,000 dalton component incorporated exogenous g2 and the 15,000 dalton component disappeared after such treatment. We concluded that the 15,000 dalton component was produced from g2 by limitted proteolysis. The subfragment-1 was separated into two protein fractions in equal yield by recycling the gel filtration. One contained the 15,000 dalton component and was able to bind Ca2+ while the other did not contain the component and was unable to bind Ca2+. According to analysis by SDS gel electrophoresis, the large polypeptide chain (the f component) of the first S-1 was approximately 5,000 dalton larger than the f component of the second S-1. The polypeptide corresponding to 5,000 dalton was designated polypeptide-C, because it was released from the C terminal of the f component. It seems to be essential for the attachment of the Ca2+-binding light chain g2. The location of g2 in myosin may thus be at the polypeptide-C which links the head to the tail of myosin.


Development and application of homologous radioimmunoassays for porcine gonadotrophins. Antisera were raised against highly purified preparations of porcine luteinizing hormone (pLH) and follicle-stimulating hormone (pFSH). Highly specific and sensitive radioimmunoassay systems were developed. The antisera to LH and FSH were used at working dilutions of 1:500,000 and 1:200,000 respectively and the sensitivities of the assays were 0.1 ng LH/ml serum (3 x 10(-12) mol/l) and 0.5 ng FSH/ml serum (1.5 x 10(-11) mol/l). The LH and FSH preparations used as standards were 1.2 and 81 times as potent as NIH-LH-S15 and NIH-FSH-P1 respectively. Both assays were validated and adapted for the measurement of the gonadotrophin content of porcine serum. The concentrations of LH and FSH in blood were measured simultaneously in prepubertal sows throughout a 24 h period, in adult sows during the oestrous cycle and in both prepubertal and adult animals after treatment with LH releasing hormone.


Suppression of the luteinizing hormone releasing effect of luteinizing hormone releasing hormone by arginine-vasotocin. The concentrations of 17-oxosteroids in the spermatic venous blood of anaesthetized dogs were used as an index of LH release to assess the effects of arginine-vasotocin on the response of the canine pituitary gland to exogenous luteinizing hormone releasing hormone (LH-RH). When injected into the carotid artery, arginine-vasotocin (1.0 microgram/kg body wt) caused no significant alterations in the testicular output of 17-oxosteroids. The administration of LH-RH (5 microgram/kg body wt, a standard dose) into the carotid artery produced typical stimulation of testicular 17-oxosteroid secretion. Administration of arginine-vasotocin (0.01, 0.1 or 1.0 microgram/kg body wt) into the carotid artery 3 h before the administration of a standard dose of LH-RH inhibited the testicular secretion of 17-oxosteroids normally induced by LH-RH. However, pretreatment with arginine-vasotocin (1.0 microgram/kg body wt) did not affect the testicular response to i.v. administration of human chorionic gonadotrophin (5 i.u./kg body wt). These results indicate that in the dog, arginine-vasotocin inhibits the LH-RH-induced release of LH by acting acting directly on the anterior pituitary gland.


Separate roles of androgen and oestrogen in the manipulation of growth and efficiency of food utilization in female rats. The roles of androgen and oestrogen in the perinatal and postpubertal stages of development on the masculinization of female patterns of growth have been investigated in several experiments in rats. A stimulatory effect of testosterone on subsequent growth and efficiency of food utilization was only observed when administered perinatally to intact females as the propionate ester. Thus females which were untreated (or androgenized) perinatally and ovariectomized at weaning failed to grow more rapidly or utilize food more efficiently when treated with mixed testosterone esters from 36 to 38 days of age. Also autoimmunity to luteinizing hormone releasing hormone (LH-RH) had little effect on the growth or efficiency of food utilization of male rats, although testicular development was grossly affected. An inhibitory effect of oestrogen on subsequent growth and efficiency of food utilization was demonstrated by surgical ovariectomy and by autoimmunity to LH-RH. Also perinatal administration of oestradiol benzoate to intact female rats depressed growth below that of untreated intact litter-mate females until about 50 days of age. Then oestradiol benzoate-treated female rats grew to a larger size than untreated intact litter-mates but not to a heavier weight than untreated litter-mate females which like the oestradiol benzoate-treated females, were ovariectomized at 18-21 days of age. Both of these groups of female rats differed markedly in weight gain from females which were perinatally androgenized and ovariectomized at weaning. The effects of androgenization and ovariectomy on weight gain were comparable and additive in female rats fed restrictedly or ad libitum. Nevertheless, androgenized + ovariectomized female rats fed restrictedly or ad libitum failed to grow as rapidly as male rats. Some additional factor(s) prevents complete masculinization of the female pattern of development. The stimulatory effects of androgenization and ovariectomy on the growth of females appear to be related to endocrine mechanisms controlling the onset of pubertal changes in somatic development.


A bioassay for inhibin using pituitary cell cultures. A bioassay for inhibin based on the suppression of gonadotrophin releasing hormone (Gn-RH)-stimulated secretion of FSH by primary monolayer cultures of rat anterior pituitary cells is described. The cultures were exposed to standard or test materials for 3 days. The levels of FSH in the media were measured by radioimmunoassay after exposure for 6 h to a maximally stimulating concentration of Gn-RH (10 nmol/l). The standard was prepared from ovine testicular lymph. Several preparations of proteins from gonadal tissues or secretions suppressed the levels of FSH in parallel with the standard. The levels of LH were also reduced but higher doses of active material were required. Non-specificity from cell damage and inactivation of Gn-RH have been excluded. The secretion of gonadotrophins by the pituitary cells was also inhibited by androgens, but not in parallel with the standard and secretion of LH was affected more than that of FSH. Control lymph protein preparations from castrated sheep had no detectable activity. The assay was sensitive and had adequate precision and practicability. It has proved useful for monitoring preliminary steps in the purification of inhibin.


Acute regulation of insulin release by the pituitary gland in relation to hyperinsulinaemia and obesity. The pituitary glands from mice rendered obese by gold thioglucose treatment and by dietary manipulation, and pituitary glands from lean mice after a high food intake or a glucose load, were shown to stimulate insulin secretion from isolated pancreatic islets. The insulin releasing activity of pituitary glands from obese (ob/ob) mice was reduced by fasting for 24 and 48 h. Results obtained with pituitary glands from ob/ob and from lean ob/+ and +/+ mice suggest that the insulin releasing property manifests a gene dosage effect. Pituitary glands from 3-week-old (young) ob/ob mice stimulated insulin secretion to the same extent as pituitary glands from 3-month-old (adult) ob/ob mice. The pancreatic islets of young ob/ob mice were shown to be somewhat more responsive to stimulation by the pituitary factor than were lean ob/+ or +/+ islets from this age group. The concept that high insulin level, partly under pituitary control, and high caloric intake may be interlinked and may, in combination, be a major factor in producing obesity is discussed. Furthermore, it is suggested that the pituitary insulin releasing factor may play a role in the early development of obesity in the animal models studied.


Monitoring of serum theophylline concentrations by a fully mechanized enzyme immunoassay (EMIT). An evaluation of an enzyme immunoassay for the determination of theophylline in serum (EMIT, Syva Corp.) is presented. The EMIT assay was fully mechanized by the use of an Eppendorf analyzer 5010. The precision of this enzyme immunoassay was satisfactory (coefficients of variation from day to day ranged from 5--6%). The EMIT assay proved to be specific, rapid and easy to perform. A comparison of the results obtained by EMIT and ultraviolet spectrophotometry in a series of 100 patients showed a good correlation between both methods.


Evaluation of the manual enzyme immunoassay (EMIT) procedure for determination of serum thyroxine. Our experience with the determination of thyroxine (T4) in serum using a homogeneous enzyme immunoassay technique (EMIT, Syva Corp.) is reported. The intra-assay precision of the EMIT Manual Thyroxine Assay was investigated with 2 different calibrator concentrations and showed coefficients of variation varying from 5--2% for thyroxine concentrations of 40 and 120 micrograms/l thyroxine respectively. The inter-assay precision was investigated with different series of calibrators and serum specimens. Coefficients of variation for the calibrators varied from 35--5% in the range of 20--200 micrograms/l thyroxine and for the serum specimens in the range of 8--232 micrograms/l thyroxine from 50--4%. The recovery of various amounts of thyroxine added to thyroxine-free serum varied between 91--103%. The cross reactivity of structurally related compounds such as: monoiodothyronine, diiodothyronine, triiodothyronine, triiodothyroacetic acid and tetraiodothyroacetic acid was investigated. Serum samples of 100 patients were analysed by EMIT and radioimmunoassay (T4 RIA (PEG), Abbott Lab.). A good correlation was found between the EMIT and RIA assay (r = 0.96, slope = 0.96 and y-intercept = 3.37 micrograms/l).


Direct evidence for formation of hybrid vesicles by fusion of plasma and outer acrosomal membranes during the acrosome reaction in boar spermatozoa. A variety of treatment procedures was utilized to identify the origin and composition of the vesicles formed during the acrosome reaction of boar spermatozoa. Whether the acrosome reaction occurred spontaneously or was induced chemically the vesicles were hybrid vesicles composed of roughly equal proportions of plasma and outer acrosomal membranes.


Aerobic and anaerobic metabolism during activity in small rodents. Analysis of oxygen consumption and lactic acid formation during five minutes of maximal activity by the rodents Microtus montanus (Cricetidae) and Dipodomys merriami (Hetermyidae) indicates that: (1) anaerobiosis provides approximately 10% of total energy utilized during the 5-minute activity period; (2) anaerobiosis may account for as much as one-third of total energy utilized during the first 30 seconds of activity. In addition, these data indicate at least one species of lizard may be capable of a higher total rate of metabolism during "burst" activity than are the rodents investigated here.


Regenerative responses in larval axolotl limbs with skin grafts over the amputation surface. Complete flaps of whole skin over the amputation surface of larval axolotl limbs did not prevent dedifferentiation, DNA synthesis, and mitosis. However, even by two weeks, insufficient cell division had occurred for blastema formation. In about 50% of the cases, small areas of wound epidermis formed over the amputated tip of the radius or ulna. At two weeks after amputation, mitotic figures and dedifferentiated cells which incorporated H3-thymidine were predominantly located in the vicinity of the small area of wound epidermis. Although delayed in relation to controls, these limbs regenerated. The results are consistent with the view that the wound epidermis is necessary to maintain dedifferentiated cells in the cell cycle.


The focal differentiation of pigment cells. A study has been made of the normal development and of the regeneration after excision of the groups of large pigment cells which form the spotted skin pattern of the gecko Eublepharis macularius, together with the effects of neonatal graft transplantation on this pattern. The results all indicate strongly that such groups of specialized pigment cells are not clones but the product of an induction process. This is then compared with the neural reflex mechanism by which the skin pattern of Chamaeoleo dilepis is formed.


Neural control of light emission in Photuris larvae: identification of octopamine-sensitive adenylate cyclase (1). Octopamine, a putative phenylethylamine neurotransmitter present in the firefly, has been found to be a potent stimulator of cyclic AMP synthesis in the larval light organ. In the same tissue, octopamine causes a small inhibition of phosphodiesterase activity. Because of the relatively simple anatomical relationships present in the larval light organ, compared with that of the adult, this preparation should offer an attractive model for studying the biochemistry of neurohumoral control of light emission.


Localization of indoleamine and protein synthesis in the mammalian pineal gland. In a table, conditions and compounds have been listed either known or suggested to influence pineal function. In the same model, systems and organs and their products are indicated the function or, respectively, the quantity of which are known or suggested to be influenced by pineal secretory substances. Of the latter, indoleamines and polypeptides are mentioned only although it is now known that also other low molecular weight biologically active compounds are produced by the gland. Problems relating to the localization of indoleamine and protein synthesis in the pinealocyte are discussed. The synthesis of indoleamines, at least of serotonin, may occur freely in the cytosol although, especially in non-mammals, the presence of serotonin seems to be preferentially, but perhaps secondarily, related to the secretory dense-core vesicles. Questions concerning the exact function of pineal melatonin are shortly dealt with. The granules of the secretory vesicles probably also contain a proteinaceous substance which, notwithstanding its unknown chemical nature, has been considered the antigonadotropic principle by some authors. Recent investigations on the production of active proteinaceous compounds in cisterns of the endoplasmic reticulum and between the layers of the nuclear envelope are reviewed. These substances are not concentrated in secretory granular vesicles, so scarce in the pinealocyte which proteinaceous active principle is produced in still largely unsolved as is their exact chemical composition.


Pineal peptides.

Pineal peptides. In pineal extracts several activities have been observed which cannot be explained on the basis of their content of melatonin or other pineal indoles to which an antigonadotropic activity is ascribed. The significance of structurally identified and unidentified substances is not fully understood. The list includes a neurohypophysial peptide, vasotocin (AVT), hypothalamic releasing factors and certain unidentified substances including the antigonadotropin under investigation in our laboratory. Our initial experiments demonstrated the presence of a non-melatonin antigonadotropin (PAG) in partially purified extracts of bovine, ovine, rat and human pineals. Purification by ion exchange chromatography and amino acid analyses have shown that this substance is not AVT. Recent purification studies include paper chromatography and high performance chromatography as final steps. It is concluded that if the active principle in a peptide, or contains a peptide moiety important for the biological activity, it may be present only in minute amounts in the puriest fractions derived and the quantities of recoverable material may be similar to those amounts of releasing factors recoverable from hypothalamic tissue. It is anticipated that large scale preparative methods will be required for structural determination. High performance chromatography may prove to be extremely useful in future studies.