Transaminase B from Escherichia coli: quaternary structure, amino-terminal sequence, substrate specificity, and absence of a separate valine-alpha-ketoglutarate activity. Transaminase B (branched-chain amino acid aminotransferase, EC 2.6.1.42), the ilvE gene product, was purified to apparent homogeneity from an Escherichia coli K-12 strain which carries the ilvE gene both on the host chromosome and on a plasmid. The oligomeric structure of the enzyme, as determined by analytical ultracentrifugation and sodium dodecyl sulfate-polyacrylamide gel electrophoresis, was confirmed to be that of a hexamer with a molecular weight of about 182,000 and apparently identical subunits. Cross-linking with dimethylsuberimidate yielded trimers, dimers, and monomers, but essentially no species of higher molecular weight. These results are consistent with a double-trimer arrangement of the subunits in native enzyme. The amino-terminal sequence was found to be: Gly Thr Lys Lys Ala Asp Tyr Ile (Trp) Phe Asn Gly (Thr) (Met) Val. Purified transaminase B catalyzed transamination between alpha-ketoglutarate and l-isoleucine, l-leucine, l-valine, and, to a lesser extent, l-phenylalanine and l-tyrosine, the latter reacting very sluggishly. The enzyme was free of aspartate transaminase and of transaminase C. The apparent K(m) values for the branched-chain alpha-ketoacids were smaller than those for the corresponding amino acids. The lowest K(m) was recorded for dl-alpha-keto-beta-methyl-n-valerate, and the highest was recorded for l-valine. The ratio of the valine- and isoleucine-alpha-ketoglutarate activities did not change significantly during purification, and both activities were quantitatively removed from crude extract by antibody raised against purified transaminase B. These observations argue against the existence of a separate valine-alpha-ketoglutarate transaminase. Anti-E. coli transaminase B antibody cross-reacted with crude extract from Salmonella typhimurium, but not with extract obtained from Pseudomonas aeruginosa.


Transposition of ampicillin resistance to an enterotoxin plasmid in an Escherichia coli strain of human origin. We examined a strain of Escherichia coli, serotype O159.H34, of human origin which produced heat-stable and heat-labile enterotoxins, was resistant to ampicillin, and produced colicin. By conjugation and transformation experiments plasmids coding for enterotoxin production (Ent), enterotoxin production and ampicillin resistance (Ap-Ent), ampicillin resistance (Ap), and colicin production were isolated. Both the Ent and Ap-Ent plasmids were autotransferring and belonged to the F-incompatibility complex. However, the Apr Ent+ transconjugants showed differences in their levels of resistance and in their abilities to propagate F-specific phages and to transfer resistance. The results suggested there was transposition from the small Ap plasmid to the Ent plasmid. The Ap-Ent plasmids were larger than the enterotoxin factor and when treated with restriction endonuclease BamHI showed an additional fragment not present in the enterotoxin plasmid. The insertion of ampicillin resistance probably occurred at different sites on the enterotoxin plasmid, resulting in the observed variation in phenotype.


Mutants defective in the 33K outer membrane protein of Salmonella typhimurium. Salmonella typhimurium LT2 lines, if phenotypically rough, are fully sensitive to bacteriocin 4-59, produced by Salmonella canastel strain SL1712. Bacteriocin-resistant mutants fell into three classes. Those resistant to phage ES18 and to albomycin proved to be mutants of class chr (equivalent to tonB of Escherichia coli); these mutants still adsorb the bacteriocin and so are classified as tolerant. Another class of (incompletely) tolerant mutants was resistant to phage PH51; their envelope fractions lacked the band corresponding to outer membrane protein 34K, known to serve for adsorption of phage PH51. A third class of mutants, which did not adsorb the bacteriocin, was unaltered in sensitivity to phages. Their envelopes lacked the 33K band, indicating absence of the outer membrane protein 33K, considered to correspond to outer membrane protein II* of E. coli, which in that species is determined at locus ompA (formerly tolG or con). Phage P22 HT105/1 cotransduced the 33K S. typhimurium gene (to be called ompA, to accord with E. coli usage) with pyrD+ at about 30% frequency when the donor allele was ompA+ or one ompA, but at only 3 to 11% when the donor allele was another ompA. When the donor carried either of two long deletions of the put (proline utilization) operon, phage P22 HT105/1 cotransduced put (and ompA+) with pyrD+ at low frequency. The cotransduction data indicate that ompA of S. typhimurium is located between pyrD and put, nearer the former. This corresponds to the map position of ompA in E. coli K-12.


Escherichia coli mutant strain with altered expression of the tryptophan operon: isolation and preliminary characterization. From a strain of Escherichia coli with two copies of the tryptophan (trp) operon and one copy of the lactose (lac) operon, under control of one of the trp regulatory elements, we have isolated a mutant which does not grow in a medium containing 19 amino acids, unless tryptophan is added, and which cannot ferment lactose. The apparent pleiotropic nature of the mutation(s) is indicated by the very slow growth of mutant bacteria on minimal-medium agar supplemented with glucose and tryptophan. The amount of the trp enzymes (anthranilate synthetase and tryptophan synthetase) and trp messenger ribonucleic acid is reduced several-fold in the mutant compared to the isogenic wild-type strain, whereas the enzymes tryptophanyl-transfer ribonucleic acid synthetase and glucose 6-phosphate dehydrogenase remain the same. The incorporation of radioactive label into pulse-labeled but not into stable ribonucleic acid is significantly lower. Our results suggest that in the mutant organism the control of transcription of some operons, including the trp operon, is modified. An alternative explanation is that mutant bacteria contain a ribonuclease with increased activity for some messenger ribonucleic acid species.


Escherichia coli mutant strain with altered expression of the tryptophan operon: ribonucleic acid synthesis in vitro. Ribonucleic acid (RNA) synthesis has been studied in vitro with a partially purified preparation of RNA polymerase from a mutant strain of Escherichia coli with a reduced rate of accumulation of tryptophan RNA (P.H. Pouwels and H.J. Scholten, J. Bacteriol. 139:393-397, 1979). The incorporation of radioactive label into RNA with polymerase from mutant bacteria is considerably lower than that with the enzyme from wild-type bacteria. These results are explained by the presence in mutant bacteria, but not in wild-type bacteria, of a factor which suppresses the accumulation of RNA. Mutant bacteria contain a factor which renders RNA synthesis with mutant and wild-type RNA polymerase resistant to various inhibitors of RNA synthesis, e.g. rifampin, streptolydigin, and heparin. We conclude that in mutant bacteria a factor is modified which suppresses the accumulation of some RNA species and lowers the sensitivity of RNA polymerase to some transcription inhibitors.


Specialized lambda transducing bacteriophage which carries hisS, the structural gene for histidyl-transfer ribonucleic acid synthetase. A number of specialized lambda transducing bacteriophages which carry the Escherichia coli gene guaB were isolated from E. coli. One of these bacteriophages, lambda cI857 Sam7 d guaB-2, also carries hisS, the structural gene for histidyl-transfer ribonucleic acid synthetase (EC 6.1.1.21). Histidyl-transfer ribonucleic acid synthetase activities in induced and uninduced lysogens carrying lambda d guaB-2 indicate that the phage carries the entire structural gene and that the gene is under the control of an E. coli promoter. These conclusions were confirmed by the in vivo production of a protein encoded by the phage which comigrates with authentic histidyl-transfer ribonucleic acid synthetase on two-dimensional polyacrylamide gels.


Isolation and partial characterization of protein E, a major protein found in certain Escherichia coli K-12 mutant strains: relationship to other outer membrane proteins. Escherichia coli outer membrane protein E was purified, and its amino acid composition and N-terminal amino acid were determined. The purified protein was shown to be immunologically and electrophoretically identical to proteins Ic (U. Henning, W. Schmidmayr, and I. Hindennach, Mol. Gen. Genet. 154:293-298, 1977) and e (W. van Alphen, N. van Selm, and B. Lugtenberg, Mol. Gen. Genet. 159:75-83, 1978). Proteins E, e, and Ic were also immunologically related to E. coli outer membrane protein Ia. Lugtenberg and co-workers (B. Lugtenberg, R. van Boxtel, C. Verhoef, and W. van Alphen, FEBS Lett. 96:99-105, 1978) have shown that electrophoretically identical peptides were generated by cyanogen bromide treatment of proteins E, e, and Ic.


Specific inactivator of flagellar reversal in Salmonella typhimurium. Specific inhibition of flagellar rotation reversal was observed after exposure of chemotactic Salmonella typhimurium to citrate autoclaved at neutral pH. The presence of a rotation reversal inactivator was established in autoclaved citrate-containing media and nutrient broth. Since modulation of flagellar rotation by attractants and repellents is the basis of chemotactic behavior, a specific inhibitor of rotation reversal, which is essential for tumble generation, provides a useful probe into the molecular mechanism of bacterial chemotaxis. The inactivator inhibits clockwise rotation without affecting counterclockwise rotation, speed of rotation, or the capacity of the cells to grow and divide. Inactivation of clockwise rotation is gradual and irreversible, differing from the transient inhibition of clockwise rotation by attractants, which is characterized by an immediate suppression followed by a return to normal rotation patterns. The rotation reversal inactivator is stable to acidification, rotary evaporation, lyophilization, and rehydration.


Inactivation of Escherichia coli by near-ultraviolet light and 8-methoxypsoralen: different responses of strains B/r and K-12. A series of Escherichia coli K-12 AB1157 strains with normal and defective deoxyribonucleic acid repair capacity were more resistant to treatment with 8-methoxypsoralen (8-MOP) and near-ultraviolet light (NUV) than a comparable series of strains from the B/r WP2 family although sensitivities to 254-nm ultraviolet light were closely similar. The difference was most marked with strains deficient in both excision and postreplication repair (uvrA recA). The hypothesis that the internal level of 8-MOP was lower in K-12 than B/r uvrA recA derivatives was ruled out on the basis of fluorometric determinations of 8-MOP content and the similar inactivation curves for phage T3 treated intracellularly within the two strains. The demonstration of liquid holding recovery with AB2480 but not WP100 (both recA uvrA strains) and the somewhat greater resistance of the former strain to inactivation by captan revealed the presence in the K-12 strain of a deoxyribonucleic acid repair system independent of the recA(+) and uvrA(+) genes. The presence of this repair system did not, however, affect the survival of T3 phage treated with 8-MOP plus NUV and probably has a relatively small effect on survival of AB2480 under normal conditions. Experiments in which 8-MOP monoadducts were converted to cross-links by a second NUV exposure in the absence of 8-MOP indicated that the level of potentially cross-linkable monoadducts immediately after 8-MOP + NUV is about eightfold lower in K-12-than in B/r-derived strains. It is therefore suggested that the photoproduct yield in the former is well below that in the latter. In agreement with this is the observation that, during the first 10 min after treatment, deoxyribonucleic acid synthesis was just over five times more sensitive to inhibition by 8-MOP plus NUV in WP100 than in AB2480. We assume that 8-MOP in K-12 bacteria is hindered in some way from adsorbing to cellular (though not to phage T3) deoxyribonucleic acid. Consistent with this, 8-MOP has been shown to act as an inhibitor of a component of repair of 254-nm ultraviolet light damage in WP2 but not in AB1157.


Spontaneous and induced rho mutants of Saccharomyces cerevisiae: patterns of loss of mitochondrial genetic markers. The deletion which leads to spontaneous rho mutants occurs preferentially at a unique region covering genes oxi3, pho1/OII, and mit175. The frequency of loss of genetic markers in this region was significantly higher than in other regions as determined with a 15- marker system. When various mutagenic treatments were applied, this specific pattern of deletion was also observed, but it was dramatically amplified. This suggests that the basic mechanism of rho production is the same in yeast mitochondrial genomes in both spontaneous and induced mutants.


Chromosomal location and expression of the structural gene for major outer membrane protein Ia of Escherichia coli K-12 and of the homologous gene of Salmonella typhimurium. The gene determining the structure of a major outer membrane protein of Escherichia coli, protein Ia, has been located between serC and pyrD, at the min 21 region of the linkage map. This is based on the isolation and characterization of E. coli-Salmonella typhimurium intergeneric hybrids as well as analyses of a mutation (ompF2) affecting the formation of protein Ia. When the serC region of the S. typhimurium chromosome was transduced by phage P1 into E. coli, two classes of transductants were obtained; one produced protein Ia like the parental strain of E. coli, whereas the other produced not protein Ia but a pair of outer membrane proteins structurally related to 35K protein, one of the major outer membrane proteins of S. typhimurium. Furthermore, a strain of S. typhimurium harboring an F' plasmid which carries the ompF region of the E. coli chromosome was found to produce a protein indistinguishable from protein Ia, beside the outer membrane proteins characteristic to the parental Salmonella strain. These results suggest that the structural genes for protein Ia (E. coli) and for 35K protein (S. typhimurium) are homologous to each other and are located at the ompF region of the respective chromosome. The bearing of these findings on the genetic control of protein Ia formation is discussed.


Mode of action of pesticin: N-acetylglucosaminidase activity. Homogeneous preparations of pesticin, a bacteriocin produced by Yersinia pestis, neither significantly inhibited net synthesis of deoxyribonucleic acid, ribonucleic acid, or protein in Escherichia coli phi nor caused detectable degradation of deoxyribonucleic acid in vivo. Accordingly, its mode of action does not resemble that of colicin E2 as suggested by others. However, incorporation of cell wall-specific label ([14C]diaminopimelic acid) into trichloroacetic acid-insoluble material of growing cells was inhibited by pesticin which also promoted release of such radioactivity from both resting cells and purified mureinlipoprotein. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis of reaction mixtures containing appropriately labeled mureinlipoprotein showed that [3H]N-acetylglucosamine comigrated either with [14C]diaminopimelic acid in the murein peptide or with [14C]isoleucine of the Braun lipoprotein. As judged by these findings and pesticin-dependent release of reducing equivalents but not 4-hydroxy-2-acetamido sugars, the bacteriocin possesses N-acetylglucosaminidase activity. Hydrolysis of murein-lipoprotein occurred over a broad pH, with an optimum of 4.7. Mureinlipoproteins from a variety of pesticin-sensitive and -resistant organisms were hydrolyzed by the bacteriocin, indicating that its antibacterial specificity resides at the level of absorption.


Adherence of bacteria to mammalian cells: inhibition by tunicamycin and streptovirudin. Group B streptococci were labeled either by growing the cells in [14C]fructose or by using the surface label 4,4'-[3H]diisothiocyano-1,2-diphenylethane-2,2'-disulfonic acid, which reacts with amino groups. A quantitative assay was developed by using these labeled bacteria to study the adherence of streptococci to canine kidney epithelial cells. The bacteria adhered to kidney cells that had been infected with influenza A virus, but did not adhere to uninfected cells. The binding of 3H-labeled group B streptococci was proportional to the number of bacteria added and showed saturation kinetics. The binding was blocked by the addition of unlabeled group B streptococci but was not affected by addition of streptococci from other groups. It was also blocked by mixing the 3H-labeled streptococci with influenza A virus before adding the bacteria to the kidney cells. When the kidney cells were infected with influenza virus in the presence of either tunicamycin or streptovirudin, these antibiotics inhibited the appearance of viral hemagglutinin in the kidney cells and also prevented the release of mature virus. In these experiments, the adherence of 3h-labeled streptococci was also inhibited. Tunicamycin was shown to block the incorporation of [14C]mannose into lipid-linked oligosaccharides and glycoprotein in both normal and virus-infected kidney cells. These data give strong support to the notion that adherence of streptococci to mammalian cells involves recognition of viral hemagglutinin, a glycoprotein whose synthesis is blocked by certain antibiotics.


Effect of temperature on translocation frequency of the Tn3 element. The effect of temperature on the translocation frequency of the Tn3 element was investigated. The temperature optimum for translocation of Tn3 was in the range from 26 to 30 degrees C. At temperatures above 30 degrees C, the translocation frequency decreased rapidly and linearly; at 36 degrees C it was only 5% of the frequency observed at 30 degrees C. The duration and reversibility of the temperature effect were utilized to demonstrate a requirement for protein synthesis in the translocation process.


Suppression of a deletion mutation in the glutamine amidotransferase region of the Salmonella typhimurium trpD gene by mutations in pheA and tyrA. Prototrophic revertants of a trpD deletion mutant that lacks the glutamine amidotransferase domain of the bifunctional component II subunit of the anthranilate synthetase-phosphoribosyltransferase complex have been found to arise by the occurrence of sublethal missense mutations in either the pheA or tyrA loci. Such suppressor mutations were obtained directly by mutation of the wild-type pheA gene as well as indirectly by partial reversion of a variety of nonleaky pheA and tyrA mutations. The suppressor strains have only a portion of the normal level of the pheA or tyrA enzyme activity and thus experience a partial limitation in the synthesis of phenylalanine or tyrosine. This limitation leads to a relaxation of end-product regulation of the phenylalanine- or tyrosine-specific enzymes of the common aromatic pathway and to the overproduction of the branch point intermediate, chorismic acid, which is one of the substrates of the anthranilate synthetase reaction. It is proposed that the high intracellular level of chorismic acid acts to elevate the non-physiological NH3-dependent anthranilate synthetase activity of the component I subunit, thereby eliminating the need for the glutamine amidotransferase activity of the component II subunit. Consistent with this is the finding that phenylalanine and tyrosine are specific inhibitors of growth of the pheA and tyrA suppressor strains, respectively, causing a shutdown of the overproduction of chorismic acid by reestablishing normal end-product control of the common pathway.


Isolation and characterization of replication-deficient mutants of ColE1 plasmids. Replication-defective mutants of plasmid ColE1 were isolated from a chimeric plasmid formed by ligating a temperature-sensitive replication derivative of pSC101, pHSG1, with a ColE1-Tn3-containing plasmid. The replication-defective ColE1 mutants isolated were all spontaneous deletion mutants that had lost the ColE1 replication origin and regions adjacent to it. The extent of a deletion was determined by analyzing restriction endonuclease-generated deoxyribonucleic acid fragments of the ColE1 plasmid component of the chimeras by both agarose and polyacrylamide gel electrophoresis. None of the chimeras containing the replication-defective ColE1 mutants was able to replicate in the presence of chloramphenicol. The expression of ColE1 incompatibility was either markedly reduced or not detectable in the replication mutants isolated.


ColE1 plasmid incompatibility: localization and analysis of mutations affecting incompatibility. Deletion mutants of plasmid ColE1 that involve the replication origin and adjacent regions of the plasmid have been studied to determine the mechanism by which those mutations affect the expression of plasmid incompatibility. It was observed that (i) a region of ColE1 that is involved in the expression of plasmid incompatibility lies between base pairs -185 and -684; (ii) the integrity of at least part of the region of ColE1 DNA between base pairs -185 and -572 is essential for the expression of ColE1 incompatibility; (iii) the expression of incompatibility is independent of the ability of the ColE1 genome to replicate autonomously; (iv) plasmid incompatibility is affected by plasmid copy number; and (v) ColE1 plasmid-mediated DNA replication of the lambda phage-ColE1 chimera lambda imm434 Oam29 Pam3 ColE1 is inhibited by ColE1-incompatible but not by ColE1-compatible plasmids.


Very slow growth of Escherichia coli.

Very slow growth of Escherichia coli. A recycling fermentor (a chemostat with 100% biomass feedback) was used to study glucose-limited behavior of Escherichia coli B. The expectation from mass transfer analysis that growth would asymptotically approach a limit mass determined by the glucose provision rate (GPR) and the culture's maintenance requirement was not met. Instead, growth proceeded at progressively lower rates through three distinct phases. After the fermentor was seeded, but before glucose became limiting, growth followed the usual, exponential path (phase 1). About 12 h postseeding, residual glucose in the fermentor fell below 1 microgram . ml-1 and the growth rate (dx/dt) became constant and a linear function of GPR (phase 2). The specific growth rate, mu, therefore fell continuously throughout the phase. Biomass yield and glucose assimilation (13%) were near the level for exponential growth, however, and independent of GPR over a broad range. At a critical specific growth rate (0.04 h-1 for this strain), phase 2 ended abruptly and phase 3 commenced. In phase 3, the growth rate was again constant, although lower than in phase 2, so that mu continued to fall, but growth rates and yields were praboloid functions of GPR. They were never zero, however, at any positive value of GPR. By inference, the fraction of metabolic energy used for maintenance functions is constant for a given GPR, although different for phases 2 and 3, and independent of biomass. In both phases 2 and 3, orcinol, diphenylamine, and Lowry reactive materials were secreted at near-constant rates such that over 50% as much biosynthetic mass was secreted as was retained by the cells.


Ultrastructure of the outer membrane of Salmonella typhimurium bacteriocin-resistant mutants deficient in the 33K protein. Outer membrane mutants of Salmonella typhimurium deficient in one, two, or three of the 33,000-dalton (33K), 34K, and 36K outer membrane proteins (7) were studied by using thin sectioning and freeze-fracturing electron microscopy techniques. The outer concave fracture face of all mutants deficient in the 33K protein had numerous particleless patches. In contrast to all previously examined 34K to 36K-deficient mutants, the 33K-deficient mutants showed marked heterogeneity in the size and distribution of such "empty" patches between cells of a culture. One mutant was deficient in both the 33K and the 34K to 36K "porin" protein complex; its outer membrane had very large particleless smooth areas. It is concluded that the 33K protein on one hand and the porin on the other are both able to form intramembraneous particles.


Inhibition of deoxyribonucleic acid repair in Escherichia coli by caffeine and acriflavine after ultraviolet irradiation. The effects of caffeine and acriflavine on cell survival, single-strand deoxyribonucleic acid break formation, and postreplication repair in Escherichia coli wild-type WP2 and WP2 uvrA strains after ultraviolet irradiation was studied. Caffeine (0.5 mg/ml) added before and immediately after ultraviolet irradiation inhibited single-strand deoxyribonucleic acid breakage in wild-type WP2 cells. Single-strand breaks, once formed, were no longer subject to repair inhibition by caffeine. At 0.5 to 2 mg/ml, caffeine did not affect postreplication repair in uvrA strains. These data are consistent with the survival data of both irradiated WP2 and uvrA strains in the presence and absence of caffeine. In unirradiated WP2 and uvrA strains, however, a high caffeine concentration (greater than 2 mg/ml) resulted in gradual reduction of colony-forming units. At a concentration insufficient to alter survival of unirradiated cells, acriflavine (2 microgram/ml) inhibited both single-strand deoxyribonucleic acid breakage and postreplication repair after ultraviolet irradiation. These data suggest that although the modes of action for both caffeine and acriflavine may be similar in the inhibition of single-strand deoxyribonucleic acid break formation, they differ in their mechanisms of action on postreplication repair.


Introduction of bacteriophage lambda into cells of Klebsiella aerogenes. We have shown that a mutation in the cro gene of phage lambda greatly reduces zygotic induction. This observation has allowed us to move this phage on an episome into cells of Klebsiella aerogenes where it grows as well as in cells of Escherichia coli. This technique should allow the introduction of various derivatives of lambda into any organism which is able to receive deoxyribonucleic acid from E. coli.


Conditional-lethal deoxyribonucleic acid ligase mutant of Escherichia coli. A new Escherichia coli deoxyribonucleic acid (DNA) ligase mutant has been identified among a collection of temperature-sensitive DNA replication mutants isolated recently (Sevastopoulos, Wehr, and Glaser, Proc. Natl. Acad. Sci. U.S.A. 74:3485-3489, 1977). At the nonpermissive temperature DNA synthesis in the mutant stops rapidly, the DNA is degraded to acid-soluble material, and cell death ensures. This suggests that the mutant may be among the most ligase-deficient strains yet characterized.


Enhancing the efficacy of lithium treatment by combined use with diuretics and low sodium diets: a preliminary report. Because reports have indicated that sodium metabolism may bear some influence on manic-depressive illness, the present study was designed to test the efficacy of sodium depletion in enhancing lithium's therapeutic effect. Twenty-seven manic patients refractory to lithium doses producing therapeutic serum lithium levels were subjected to lithium alone, lithium in combination with a thiazide diuretic, and lithium in combination with a low sodium diet. Results indicated that such combinations could be helpful in reducing manic symptoms in the majority of patients without engendering undue toxicity. While mechanisms of action remain to be elucidated, it appears that such combinations may be safely administered to those manic patients not responding to lithium alone or to those requiring lithium but suffering either a lithium-induced nephrogenic diabetes insipidus or other severe side effects. However, caution is still advised in employing these combinations until further, better controlled research substantiates these preliminary data.


A new structural protein located in the Z lines of chicken skeletal muscle. A new structural protein was purified from a prolonged 0.6 M KI extract of residues of chicken skeletal myofibrils, from which myosin, actin, and some other proteins had been removed. The protein had a chain weight of 55,000. The indirect immunofluorescence technique using antiserum against the 55,000 dalton protein revealed that the protein was exclusively located in the Z lines of a myofibril. The new protein formed lattice structures in vitro which were similar to those observed in the Z lines in situ.


Comparison of denaturation by guanidine hydrochloride of the wild type tryptophan synthase alpha-subunit of Escherichia coli and two mutant protein (Glu 49 replaced by Met or Gln). In order to elucidate the roles of individual amino acid residues in the conformational stability of proteins, the denaturation by guanidine hydrochloride of the wild-type trytophan synthase alpha-subunit of Escherichia coli and two mutant proteins, trpA33 (Glu 49 leads to Met) and trpA11 (Glu 49 leads to Gln), has been compared by means of CD measurements at pH 7.0 and various temperatures. CD spectra of the two mutant proteins were similar to that of the wild-type protein. The trpA33 and the trpA11 proteins were more and less resistant, respectively, to guanidine hydrochloride than the wild-type protein at 9.7 to 49.6 degrees C. The free energy change of unfolding in water delta delta Gnd H2O, was evaluated assuming a three state denaturation, since the denaturation curves of three proteins suggested the presence of one stable intermediate. The values of delta Gnd H2O for the trpA33, the wild-type, and the trpA11 proteins at 25.8 degrees C and pH 7.0 were 13.4,8.8, and 6.3 kcal/mol, respectively. The delta Gnd H2O of the trpA11 protein was almost independent of temperature, though that of the trpA33 protein was remarkably dependent on temperature. The conformation stabilities of the three proteins were correlated with the hydrophobicities of the substituted amino acid residues.


Kinetics of selective acylation of sn-glycerol 3-phosphate in the synthesis of phospholipid molecular species. The kinetics of the sn-glycerol 3-phosphate acyltransferase [EC 2.3.1.15] reaction support the view that the selective acylation primarily depends on the differences in the affinity of the enzyme for acyl-CoAs.


Studies on delta8-delta7 isomerization and methyl transfer of sterols in ergosterol biosynthesis of yeast. The formation of cholesta-7,24-dien-3 beta-ol and its activity as a substrate for the sterol side-chain methyltransferase in yeast have not previously been studied. Experiments with acetone-powder extracts of yeast showed that the sterol is formed from zymosterol by delta8-delta7 isomerization. However, direct conversion of cholesta-7,24-dien-3 beta-ol into zymosterol could not be demonstrated. The reversibility of the reaction was proved by the detection of 3H-incorporation into cholesta-8-en-3 beta-ol (with lathosterol as a carrier) from [3H]H2O in the medium. Incubation of cholesta-7,24-dien-3 beta-ol and S-adenosyl-L-[methyl-14C]methionine with the acetone-powder extract resulted in methylation of the sterol to form episterol. Similar  incubation of zymosterol gave fecosterol and episterol, suggesting that fecosterol initially formed by the methylation was isomerized to episterol. In intact cells, however, an alternative pathway (zymosterol yields cholesta-7,24-dien-3 beta-ol yields episterol) may also operate. The relative importance of the two pathways is not known.


Resolution and reconstitution of active transport of calcium by a protein(s) from Mycobacterium phlei. Membrane protein(s) responsible for the active transport of calcium in membrane vesicles from Mycobacterium phlei have been solubilized from membranes by sodium cholate treatment and partially purified using a hydrophobic resin. Reconstitution of calcium transport was demonstrated by reconstitution of detergent extracted membranes with the partially purified protein. The uptake of calcium in the reconstituted system was sensitive to proton-conducting uncouplers. Liposomes prepared with partially purified calcium translocating protein were capable of accumulating calcium. The uptake of calcium in this system occurred as a result of an artificial proton gradient generated by the reduction of entrapped ferricyanide with ascorbate-benzoquinone serving as a hydrogen carrier. The addition of the ionophore A23187 caused efflux of accumulated calcium in both native and proteoliposomal-reconstituted system.


Transfer RNA control of the activation of isomeric tRNATrp's. Previous studies of the homologous aminoacylations of Escherichia coli and yeast tRNATrp's terminating in 2'- and 3'-deoxyadenosine established that E. coli tryptophanyl-tRNA synthetase activates its cognate tRNA preferentially on the 2' position, while the corresponding yeast enzyme utilizes the 3' position on its homologous substrate tRNA. As this seemed to be the only change in positional specificity during evolution, the heterologous activations were investigated in an effort to determine the basis for this change. Remarkably, E. coli tRNATrp terminating in 3'-deoxyadenosine was found to be the preferred substrate for both the E. coli and yeast activating enzymes, while the same tryptophanyl-tRNA synthetase preparations both activated the isomeric yeast tRNATrp's preferentially on the 3' position. Thus, the preferred position of activation was found to be specified by the tRNA rather than the activating enzyme and, additionally, to be due to some process not reflected in initial velocity measurements. The variable utilization of individual modified aminoacyl-tRNA's as substrates in an enzyme-catalyzed deacylation process appears to provide the most likely explanation for the experimental observations.


Hemoglobin switching in sheep. Synthesis, cloning, and characterization of DNA sequences coding for the beta B, beta C, and gamma-globin mRNAs. Synthetic double-stranded DNAs (sDNAs) were prepared from sheep globin mRNA templates isolated from reticulocytes producing either hemoglobin B (HbB) (alpha 2 beta B2), HbC (alpha 2 beta C2), or HbF (alpha 2 gamma 2). These DNAs were inserted into the Eco RI site of plasmid pMB9 by the homopolymer tailing method and used to transform Escherichia coli X1776 to tetracycline resistance. Recombinant clones were identified by colony hybridization and further characterized by molecular hybridization and restriction endonuclease analysis. All plasmids analyzed thus far contained either beta- or gamma-globin DNA sequences. Moreover, sDNAs used for cloning yielded restriction endonuclease fragments consistent with the presence of predominantly beta- or gamma-sDNA, indicating that formation of double-stranded alpha-sDNA proceeds much less efficiently under our conditions than the formation of non-alpha-sDNAs. Three recombinant plasmids, pS beta B2, pS beta C69, and pS gamma 56, were selected for detailed study. These were shown to contain, respectively, beta B-, beta C-, and gamma-DNA sequences by molecular hybridization and by protection of the appropriate cDNAs from S1 nuclease digestion. Each contained all of the restriction endonuclease sites defined for the synthetic sDNAs and protected at least 90% of the sequence length of homologous cDNA. Restriction endonuclease maps of the beta B- and beta C-globin genes were identical at all 12 sites that were mapped, whereas four differences were identified in the gamma gene compared to the two others; three of these corresponded to differences in amino acid sequence of the globins. A method was developed to isolate the anti-mRNA strand of the insert for use as a specific molecular hybridization probe analogous to complementary DNA.


Both positional isomers of aminoacyl-tRNA's are bound by elongation factor Tu. Six purified Escherichia coli and yeast tRNA's were converted to positionally defined tRNA's terminating in 2'- and 3'-deoxyadenosine; the modified (amino-acyl) tRNA's were compared for their abilities to bind to elongation factor Tu (EF-Tu) in the presence both of GTP and guanylylimidodiphosphate (GMP-P(NH)P). Formation of aminoacyl-tRNA . EF-Tu . guanine nucleotide ternary complexes was monitored by gel filtration on Sephadex G-100 and Ultrogel ACA 44 columns and also by measurement of the ability of the factor to diminish the rate of chemical hydrolysis of the aminoacyl-tRNA's. The apparent positional specificity of the factor was found to be affected substantially both by the choice of guanine nucleotide and gel filtration resin utilized, but not in any systematic fashion. Likewise, assay of ternary complex formation by diminution of the rate of chemical deacylation failed to reveal any consistent positional preference from one isoacceptor to another. It is worthy of note that each modified aminoacyl-tRNA tested did form a ternary complex with EF-Tu under each of the experimental conditions used for assay, but that in each case the difference in affinity of the factor for isomeric aminoacyl-tRNA's was less than that between either of the modified aminoacyl-tRNA's and the corresponding unmodified species. On the basis of the experiments performed, we conclude that (i) EF-Tu has remarkable conformation flexibility, possibly reflecting its physiological role in recognizing 20 tRNA isoacceptors and (ii) the factor has no obvious preference for a single positional isomer of aminoacyl-tRNA and it is not clear that any preference that might exist could be established convincingly using tRNA's terminating in 2'- and 3'-deoxyadenosine.


The effect of chemical modification of 3-(3-amino-3-carboxypropyl)uridine on tRNA function. The minor base 3-(3-amino-3-carboxypropyl)uridine (acp3U) in Escherichia coli tRNAPhe was acylated with the N-hydroxysuccinimide esters of acetic, phenoxy-acetic, and naphthoxyacetic acid, as well as the ester of 5-dimethylaminonaphthalene-1-sulfonyl (dansyl)-glycine. The derivatives of tRNAPhe formed were all capable of accepting phenylalanine. There were only minor effects on the kinetic parameters of these derivatives for E. coli phenylalanyl-tRNA  synthetase. There was no effect on the ability of tRNAPhe to participate in poly(U)- or poly(ACU)-directed polypeptide synthesis or in the poly(U)-stimulated binding to E. coli ribosomes. The rate of photodynamic cross-linking of 4-Srd 8 to Cyd 13 was decreased in tRNAs containing the acetyl and dansyl-glycyl derivatives of acp3U, indicating that acylation of this base may perturb the tertiary structure of the tRNA. This base in tRNAPhe does not appear to play any role in the known biological functions of tRNAPhe.


Preparative enzymatic synthesis and hydrophobic chromatography of acyl-acyl carrier protein. We have used purified preparations of acyl-acyl carrier protein synthetase to prepare pure, native acyl-acyl carrier proteins (acyl-ACP) ranging in chain lengths from C10:0 to C delta 9 18:1. Factors affecting yield are explored and reaction conditions are presented that yield 0.8 to 0.9 mg of C16:0-ACP/ml of reaction mix. Ohter acyl groups, such as C10:0 and C delta 9 18:1 are poorer substrates and gave correspondingly lower yields. Acyl-Acp synthetase may be recovered from the reaction mixture using blue-Sepharose CL-6B and recycled. ACP and acyl-ACP are separated by hydrophobic chromatography on octyl-Sepharose CL-4B. Mixtures of acyl-ACPs could be resolved according to acyl chain length using octyl-Sepharose CL-4B columns eluted with a 2-propanol gradient. The high resolution obtained using 2-propanol gradients to separate acyl-ACP species suggests that similar techniques would be applicable to the chromatography of protein mixtures on hydrophobic supports.


Competitive inhibitors of rabbit hepatic microsomal 12 alpha-steroid hydroxylase. Rabbit hepatic microsomal 12 alpha-steroid hydroxylase which is stable to storage at -70 degrees C in the pellet form was assayed for activity with [5 alpha,6 alpha-3H2]cholestane-3 alpha,7 alpha-diol solubilized with Tween 80 since methanol was incapable of maintaining the sterol in aqueous solution. Under optimized conditions in phosphate buffer, pH 7.4, containing nicotinamide, magnesium chloride, and NADPH, the enzyme conversion appeared linear for the initial 10 min. The rate of hydroxylation was proportional to protein concentration up to 4 mg/ml. Apparent Km and Vmax were 71 microM and 323 pmol of product/mg of protein/min. Based on the known structural requirements of the enzyme system, competitive inhibitors were prepared with the C-12 position derivatized as an alkene, hydroxyl, or oxo functional group. A Dixon plot revealed that 5 alpha-cholest-11-ene-3 alpha,7 alpha,26-triol was the best inhibitor with an apparent Ki of 26 microM.


Role of a membranous sialyltransferase complex in the synthesis of surface polymers containing polysialic acid in Escherichia coli. Temperature-induced alteration in the assembly process. Membrane-associated sialyltransferase complexes of Escherichia coli K-235 catalyze the synthesis of sialyl polymers which remain associated with the cell envelope. Sialyl monophosphorylundecaprenol is an intermediate in the formation of these unique surface structures, and fluidity of the lipid phase is required for the proper function of the enzyme complex (Troy, F.A., Vijay, I.K., and Tesche, N. (1975) J. Biol. Chem. 250, 156-163, 164-170). In membranes containing an increased unsaturated fatty acid content of the phospholipids, obtained by growing cells at 15 degrees C, synthesis of polysialic acid was uncoupled from synthesis of the sialyl lipid-linked intermediate. Using reconstruction experiments, the importance of the role of an endogenous acceptor in polymer formation was suggested by the unexpected finding that polysialic acid synthesis could be reactivated in inactive membranes by the addition of an exogenous acceptor which contained sialic acid. Concomitant with polymer synthesis was a rapid loss of labeled sialic acid from the lipid phase. The activated sialic acid was shown to be transferred directly to the exogenous acceptor. These results establish: 1) that the temperature-induced alteration in polymer synthesis resulted from the inability of cells grown at 15 degrees C to either synthesize or assemble a functional endogenous acceptor and not from a defect in the synthesis of the sialyltransferase; 2) the intermediate precursor role of lipid-soluble sialic acid in sialyl polymer synthesis; and 3) that the exogenous acceptor served directly as an "acceptor" and not as a catalytic "effector" which stimulated an inactive membrane-enzyme complex. These results are in accord with the possibility that the low temperature-induced derangement in polymer formation is a consequence of the altered lipid structure resulting from the greater unsaturated fatty acid content in the membrane phospholipids. U-14C-labeled exogenous acceptor was isolated from the culture filtrate of cells grown at 37 degrees C and purified to homogeneity by preparative polyacrylamide gel electrophoresis. The pure acceptor was characterized structurally as a homopolymer of sialic acid with a degree of polymerization of approximately 12. Potassium borohydride reduction of the acceptor prior to complete hydrolysis with neuraminidase established that the polymer possessed a free reducing terminus of sialic acid. Subsequent structural studies showed that these oligomers of sialic acid appeared in the culture filtrate as a result of acid-catalyzed hydrolysis from membrane-associated polysialic acids of about 150 to 200 sialyl residues. Marked diminution of several membrane proteins was observed for cells grown at 15 degrees C. The possible relationship of these alterations to the upward shift in unsaturated lipids and to the loss of a functional endogenous acceptor is currently under study.


The product of the his4 gene cluster in Saccharomyces cerevisiae. A trifunctional polypeptide. The his4 region of yeast encodes the information for the third (phosphoribosyl-AMP cyclohydrolase), second (phosphoribosyl-ATP pyrophosphohydrolase), and tenth (histidinol dehydrogenase) steps in the histidine biosynthetic pathway. These three activities co-purify with a single protein which has a subunit molecular weight of 95,000 (95,000 protein), as determined by electrophoresis on polyacrylamide gels in the presence of sodium dodecyl sulfate. Extracts of yeast strains which carry nonsense or deletion mutations in various portions of the his4 region, purified in parallel by affinity chromatography on AMP-agarose columns, were examined on sodium dodecyl sulfate-polyacrylamide gel electrophoresis slabs. All such mutant extracts examined were found to lack the 95,000 protein found in a strain carrying a wild type his4 allele. The presence of a protease inhibitor, phenylmethylsulfonyl fluoride, during the purification of the trifunctional enzyme prevented the degradation of the 95,000 protein to polypeptides of lower molecular weight. Monospecific antibody prepared against the 95,000 protein removed all three of the activities specified by his4 from solution; active 95,000 protein was recovered in the resuspended immunoprecipitates. All this evidence shows that the product of the his4 region is a trifunctional, 95,000-dalton protein. Preliminary evidence from two-dimensional gel electrophoresis, NH2-terminal analysis, and gel filtration column chromatography indicates that the native trifunctional enzyme is a dimer of identical 95,000-dalton subunits.


Expression of the major red cell sialoglycoprotein, glycophorin A, in the human leukemic cell line K562. We have found that the human leukemic cell line K562 (Lozzio, C.B., and Lozzio, B.B. (1975) Blood 45, 321-334) synthesizes a surface membrane glycoprotein which is identical or closely similar to the major red cell sialoglycoprotein, glycophorin A. The protein can be precipitated by specific anti-glycophorin A antiserum both from surface-labeled and metabolically labeled K562 cells. Cyanogen bromide cleavage of glycophorin A from red cells and the K562 cell protein gives apparently identical fragments, and the glycopeptides and oligosaccharides obtained after Pronase and mild alkaline treatment are closely similar. An antiserum made against intact K562 cells and absorbed with normal human white blood cells precipitated surface-labeled glycophorin A from erythrocytes. The amount of glycophorin A per cell in erythrocytes and K562 cells was very similar when determined by radioimmunoassay. The K562 cells contained blood group MN activity when tested with rabbit anti-M and anti-N sera. When incubated at 37 degrees C with rabbit anti-glycophorin A F(AB)2 fragments and fluorescent sheep anti-rabbit IgG, partial redistribution of glycophorin A (patching and capping) was seen in K562 cells but not in erythrocytes.


A new method of patellectomy for patellofemoral arthritis. Total patellectomy, although it has a biomechanical disadvantage in that it may lead to a degree of quadriceps weakness, is frequently indicated in selected patients. Numerous different techniques of performing patellectomy have been described. In this paper, we present a technique of performing a patellectomy in which the continuity of the quadriceps mechanism is not disrupted and the vastuc medialis is advanced. Twenty-six patients (twenty-nine knees) in whom the procedure was performed were studied retrospectively. The findings in this series showed 90 per cent good or excellent results, two cases of extensor lag, an average of 118 degrees of knee flexion, minimum quadriceps atrophy with good strength, and minimum postoperative immobilization. The method presented has the important advantage of an easier, smoother postoperative knee-rehabilitation period, which is extremely advantageous in elderly patients.


The design of clinical trials comparing pi-meson therapy with conventional radiotherapy. Comparative clinical trials of pi-meson therapy for several tumor sites will be performed in Vancouver using the TRIUMF facility. The design of these trials poses some particular problems related to the limited numbers of patients available. In this paper, some principles of study design are set forth and applied to the issue of randomised and non-randomised designs in general, and to the pi-meson trials in particular.


[Radiological study of 305 cases of spondylolysis with or without spondylolisthesis]. Three hundred and five cases of spondylolysis with or without spondylolisthesis were reviewed. The concept of an acquired lesion must be retained even though a dysplastic lesion may be postulated to account for the familial incidence. The examination in erect position facilitates the diagnosis by favoring slipping. The vast majority of lesions are found at L5-S1. Spina bifida occulta is often found in association with spondylolisthesis. This lesion leads to apophyseal arthrosis and disc degeneration at the involved level. The accentuation of the lombosacral angle is not a predominent factor leading to spondylolysis. This angulation will increase with duration and degree of slipping. CT scan provides an excellent account of the state of the spinal canal at the affected level. Could pseudospondylolisthesis be a headed stage of spondylolysis?


Sexual conjugation in yeast. Cell surface changes in response to the action of mating hormones. In the yeast Saccharomyces cerevisiae, sexual conjugation between haploid cells of opposite mating type results in the formation of a diploid zygote. When treated with fluorescently labeled concanavalin A, a zygote stains nonuniformly, with the greatest fluorescence occurring at the conjugation bridge between the two haploid parents. In the mating mixture, unconjugated haploid cells often elongate to pear-shaped forms ("shmoos") which likewise exhibit asymmetric staining with the most intense fluorescence at the growing end. Shmoo formation can be induced in cells of one mating type by the addition of a hormone secreted by cells of the opposite mating type; such shmoos also stain asymmetrically. In nearly all cases, the nonmating mutants that were examined stained uniformly after incubation with the appropriate hormone. Asymmetric staining is not observed with vegetative cells, even those that are budded. These results suggest that, before and during conjugation, localized cell surface changes occur in cells of both mating types; the surface alterations facilitate fusion and are apparently mediated by the hormones in a manner that is mating-type specific.


Studies on the organization and localization of actin and myosin in neurons. The organization of actin in mouse neuroblastoma and chicken dorsal root ganglion (DRG) nerve cells was investigated by means of a variety of electron microscope techniques. Microspikes of neuroblastoma cells contained bundles of 7- to 8-nm actin filaments which originated in the interior of the neurite. In the presence of high concentrations of Mg++ ion, filaments in these bundles became highly ordered to form paracrystals. Actin filaments, but not bundles, were observed in growth cones of DRG cells. Actin was localized in the cell body, neurites, and microspikes of both DRG and neuroblastoma nerve cells by fluorescein-labeled S1. Myosin was localized primarily in the neurites of chick DRG nerve cells with fluorescein-labeled anti-brain myosin antibody. This antibody also stained stress fibers in fibroblasts and myoblasts but did not stain muscle myofibrils.


Role of calcium ions in the control of embryogenesis of Xenopus. Changes in the subcellular distribution of calcium in early cleavage embryos after treatment with the ionophore A23187. Treatment of stage 5 Xenopus embryos with the ionophore A23187 for only 10 min, in the absence of extracellular Mg2+ and Ca2+, causes cortical contractions and a high incidence of abnormal embryos during subsequent development. Cation analysis shows that divalent ions are not lost from the embryos, but that Ca2+ is redistributed within the subcellular fractions. Ca2+ is probably released from yolk platelets and/or pigment granules by the action of A23187, [Ca2+] rises in the cytosol, and the mitochondria attempt to take up this free Ca2+. The mitochondria concomitantly undergo characteristic ultrastructural transformations, changing towards energized-twisted and energized-zigzag conformations. A23187 allows these changes to be demonstrated in situ. Extracellular divalent cations (10(-4) M) interfere with this intracellular action of A23187. Intracellular accumulation of Na+ (by treatment with ouabain) or Li+ also causes abnormal development, probably by promoting a release of Ca2+ from the mitochondria. It is suggested (a) that all these treatments cause a rise in [Ca2+]i which interferes with normal, integrated cell division, so causing, in turn, abnormal embryogenesis, (b) that levels of [Ca2+]i are of importance in regulating cleavage, (c) that the mitochondria could well have a function in regulating [Ca2+]i during embryogenesis in Xenopus, and (d) that vegetalizing agents may well act by promoting a rise in [Ca2+]i in specific cells in the amphibian embryo.


Nerve fibers in culture and their interactions with non-neural cells visualized by immunofluorescence. Cultures of embryonic mouse spinal cord explants, alone or in combination with rat myotubes, were stained by indirect immunofluorescence using antibodies against three structural proteins to: (a) reveal the distribution of these proteins among different cell types, and (b) test the usefulness of antibody staining to reveal the gross morphology of the neurite network in complex cultures. Affinity column purified antibodies were used against chicken gizzard actin, porcine brain tubulin, and skeletal muscle alpha-actinin. Neurites were stained intensely by anti-actin as was the stress fiber pattern of underlying fibroblasts. With anti-tubulin, the staining of neurites was an order of magnitude more intense than the staining of the microtubule pattern of background fibroblasts. Neurite cell bodies and astrocyte-like glia cells were stained with anti-tubulin and their nuclei remained unstained. Anti-tubulin could thus be used to trace even the finest extensions of nerve processes in spinal cord and spinal cord-muscle cultures. Furthermore, it could be combined with the histochemical reaction for acetylcholinesterase (AChE, EC 3.1.1.7) to demonstrate AChE-positive neurons and specialized nerve-muscle contact sites. The staining of neural elements with anti-alpha-actinin was generally much weaker than with anti-actin and anti-tubulin. Neurites were stained only moderately in comparison to myotube Z lines in the same culture. However, a distinct staining of the periphery of dorsal root ganglion cells was observed. Thus, a protein immunologically related to muscle alpha-actinin is present in the nervous system. In myotubes, Z lines were stained intensely with anti-alpha-actinin while I bands were only faintly stained with anti-actin. In isolated myofibrils, both structures were stained intensely with the same antibody preparations.


High resolution microchemical analysis using soft X-ray lithographic techniques. High resolution x-ray lithographic studies of cells from chick embryo hearts dried by the CO2 critical point method have been made with soft x-ray radiation of different wavelengths. A marked difference in the relief replica in polymethyl methacrylate (PMMA) resulting from the differential absorption by the dried cells of carbon K alpha radiation at 4.48 nm and broad band synchrotron radiation (SR) with lambda is greater than 1.5 nm demonstrates the potential usefulness of the technique in making high resolution (approximately or equal to 10 nm) chemical identification of the constitutents which make up the various parts of the cell.


Ultrastructure of microfilament bundles in baby hamster kidney (BHK-21) cells. The use of tannic acid. After standard glutaraldehyde-osmium tetroxide fixation procedures, the majority of microfilament bundles in BHK-21 cells exhibit relatively uniform electron density along their long axes. The inclusion of tannic acid in the glutaraldehyde fixation solution results in obvious electron density shifts along the majority of microfilament bundles. Striated patterens are frequently observed which consist of regularly spaced electron dense (D) and electron lucid (L) bands. A striated pattern is also observed along many BHK-21 stress fibers after processing for indirect immunofluorescence utilizing BHK-21 myosin antiserum. A direct correlation of these periodicities seen by light and electron microscope techniques is impossible at the present time. However, comparative measurements indicate that the overall patterns seen in the immunofluorescence and electron microscope preparations are similar. The ultrastructural results provide an initial clue for the ultimate determination of the supramolecular organization of contracile proteins other than actin within the microfilament bundles of non-muscle cells.


Cellular interaction between fixed and living cells. Transfer of radioactive materials from living cells to fixed cells. Transfer of radioactive materials to fixed cells from an overlying layer of living cells has been examined to determine whether fixed cells can act as acceptors of glycosyltransferases of living cells. After the incubation of living cells were removed by EDTA treatment, and the radioactivity associated with the fixed cells was determined. Lipids, proteins and carbohydrates were found to be transfered from the living cells to the fixed cells. The amount of radioactivity transferred to the fixed cells was dependent on the number of both fixed and living cells and increased with the time of incubation. When fixed cells were treated with chloroform-methanol before the addition of living cells, the transfer of both lipids and proteins to the fixed cells decreased drastically, but only a slight decrease incarbohydrate transfer was observed. Most of the radioactive materials transferred from living cells labeled with glucosamine or fucose to chloroform-methanol-treated fixed cells were solubilized by trypsin but not by the detergents tested. Approximately 55% of the materials transferred from the cells labeled with glucosamine could be solubilized by hyaluronidase and chondroitinase, and the rest was solubilized by neuraminidase and a glycosidase mixture. The treatment of chloroform-methanol-extracted fixed cells with trypsin caused a significant decrease in the transfer from cells labeled with glucosamine. When nucleotide sugars were used as the radioactive precursor, no significant amount of radioactivity was transferred to the fixed cells.


Vertebrate lectins, Comparison of properties of beta-galactoside-binding lectins from tissues of calf and chicken. Beta-galactoside-binding lectins were isolated from various calf tissues and from chicken hearts by affinity chromatography on asialofetuin-Sepharose, and were compared with respect to biochemical characteristics, binding properties, antigenic cross-reactivity, and cellular localization. The lectins are all thiol group-requiring, divalent cation-independent dimers, of apparent monomer mol wt 12,000 (calf lectins) or 13,000 (chicken lectin), and acidic pI. The calf lectins appear essentially identical by dodecyl sulfate-polyacrylamide gel electrophoresis, isoelectric focusing, amino acid composition, and radioimmunoassay, while the chicken lectin is distinctly different by these criteria. However, all of the lectins competed for the same binding sites on rabbit erythrocytes, and could be inhibited by the same saccharide haptens (notably lactose and thiodigalactoside). Immuno-fluorescence studies on several cultured cell lines revealed that the bovine and chicken lectins had primarily an intracellular cytoplasmic localization. The beta-galactoside-binding lectins of vertebrates appear to be species-specific rather than tissue-specific.


Intermediate-sized filaments of human endothelial cells. Human endothelial cells prepared from unbilical cords are characterized in parallel by electron microscopy and indirect immunofluorescence microscopy using specific antibodies against different classes of intermediate-sized filaments. The strongly developed, loose bundles of intermediate-sized filaments typically found in these cells are not decorated by antibodies against prekeratin or antibodies against smooth muscle desmin. They are, however, strongly decorated by antibodies directed against murine "vimentin," i.e., the 57,000 mol wt polypeptide which is the major protein of the intermediate-sized filaments predominant in various cells of mesenchymal origin. Cytoskeletal preparations greatly enriched in intermediate-sized filaments show the enrichment of a polypeptide band comigrating with murine vimentin. This shows that the intermediate-sized filaments that are abundant in human endothelial cells are predominantly of the vimentin type and can be demonstrated by their cross-reaction with the vimentin of rodents. These data also strengthen the evidence for several subclasses of intermediate-sized filaments, which can be distinguished by immunological procedures.


Tubulin and calmodulin. Effects of microtubule and microfilament inhibitors on localization in the mitotic apparatus. Indirect immunofluorescence was used to determine the distribution of calmodulin in the mitotic apparatus of rat kangaroo PtK2 and Chinese hamster ovary (CHO) cells. The distribution of calmodulin in PtK2 cells was compared to the distribution of tubulin, also as revealed by indirect immunofluorescence. During mitosis, calmodulin was found to be a dynamic component of the mitotic apparatus. Calmodulin first appeared in association with the forming mitotic apparatus during midprophase. In metaphase and anaphase, calmodulin was found between the spindle poles and the chromosomes. While tubulin was found in the interzonal region throughout anaphase, calmodulin appeared in the interzone region only at late anaphase. The interzonal calmodulin of late anaphase condensed during telophase into two small regions, one on each side of the midbody. Calmodulin was not detected in the cleavage furrow. In view of the differences in the localization of calmodulin, tubulin, and actin in the mitotic apparatus, experiments were designed to determine the effects of various antimitotic drugs on calmodulin localization. Cytochalasin B, an inhibitor of actin microfilaments, had no apparent effect on calmodulin or tubulin localization in the mitotic apparatus of CHO cells. Microtubule inhibitors, such as colcemid and N2O, altered the appearance of tubulin- and calmodulin-specific fluorescence in mitotic CHO cells. Cold temperature (0 degrees C) altered tubulin-specific fluorescence of metaphase PtK2 cells but did not alter calmodulin-specific fluorescence. From these studies, it is concluded that calmodulin is more closely associated with the kinetichore-to-pole microtubules than other components of the mitotic apparatus.


Ultrastructural localization of wheat germ agglutinin-binding sites on surfaces of chick embryo cells during early differentiation. The objective of this work was to examine changes in a surface component of cells from the chick embryo during morphogenetic migrations of gastrulation. Two electron microscope techniques were used to localize cell-bound wheat germ agglutinin (WGA), a lectin which specifically binds N-acetyl glucosamine residues. One technique involved conjugation of peroxidase to WGA before reaction with the cells; the other technique used glucose oxidase to mark WGA which was already cell-bound. In both cases, binding was revealed using diaminobenzidine. Before formation of the primitive streak, all surfaces of the two-layered embryo bound WGA. After migration of cells through the streak, to form the three-layered embryo, not all cell surfaces bound WGA equally. Epiblast cells generally bound WGA lateral to the primitive streak but not during passage through the streak. Mesenchyme cells, after passage through the streak, bound WGA increasingly as they migrated away from the streak. A WGA-binding matrix was observed in the vicinity of the mesenchyme cells and on the dorsal surface of the endoblast. The ventral surface of the endoblast bound the lectin very poorly. In some instances, a peroxidase reaction product was consistently seen on certain surfaces which was not removable by addition of the simple hapten N-acetyl glucosamine. In these cases, the density of the deposit was lessened by use of diacetyl chitobiose as a hapten. This result, together with the reduction of reaction product following certain hyaluronidase treatments, suggests that WGA may be binding to hyaluronic acid as well as membrane glycoproteins.


Repair and survival after UV in quiescent and proliferating Microtus agrestis cells: different rates of incision and different dependence on DNA precursor supply. Cultured cells of Microtus agrestis, the common field vole, perform unscheduled DNA synthesis after UV irradiation. They respond to incubation with a DNA synthesis inhibitor (1-beta-D-arabinofuranosylcytosine) following UV in ways typical of cells capable of excision repair, with reduced survival and an accumulation of breaks in pre-existing DNA. Microtus cells irradiated with UV in a quiescent pre-S-phase state are more sensitive to UV than are proliferating cells, in terms of survival. Adding DNA precursors (deoxyribonucleosides), and--in case of proliferating cells--growing in complete rather than dialysed serum, enhance UV survival. Quiescent cells show a higher rate of endonucleolytic incision of DNA after UV than do proliferating cells. The balance between incision (producing single-strand DNA breaks) and repair DNA synthesis (leading to rejoining of breaks) is shifted by the addition of deoxyribonucleosides, which suggests that DNA precursor supply is a rate-limiting factor in repair. The lower survival of quiescent cells (in the absence of added deoxyribonucleosides) may be due to insufficient precursor supply to meet the demands of the high incision rate.


Defective transient endogenous spleen colony formation in S1/S1d mice. WCB6F1 mice of the genotype S1/S1d did not form transient 5-day endogenous spleen colonies following midlethal irradiation, either spontaneously or in response to postirradiation bleeding. Their hematologically normal (+/+) littermates produced colonies equivalent in number and morphologic type to a normal strain (D2B6F1), as evaluated by both macroscopic and microscopic criteria. Bone marrow cells from S1/S1d mice, when transplanted into lethally irradiated +/+ mice, were able to generate equivalent numbers of transient endogenous spleen colonies (TE-CFUs), as compared to that obtained when syngeneic +/+ marrow cells were injected into lethally irradiated +/+ recipients. A defective growth of an early class of hematopoietic progenitor cells, resulting in the clinical course of the S1/S1d anemia is suggested and confirms previous reports on the microenvironmental nature of this abnormality.


Ultrastructural-immunocytochemical localization of growth hormone and prolactin in human pituitaries. Immunocytochemical staining using the unlabeled antibody peroxidase-antiperoxidase method was undertaken to localize and characterize in ultrathin sections of human pituitaries the cells responsible for the secretion of GH and PRL. Somatotrophs in seven pituitaries stained with human (h) PRL-absorbed antiserum to hGH, were abundant, round to ovoid, densely granulated cells, whose mean (+/-SD) granule diameter was 368 +/- 60 nm. Lactotrophs immunostained with antiserum to hPRL were less numerous, angular or branching cells, with fewer round to ovoid granules, the mean diameter (+/-SD) of which was 185 +/- 35 nm in six pituitaries. The somewhat larger PRL granules (up to a mean diameter of 360 nm) seen in two of three additional pituitaries may have been related to the previous therapeutic administration of estrogen. Whereas the immunostained GH-secreting cells resemble the presumed somatotrophs identified in other studies on the basis of nonimmunological staining, the immunostained PRL-secreting cells differ considerably from the cells with large (600--1000 nm) granules designated as lactotrophs by several previous investigators. The hazards of ultrastructural identification of human pituitary cell types on purely morphological (as opposed to immunocytochemical) grounds are emphasized.


Measurement of immunoglobulin M (IgM), IgG, and IgA antibodies against Yersinia enterocolitica by enzyme-linked immunosorbent assay: persistence of serum antibodies during disease. An enzyme-linked immunosorbent assay for the detection and quantitation of human immunoglobulin M (IgM), IgG, and IgA antibodies against Yersinia enterocolitica is described. Formalinized or heat-treated bacteria were adsorbed onto specially designed microcuvettes, and antibodies were allowed to attach to the antigen-coated cuvettes. Rabbit anti-human mu, anti-human gamma, and anti-human alpha antisera were allowed to react with human antibodies, and these class-specific anti-immunoglobulins were detected by alkaline phosphatase-labeled swine anti-rabbit IgG. A total of 423 sera were tested. The results obtained with the enzyme-linked immunosorbent assay were compared with the results of the conventional tube agglutination test. Persistence of different antibodies was studied in six patients. Antibodies of the IgM class persisted only for 1 to 3 months after onset of the disease; thus the occurence of IgM-class Yersinia antibodies in a single sample indicates a recently acquired infection. The persistence of the IgG- and IgA-class antibodies was variable and not parallel with each other. Remarkably, all three patients in which the disease was complicated with arthritis had IgA-class Yersinia antibodies at the end of the follow-up period of 9 to 14 months, and in those without arthritis the IgA-class antibodies disappeared within 3 months after onset of the disease.


Tyrosine degradation in presumptive identification of Peptostreptococcus anaerobius. A new tyrosine medium was developed and evaluated for the differentiation of Peptostreptococcus anaerobius from other anaerobic, gram-positive cocci. The strains included 159 originating from clinical specimens and 13 reference strains received from other workers in the field. Only one strain of each species was included in the study from multiple cultures from the same patient. This medium is simple to prepare and can be used in a small clinical laboratory. One hundred seventy-two strains of anaerobic gram-positive cocci were grown and evaluated with the new tyrosine medium; 36 strains (100%) of P. anaerobius degradated the tyrosine crystals when incubated at 37 degrees C under anaerobic conditions in a GasPak jar (Baltimore Biological Laboratory) for approximately 72 h. On the other hand, 135 of 136 other anaerobic gram-positive cocci were negative for tyrosine degradation, but grew on the tyrosine agar plate when incubated anaerobically. The single strain that degraded tyrosine was 1 of the 13 isolates of Peptostreptococcus micros studied.


Partially purified antibodies used in a solid-phase radioimmunoassay for detecting candidal antigenemia. The development of a solid-phase radioimmunoassay procedure for the detection of Candida albicans antigens in serum of mice is described. Antibodies against C. albicans that were used in the radioimmunoassay procedure were partially purified from immune serum by a C. albicans antigen-coupled affinity column. Elution of anti-C. albicans antibodies from the column was by glucose and mannose; 4 mg of protein was recovered per ml, which contained 50% of the candidal agglutinin activity of immune serum. Also, 81% of the protein (partially purified antibody) recovered was adsorbed by whole C. albicans cells. Anti-C. albicans antibodies were either coupled to Sepharose 4B for use as the solid phase to bind candidal antigen in serum of infected animals, or radioiodinated (125I) for use as a tracer molecule to bind to the candidal antigen solid-phase complex. Although control experiments indicated that at least 100 ng of candidal antigen should be present in a serum specimen for a positive radioimmunoassay test, candidal antigenemia was detected in 70.4% of infected mice even in cases where blood cultures for C. albicans were negative. With further refinement and adaptability to human serum, the radioimmunoassay test may become a helpful tool for use in the diagnosis of systemic candidiasis.


False-positive reactions in the rapid plasma reagin-card, fluorescent treponemal antibody-absorbed, and hemagglutination treponemal syphilis serology tests. Sera from 628 nonsyphilitic individuals were tested with the Rapid Plasma Reagin-Card, Fluorescent Treponemal Antibody-Absorbed, and Hemagglutination Treponemal Test for Syphilis tests to ascertain the comparative specificity of these tests. Many sera were also tested with the quantitative Venereal Disease Research Laboratory test. Sera included in the study were from both normal individuals and patients with a variety of illnesses and conditions. The Hemagglutination Treponemal Test for Syphilis gave the lowest overall percentage of false-positive reactions (1.6%), followed by the Fluorescent Treponemal Antibody-Absorbed test (3.3%) and the Rapid Plasma Reagin-Card test (10.8%).


Improved blood culture technique based on centrifugation: clinical evaluation. A total of 3,335 blood samples from 1,180 patients suspected of having bacteremia were analyzed concurrently by two methods: (i) supplemented peptone broth with sodium polyanethanol sulfonate and a CO2 atmosphere; and (ii) lysis centrifugation at 3,000 X g for 30 min onto a high-density, hydrophobic cushion. The centrifugation technique recovered 80% of the positive cultures as compared with 67% for the broth method. The centrifugation technique showed an apparent increase in the isolation of staphylococcus aureus, Pseudomonas, and yeasts. In almost every instance, the time required for detection of a positive culture was shortest for the centrifugation method. Contamination rates for both systems were comparable (1.4%). Quantitation, offered only by the centrifugation method, proved useful on several occasions in discriminating between an opportunistic infection versus a skin contaminant and in judging efficacy of antimicrobial therapy.


Achromobacter species (CDC group Vd): morphological and biochemical characterization. Twenty-three isolates of Achromobacter species (CDC group Vd) were examined morphologically and biochemically. Gram stains revealed gram-variable bacilli frequently curved or hooked at one pole and often coryneform in shape and arrangement. Electron microscopy revealed the presence of extracellular material in polar accumulations and demonstrated the polar flagella arrangement seen by light microscopy to be lateral. Two colony types were produced; one was minute and watery at 24 h (35 degrees C) progressing to large, mucoid colonies at 48 h, and the other type was shiny, glistening, opaque but nonmucoid. All isolates grew on MacConkey agar and produced catalase, oxidase, and urease. Most grew on salmonella-shigella agar, reduced nitrate to nitrite and gas, hydrolyzed esculin, deaminated phenylalanine (2 to 4 days) and produced H2S in triple sugar iron agar (4 to 12 days). Oxidation of carbohydrates was weak, delayed, and limited to glucose and xylose. Two isolates also oxidized maltose, mannitol, and sucrose. The ability of miniaturized "nonfermenter" kits to identify Achromobacter species was tested. The Minitek (Baltimore Biological Laboratory, Cockeysville, Md.) and N/F (Corning, Roslyn, N.Y.) systems, respectively, identified 21 and 19 of the 23 isolates, whereas the Oxi/Ferm (Roche, Nutley, N.J.) identified 13 and the API 20E (Analytab Products, Plainview, N.Y.) identified only 3.


Solid-phase enzyme-linked immunosorbent assay for detection of hepatitis A-specific immunoglobulin M. A solid-phase enzyme linked immunosorbent assay was developed for the detection of immunoglobulin M antibody to hepatitis A virus. The system was capable of detecting hepatitis A-specific immunoglobulin M in a single dilution of serum and appears to be a reliable and rapid means of establishing a diagnosis of hepatitis A infection. Specific immunoglobulin M was only detected in patients with serologically confirmed hepatitis A and not in patients with other forms of hepatitis, chronic liver disease, or autoimmune disease. In patients with hepatitis A, specific immunoglobulin M was usually detectable for 6 weeks after the onset of dark urine, and the longest period for which it was present in any patient was 115 days. This enzyme-linked immunosorbent assay is rapid, simple to perform, and does not require complicated equipment. Provided adequate supplies of purified reagents can be obtained, this enzyme-linked immunosorbent assay procedure is likely to simplify hepatitis A serology, because the same antibody-coated plates can be utilized to detect hepatitis A virus, anti-hepatitis A virus, and hepatitis A-specific immunoglobulin M.


Laboratory and field evaluation of selective media for isolation of group B streptococci. Problems encountered with currently recommended selective media for group B streptococci (GBS) (selective broth medium and CNA agar) prompted a searach for alternative culture methods in ongoing epidemiological studies. Previously recommended inhibitory agents were tested in vitro. Gentamicin, alone or in combination with nalidixic acid, proved inhibitory for many GBS strains. Among other agents tested, polymyxin was most complementary to the gram-negative spectrum of nalidixic acid, without compromising GBS growth. Crystal violet provided the simplest, most economical staphylococcal inhibitor. Broth and agar media, constituted with these three agents and designated NPC, were evaluated in vitro and in field studies. This investigation represents the first direct comparison of broth media containing inhibitory agents for the preferential isolation of GBS. In maternal colonization studies, NPC broth proved superior to Todd-Hewitt broth containing nalidixic acid and gentamicin at concentrations employed in the previously described selective broth medium (95% versus 59% recovery). Our comparisons were done without added sheep blood since GBS grow well in Todd-Hewitt broth. NPC broth proved more sensitive than NPC agar for detecting GBS colonization in newborns. The NPC agar medium was useful for further purification of broth cultures and quantitative culture techniques.


Use of an enzyme immunoassay test for characterizing the A and M antigens of Brucella. An enzyme immunoassay test was developed for detecting the A and M antigens of brucellae. One hundred fifteen isolates, including 96 strains of Brucella abortus, 5 strains of B. suis, 1 strain of B. melitensis and 1 strain of B. neotomae, were accurately serotyped with the enzyme immunoassay test. No reactions were observed with 11 isolates of B. canis and 1 isolate of B. ovis. This rapid serological test provides for considerable savings in time and materials as compared with the standard tube agglutination test.


Use of colony pools for diagnosis of enterotoxigenic Escherichia coli diarrhea. Diagnosis of enterotoxigenic Escherichia coli diarrhea was made in 109 adult males with an acute dehydrating cholera-like syndrome in Dacca, Bangladesh, by testing 10 colonies isolated from admission stool specimens for production of heat-labile and heat-stable toxins. Toxin testing of one colony yielded a diagnosis in 92% of the cases, testing of two colonies yielded a diagnosis in 95% of the cases, testing of a pool of 5 colonies yielded a diagnosis in 95% of the cases, and testing of a pool of 10 colonies yielded a diagnosis in 96% of the cases. From stool cultures obtained on subsequent days, toxin testing of individual colonies and pools revealed diminished efficacy of pooling with decreasing numbers of enterotoxin-positive isolates in the pool. To detect the presence of enterotoxigenic E. coli in stools, toxin testing of 5 individual isolates and a pool of 10 colonies was found to be almost as effective as the testing of 10 individual isolates.


Immunoglobulin G- and M-specific enzyme-linked immunosorbent assay for detection of dengue antibodies. An enzyme-linked immunosorbent assay for the detection of antibodies to dengue virus is described. This method correlates well with a hemagglutination inhibition technique. The enzyme-linked immunosorbent assay can also be specific for human immunoglobulin M antibodies when a mu-chain-specific antiglobulin-enzyme conjugate and fractionated serum are employed. By using this technique, dengue immunoglobulin M antibodies were demonstrated in an infant suspected of having a recent dengue infection.


Comparison of the Minitek system with conventional methods for identification of nonfermentative and oxidase-positive fermentative gram-negative bacilli. The Minitek system was compared with conventional test methods for identifying a wide range of nonfermentative and oxidase-positive fermentative gram-negative bacilli. A total of 230 isolates representing 33 species and biotypes were tested against 12 Minitek substrates and the corresponding conventional tube media. In addition, supplementary tests were included for 141 (61.3%) of the isolates. Overall, 88% of the positive reactions agreed, and negative reactions agreed 95.5%. Anaerobic dextrose, maltose, lactose, and citrate were responsible for 62.4% of the 93 discrepant positive reactions, and 51.8% of the 83 discrepant negative reactions involved the aerobic dextrose, nitrate, and citrate disks. Some discrepancies were related to specific organisms. The system and supplementary tests correctly identified 88.3% of the isolates to species level and 92.6% to correct genus. No particular organism or substrate was responsible for misidentification errors. In a test challenge with 19 selected organisms, 3 were incorrectly identified to species and 4 were misidentified to genus level. Most of the errors responsible for these incorect identifications were due to factors other than the Minitek disks and pointed out the importance of using appropriate supplemental data, and the need for a coding manual and a more enriched broth for certain types of isolates.


Pooling method for screening large numbers of Escherichia coli for production of heat-stable enterotoxin, and its application in field studies. A modified suckling mouse assay was developed for use in field studies whereby five E. coli isolates could be tested as a pool for heat-stable enterotoxin.


New medium for differentiation of Candida albicans from Candida stellatoidea. A simple medium effectively differentiated Candida stellatoidea from Candida albicans on the basis of a new criterion, relative sensitivity to cycloheximide.


Constricted tube system for presumptive identification and differentiation of group D streptococci. A single, constricted tube containing two differential media to identify and differentiate group D streptococci was developed. Test results with a limited number of group D streptococcal isolates were in complete agreement with results of conventional procedures.


Reconstitution of spectrin-deficient, spherocytic mouse erythrocyte membranes. To study directly the role of spectrin in erythrocyte membrane function, we have designed a reconstituted membrane system using erythrocyte membranes from spectrin-deficient mice and purified spectrin from normal mice. The normal spectrin is inserted into the spectrin-deficient spherocytes by exchange hemolysis. Thereafter, raising the ionic strength and temperature reseals the cells and, with time, facilitates binding of the spectrin to the spectrin-deficient membranes. The binding is apparently specific as shown by its dependence upon the concentration of undenatured spectrin and the concentration of salt used, as well as by the immunofluorescent appearance of the reconstituted cells after treatment with specific antispectrin antibody. In terms of in vitro cellular behavior, the reconstituted preparations show marked changes in comparison to the untreated spherocytes. In particular, membrane stability, as measured by the reduction of myelin figure formation and lipid loss, is considerably enhanced. In addition, membrane fusion, which occurs readily with the untreated spherocytes, is virtually eliminated. Finally, the osmotic behavior of the native spherocytes is appreciably altered, such that the early phase of osmotically induced swelling, as measured in a high-speed stop-flow apparatus, is delayed and modified. Taken together, these findings indicate specific roles for spectrin in the stabilization of the erythrocyte membrane, in the limitation of membrane fusion, and in the modulation of the membrane's response to osmotic stress.


Effect of age on glucose-stimulated insulin release by the beta-cell of the rat. To assess the effect of age on beta-cell insulin release, collagenase-isolated islets of Langerhans were obtained from rats aged 2--18 mo and incubated with increasing concentrations of glucose. Similar islets were analyzed for insulin content or subjected to morphometric measurements to identify both the number of beta-cells and the volume of beta-granules per islet. In parallel studies, the islet content of intact pancreata was also determined. The results showed that beta-cell number increased from 2,300 t0 5,000 cells as rats aged from 2 to 18 mo and islet insulin content doubled. However, glucose-stimulated insulin release decreased progressively with age, and this was especially striking when considered in terms of the increase in number of beta-cells/islet; e.g., mean (+/- SEM) insulin secretion (nanounits per minute per beta-cell) of islets incubated with 450 mg/dl of glucose was 1.3 (+/- 0.02), 1.0 (+/- 0.1), 0.4 (+/- 0.05), and 0.3 (+/- 0.01), respectively for 2-, 6-, 12-, and 18-mo-old rats. Thus, insulin secretion per beta-cell was decreased, despite increased stores of insulin per cell. These findings demonstrate that the aging process leads to a profound defect in glucose-stimulated insulin release from the beta-cell. Whether this is a global secretory defect, or solely a failure of the beta-cell to respond to glucose, remains to be defined.


Changes in sedimentation of surfactant in ventilated excised rat lungs. Physical alterations in surfactant associated with the development and reversal of atelectasis. We ventilated excised rat lungs at a constant tidal volume (CTV); they developed areas of atelectasis which could be reversed by a large inflation (CTV + I) or prevented by the addition of positive end-expiratory pressure to the CTV. To explore the possibility that these modes of ventilation led to changes in surfactant, we lavaged the lungs and centrifuged the returns at 500 g; we measured the amount of disaturated phosphatidylcholine (DSPC) in the resultant pellet and supernatant fluid as a marker for surfactant. We found 16.9+/-1.5 (mean+/-SE), 38.0+/-2.4, 18.3+/-1.6, and 21.7+/-2.3% of the total lavage DSPC, in the pellet from freshly excised, CTV, CTV + I, and positive end-expiratory pressure to the CTV lungs, respectively. The total amount of lavage DSPC was the same in all groups. The ultrastructure of acellular material pelleted by sequential centrifugation of lavage returns at 500, 1,000, and 60,000 g was examined. We found mostly tubular myelin in the 500-g and 1,000-g pellets, but no tubular myelin in the 60,000-g pellet. Air inflation pressure-volume measurements from the degassed state revealed that the opening pressure and recoil pressures up to 75% of total lung capacity were significantly higher in the CTV than in the CTV + I lungs. There were no differences between these groups in air deflation or in saline inflation and deflation pressure-volume measurements. Our findings suggest that CTV leads to increases in the tubular myelin form of surfactant and that this leads to increased surface tension in alveoli which results in alveolar collapse.


Ketamine-facilitated induced anxiety therapy and its effect upon clients' reactions to stressful situations. Evaluated the role of physiological arousal in the Induced Anxiety therapy procedure. Assigned 21 normal Ss to either conventional Induced Anxiety Induced Anxiety supplemented by the drug ketamine during arousal, or a no-treatment condition. The ketamine group was superior to the conventional group which was superior to the no-treatment group in reducing negative affect experienced during stressful situations. Most of the reduction was in depressive affect. The superior results obtained by increasing physiological arousal could not be accounted for by increased subjective emotional arousal.


Effect of hydrogen peroxide on developing plaque and gingivitis in man. The present experiment was undertaken to assess the effect of hydrogen peroxide release during mouth rinsings on the composition of the microbiota of developing plaque in humans and the amount and pathogenecity of the plaque formed. The trial was designed as a double-blind crossover study of the effect of a mouthwash (Amosan) had a placebo rinse on the development of plaque and gingivitis in young adults. The active compound was available as a powder; the rinse consisted of 1.7 g powder dissolved in 30 ml hot tap water. Fourteen dental students participated in the trial. The students were examined during two consecutive periods, each consisting of one preparatory (during which active tooth cleaning measures were carefully practiced) and one main test period (during which mouth rinsings were the only plaque control measure). Each of the two test periods was initiated by a baseline examination following which the participants rinsed either with the active or the placebo mouthwash. The rinsings were performed immediately after breakfast, after lunch and after dinner. Measurements of Plaque and Gingival Index scores were performed 4, 7 and 14 days after the start of the no-toothbrushing period. Bacteria were sampled and examined after 7 and 14 days of trial. The results demonstrated that a mouthwash which released hydrogen peroxide effectively prevented the colonization of filaments, fusiforms, motile and curved rods as well as spirochetes in developing plaque. The mouthwash which was used as the only oral hygiene measure during a 2-week period furthermore markedly reduced the amount of plaque formed and significantly retarded gingivitis development. It is suggested that H202 released by mouthwashes during rinsing may prevent or retard the colonization and multiplication of anaerobic bacteria.


Periodontal therapy by local delivery of tetracycline. The present investigation assessed the feasibility of treating periodontal disease by controlled delivery of antibacterial agents from within periodontal pockets. Tetracycline-filled hollow fibers placed in the gingival sulcus were shown to have a dramatic effect both on the periodontal microflora and clinical manifestations of disease. Furthermore, it was found that drug-filled cellulose acetate hollow fibers are biologically compatible with the environment and can be manipulated by dental personnel to provide drug therapy with less than 1/1000 the amount of tetracycline that would have been used for systemic therapy. Of theoretical importance is the observation that virtual elimination of spirochetes from the gingival sulcus is possible by a single placement of tetracycline-filled hollow fibers, and spirochetes, once eliminated from a site, do not rapidly recolonize despite the persistence of viable organisms elsewhere in the mouth.


Amalgam restorations, plaque removal and periodontal health. A total of 156 approximal subgingival amalgam overhanging margins were assessed in the buccal segments of 13 patients for plaque accumulation, gingival inflammation, pocket depth and gingival shrinkage. Recordings were made immediately before and 2, 4 and 8 weeks following scaling, removal of overhangs, and oral hygiene instruction. Surfaces with overhangs were compared with control surfaces, which were either intact or contained supragingival amalgams. Initially it was found that gingival inflammation and pocket depth were more extensive adjacent to subgingival amalgam overhangs than to sound teeth or those with supragingivally located amalgams. This appeared to be due to preferential plaque accumulation in relation to subgingival overhangs. For all parameters compared, differences apparent at the baseline had disappeared by the end of the 8-week study period. Contouring of defective subgingival restorations, followed by effective scaling and oral hygiene instruction produced approximately 1 mn of gingival shrinkage, sufficient in most cases to produce clinical gingival health. Of the 156 subgingival fillings 83 (53%) became supragingival and 43 (28%) reached the gingival crest by the end of the 8-week study period.


Clinical improvement of gingival conditions following ultrasonic versus hand instrumentation of periodontal pockets. A study utilizing a split-mouth design in 18 subjects and with four operators was performed in order to compare the effect of hand and ultrasonic instrumentation of periodontal pockets. Contralateral pairs of teeth with pockets of comparable depth were treated by either hand or ultrasonic instruments. Instrumentation was repeated after 4 weeks. Pocket depth, bleeding on probing and gingival fluid measurements were used for evaluation. A gradual reduction of pocket depth and number of bleeding points took place throughout the 8-week experimental period. Clinically significant differences were not observed comparing the effect of hand instruments to ultrasonics for any of the operators. The amounts of gingival fluid were similar for both methods of instrumentation at the final examination. Thus, the present study failed to demonstrate any significant differences between the effect of ultrasonic root debridement and hand instrumentation in the treatment of 4-6 mm deep periodontal pockets.


Universal health insurance in Canada: history, problems, trends. This paper describes the universal health insurance program in Canada and identifies the historical events and social values leading to its adoption. Universal hospital insurance was adopted in 1958, ten years before medical insurance, as a result hospital-based patterns of practice were solidified. Through cost sharing, the federal government influenced the provinces to enact relatively uniform universal plans. From 1951 to 1971 health care expenditures rose rapidly to 7.3% of the gross national product (GNP), but have since decreased and stabilized at about 6.9%. In contrast, health care in the United States represents 8.6% of GNP. Hospital use also increased rapidly in Canada to 1970 but appears to have stabilized and decreased slightly in this decade. Physician incomes rose rapidly before 1971, but since then the increases have slowed and relative incomes of physicians have fallen. Althouth the percent of GNP spent for health care has leveled, there are still substantial annual increases in expenditures that are paid for by government. Two federal initiatives, Bill C-37 and the Lalonde Report, have their roots in cost containment; Bill C-37 transfers greater taxing authority from the federal government to the provinces. To meet the goal of containing costs, provincial governments are moving in the direction of regionalization, decentralization, and greater coordination. In the short term, the provinces have limited hospital budgetary increases to percentages less than the rate of inflation. Cost constraints may be long overdue. Imposing fiscal limits encourages rational planning. It does not appear that the health of Canadians will be adversely affected or essential benefits curtailed by present budgetary restrictions or reorganization.


Display and visualization of three-dimensional reconstructed anatomic morphology: experience with the thorax, heart, and coronary vasculature of dogs. A new method, termed reprojection, is used to visualize anatomic morphology contained within three-dimensional reconstructions made up of images of multiple parallel cross sections. This method involves the projection, either orthographically into a plane or radially onto a cylinder, of the volume picture elements (voxels) of the reconstruction. Orthographic reprojection images, formed by mathematically summing the magnitudes of the voxels along selected parallel paths through the reconstructed volume, are analagous to conventional radiographs formed by the passage of an X-ray beam through the volume. The reprojection image is a two-dimensional array of picture elements that is displayed on a television monitor using a digital-to-video scan converter. Also described are the techniques of noninvasive selective tissue dissolution and numerical dissection, whereby obscuring portions of the reconstructed volume are either partially "dissolved" or totally eliminated before reprojection. Utilizing these methods, anatomic information present in a three-dimensional reconstruction but not clearly seen in a reprojection image is rendered visible after removal of superposed structures. The usefulness of these methods is demonstrated utilizing three-dimensional reconstructions of the thorax, heart, and coronary arteries of dogs.


Economic losses from and the national research program on mastitis in the United States. Scientific papers estimating economic losses from mastitis were reviewed. Reduced milk production from cows with subclinical mastitis was responsible for the largest losses. Losses from mastitis in the United States in 1976 were estimated by usable responses to a survey from 33 states representing 9.5 million cows or 86% of the dairy cow population. Losses of milk yields caused by mastitis were 386 kg/cow per yr and losses of discarded milk 62 kg/cow per yr. Annual losses per cow from mastitis were a) reduced milk production, $81.32; b) discarded milk, $12.88; c) cost of veterinary services, $1.97; d) cost of drugs, !3,86; e) increased labor, $2.28; f) decreased sale value, $5.72; g) increased replacement costs, $9.32; h) total, $117.35. For the 11 million cows in the United States 1976 losses from mastitis were $1.294 billion. Mastitis research was supported publicly at 22 locations under 43 projects. Approximately 24 scientist years were involved and $2.7 million of public funds were expended annually. If economic losses from mastitis were reduced 2% per year for 10 yr by research and expenditures for mastitis research remained the same, the benefit to cost ratio from mastitis research would be approximately 9.6 to 1.