Electron acceptor taxis and blue light effect on bacterial chemotaxis. Salmonella typhimurium and Escherichia coli from anaerobic cultures displayed tactic responses to gradients of nitrate, fumarate, and oxygen when the appropriate electron transport pathway was present. Such responses were named "electron acceptor taxis" because they are elicited by terminal electron acceptors. Mutant strains of S. typhimurium and E. coli were used to establish that functioning electron transport pathways to nitrate and fumarate are required for taxis to these compounds. Aerotaxis in S. typhimurium was blocked by 1.0 mM KCN, which inhibited oxygen uptake. Similarly, a functioning electron transport pathway was shown to be essential for the tumbling response of S. typhimurium and E. coli to intense light (290 to 530 nm). Some inhibitors and uncouplers of respiration were repellents of S. typhimurium. We propose that behavioral responses to light or electron acceptors involve electron transport-mediated perturbations of the proton motive force.


Deoxyribonucleic acid adenine and cytosine methylation in Salmonella typhimurium and Salmonella typhi. The methylations of adenine in the sequence -GATC- and of the second cytosine in the sequence - [Formula: see text] - were studied in Salmonella typhimurium and in Salmonella typhi. The study was carried out by using endonucleases which restrict the plasmid pBR322 by cleavage at the sequences -GATC- (DpnI and MboI) and - [Formula: see text] - (EcoRII). The restriction patterns obtained for this plasmid isolated from transformed S. typhimurium and S. typhi were compared with those of pBR322 isolated from Escherichia coli K-12. In E. coli K-12, adenines at the sequence -GATC- and the second cytosines at - [Formula: see text] - are met hylated by enzymes coded for by the genes dam and dem, respectively. From comparison of the restriction patterns obtained, it is concluded that S. typhimurium and S. typhi contain genes responsible for deoxyribonucleic acid methylation equivalent to E. coli K-12 genes dam and dcm.


Pyridine nucleotide cycle of Salmonella typhimurium: in vitro demonstration of nicotinamide mononucleotide deamidase and characterization of pnuA mutants defective in nicotinamide mononucleotide transport. The enzyme nicotinamide mononucleotide deamidase, an integral component of the proposed four-membered pyridine nucleotide cycle (PNC IV), has been demonstrated in extracts of Salmonella typhimurium LT2. The enzyme has an optimum pH of 8.7 and deamidates nicotinamide mononucleotide, forming nicotinic acid mononucleotide. Sigmoidal kinetic data suggest that this enzyme may be allosteric and therefore an important regulatory component of pyridine nucleotide cycle metabolism. Mutants previously designated pncC in anticipation of their lacking nicotinamide mononucleotide deamidase were examined and found to have normal levels of this enzyme. [14C]nicotinamide mononucleotide uptake studies, however, revealed a defect in the transport of this compound. Accordingly, the genetic designation for this locus was changed to pnuA to reflect its involvement in pyridine nucleotide uptake. Evidence is presented for the existence of two separate nicotinamide mononucleotide transport systems.


Regulated breakdown of Escherichia coli deoxyribonucleic acid during intraperiplasmic growth of Bdellovibrio bacteriovorus 109J. During growth of Bdellovibrio bacteriovorus on [2-14C]deoxythymidine-labeled Escherichia coli, approximately 30% of the radioactivity was released to the culture fluid as nucleoside monophosphates and free bases; the remainder was incorporated by the bdellovibrio. By 60 min after bdellovibrio attack, when only 10% of the E. coli deoxyribonucleic acid (DNA) had been solubilized, the substrate cell DNA was degraded to 5 X 10(5)-dalton fragments retained within the bdelloplast. Kinetic studies showed these fragments were formed as the result of sequential accumulation of single- and then double-strand cuts. DNA fragments between 2 X 10(3) and 5 X 10(5) daltons were never observed. Chloramphenicol, added at various times after initiation of bdellovibrio intraperiplasmic growth on normal or on heated E. coli, which have inactivated deoxyribonucleases, inhibited further breakdown and solubilization of substrate cell DNA. Analysis of these intraperiplasmic culture deoxyribonuclease activities showed that bdellovibrio deoxyribonucleases are synthesized while E. coli nucleases are inactivated. It is concluded that continuous and sequential synthesis of bdellovibrio deoxyribonucleases of apparently differing specificities is necessary for complete breakdown and solubilization of substrate cell DNA, and that substrate cell deoxyribonucleases are not involved in any significant way in the degradation process.


Polyamine biosynthesis during germination of yeast ascospores. The role of the diamine putrescine during germination and outgrowth of ascospores of Saccharomyces cerevisiae was examined. Ornithine decarboxylase activity increased and declined rapidly during germination and outgrowth; peak activity was attained after the cells had proceeded through the G1 interval of the cell cycle, whereas minimal activity was present at the completion of the first cell division. alpha-Methylornithine inhibited both ornithine decarboxylase activity and the in vivo accumulation of putrescine. In the presence of alpha-methylornithireak dormancy and proceed through one cell division. Subsequent cellular growth, however, was retarded but not completely inhibited. The supplementation of Methylglyoxal bis(guanylhydrazone) to sporulation medium greatly inhibited this sexual process. These data suggest that the synthesis of putrescine is not required for the breaking of spore dormancy, but that polyamine biosynthesis may be essential for meiosis and sporulation.


Regulation and solubilization of Candida albicans chitin synthetase. A cytoplasmic component which inhibited the activation of chitin synthetase was studied in the dimorphic fungus Candida albicans. The inhibitor was found to be heat stable and trypsin sensitive and was only effective when incubated with a vacuolar protease, an activator of chitin synthetase, before the activation of chitin synthetase. In addition, the particulate chitin synthetase from the yeast form of C. albicans was solubilized by a sodium cholate-digitonin extraction and subsequently was purified approximately 30-fold by Sepharose column chromatography and Amicon XM 100 filtration. Activity of the soluble enzyme was increased by the addition of trypsin or phosphatidyl serine. The molecular weight of the enzyme was estimated to be 400,000.


Interaction of bacteriophage K10 with its receptor, the lamB protein of Escherichia coli. The lamB protein of Escherichia coli was initially recognized as the receptor for bacteriophage lambda. It is now shown also to constitute the receptor for phage K10. The lamB protein interacts with phage K10 in vitro, but this interaction does not lead to phage inactivation. Most lambda-resistant labB mutants are also resistant to K10, and vice versa. However, a significant proportion of the mutants resistant to one of the phages is sensitive to the other. Nineteen K10-resistant lambda-sensitive mutants have been studied. Only six of them produce a lamB protein which seems totally unimpaired in its ihe same deletion interval of the lamB gene. The corresponding region of the lamB polypeptide must be specifically involved in the interaction with phage K10. An unusual pattern of K10 host range mutants has been obtained; two calsses of such mutants could be defined, growing on two distinct classes of K10-resistant lamB mutants.


Evolution of multiple-antibiotic-resistance plasmids mediated by transposable plasmid deoxyribonucleic acid sequences. Two plasmid deoxyribonucleic acid sequences mediating multiple antibiotic resistance transposed in vivo between coexisting plasmids in clinical isolates of Serratia marcescens. This event resulted in the evolution of a transferable multiresistance plasmid. Both sequences, designated in Tn1699 and Tn1700, were flanked by inverted deoxyribonucleic acid repetitions and could transpose between replicons independently of the Excherichia coli recA gene function. Tn1699 and Tn1700 mediated ampicillin, carbenicillin, kanamycin, and gentamicin resistance but differed in the type of gentamicin-acetyltransferase enzymes that they encoded. The structural genes for these enzymes share a great deal of polynucleotide sequence similarity despite their phenotypic differences. The transposition of Tn1699 and Tn1700 to coresident transferable plasmids has contributed to the dissemination of antibiotic resistance among other gram-negative bacteria. These organisms have recently caused nosocomial infections in epidemic proportions.


Selection for Escherichia coli mutants with proteins missing from the ribosome. Antibiotic-independent revertants of an erythromycin-dependent strain of Escherichia coli were isolated by spontaneous selection. Their ribosomal proteins were analyzed by two-dimensional polyacrylamide gel electrophoresis. In contrast to most ribosomally targeted selections, the specific absence of a certain protein from the ribosome, rather than alterations in ribosomal proteins, was observed. Mutants were found with protein S20, L11, L15, L28, L29, or L30 missing.


Kietics of thermal unfolding and refolding of thermostable phosphoglycerate kinase. The kinetics of denaturation by guanidine hydrochloride (GuHCl) of a thermostable phosphoglycerate kinase (PGK) extracted from Thermus thermophilus and of yeast PGK at neutral pH were studied by circular dichroism. Denaturation by GuHCl proceeded as a first-order reaction. The activation free energy of the denaturation reactions (delta Gf not identical to ) in the absence of GuHCl was estimated to be 32.7 kcal/mol for T. thermophilus PGK and 27.9 kcal/mol for yeast PGK (at 25 degrees C). Measurements of the rate constants at various temperatures indicated that delta Gf not identical to has maximum values at 29 degrees C for T. thermophilus PGK and at 20 degrees C for yeast PGK, and that the temperature dependences of delta Gf not identical to, delta Hf not identical to, and delta Sf not identical to for T. thermophilus PGK are smaller than those of yeast PGK. Values of delta Sf not identical to for thermal denaturation for both PGK's are approximately 200 e.u.


Properties of purified detergent-resistant phospholipase A of Escherichia coli K-12. Inactivation, and protection with detergents and phospholipids. A crude preparation of membrane-bound phospholipase A (detergent-resistant) in Escherichia coli K-12 cells was found to be quite stable or even apparently activated on incubation at 100 degrees C, but became strikingly thermolabile when it was highly purified and Triton X-100 was removed from the purified enzyme preparation. The rate of inactivation showed a biphasic temperature dependence: inactivation was rapid at 37 degrees C and also above 70 degrees C. Inactivation above 70 degrees C changed the mobility of the enzyme on sodium dodecyl sulfate/polyacrylamide gel electrophoresis, but inactivation at 37 degrees C did not affect the electrophoretic mobility. Triton X-100 effectively protected the enzyme against inactivation at 37 degrees C. The concentration required for the protection of the enzyme was more than its critical micelle concentration. Phospholipids, such as phosphatidylethanolamine, phosphatidylglycerol, cardiolipin, phosphatidylcholine, lysophosphatidylethanolamine, and lysophosphatidylcholine, also protected the enzyme against inactivation at 37 degrees C. These results suggest that the binding of hydrophobic compounds stabilizes the enzyme.


On the role of the sterol hydroxyl group in membranes. The adequacy of sterol derivatives containing a blocked 3-hydroxyl group for sustaining the growth of two sterol auxotrophs has been investigated. Mycoplasma capricolum, a cholesterol-requiring bacterium, grows nearly as well on media supplemented with cholesteryl methyl ether or cholesteryl acetate as on free cholesterol. The two derivatives are recovered unchanged from the bacterial cells. Similarly, cholesteryl methyl ether or ergosteryl methyl ether replace cholesterol or ergosterol as sterol sources for a yeast mutant, strain GL7, defective in 2,3-oxidosqualene-lanosterol cyclization. During aerobic or semianaerobic growth, yeast cells demethylate some of the cholesteryl methyl ether to free cholesterol. However, cells growing on cholesterol methyl ether under strict anaerobic conditions do not produce free sterol. The bearing of these results on the postulated requirement of a free sterol hydroxyl group for membrane function is discussed. Sterol esterification does not appear to be essential for the two microbial systems.


Nucleotide sequence of the intercistronic region preceding the gene for RNA polymerase subunit alpha in Escherichia coli. The gene for RNA polymerase subunit alpha is a co-transcribed with several ribosomal protein genes (Jaskunas, S.R., Burgess, R.R., and Nomura, M. (1975) Proc. Natl. Acad. Sci. U.S.A. 72, 5036-5040). The DNA sequence whicch codes for the COOH-terminal amino acids of S4 and the NH2-terminal amino acids of alpha, and the 25 nucleotide intercistronic sequence have been determined. This short distance supports the idea that some post-transcriptional regulation determines the differential synthesis of alpha and ribosomal proteins.


A synthetic tyrosine suppressor tRNA gene with an altered promoter sequence. Its cloning and relative expression in vivo. The total synthesis of a tyrosine suppressor tRNA gene with a modified promoter is described. The alteration involves the replacement of the four G:C base pairs immediately preceding the start point of transcription by A:T base pairs. The new sequence contains the recognition sequence for the HindIII restriction endonuclease at the transcriptional start point, thus permitting fusion of the structural gene with promoters containing independent sequence modifications. The construction, cloning, and biological activity of several recombinant DNAs containing the tRNA gene with the modified promoter are described. The expression of this gene in vivo is compared with that of both the unmodified synthetic suppressor gene and a naturally occurring tyr su3+ gene cloned onto a multicopy plasmid.


Inhibition of the recBC enzyme of Escherichia coli by specific binding of pyridoxal 5'-phosphate to DNA binding site. Pyridoxal 5'-phosphate rapidly abolished the DNA-hydrolyzing activities as well as DNA-dependent ATP-ase activity of the recBC enzyme of Escherichia coli. Pyridoxal also had an inhibitory effect on the enzyme but less effective than that of pyridoxal 5'-phosphate. Pyridoxamine 5'-phosphate, pyridoxamine, or pyridoxine had no effect on the activities of the enzyme. The inhibition was rapidly reversed by dilution but could be made irreversible by reduction with sodium borohydride prior to dilution. This suggests the formation of Schiff base between pyridoxal 5'-phosphate and an epsilon-amino group of a lysine residue which is essential for the enzyme activity. Pyridoxal 5'-phosphate is a competitive inhibitor of DNA substrate but not of ATP. Furthermore, the presence of DNA substrate protected the enzyme from inactivation by the reduction but the presence of ATP showed no effect. Thus, the recBC enzyme appears to have an essential lysine residue at or near the DNA binding site of the enzyme, and the enzyme possesses two independent catalytic sites, such as a DNA binding site and an ATP binding site.


The amino acid sequence of the trimethoprim-resistant dihydrofolate reductase specified in Escherichia coli by R-plasmid R67. The amino acid sequence of a trimethoprim-resistant dihydrofolate reductase (EC 1.5.1.3) specified by the R-plasmid R67 is described. The sequence was deduced from automatic and manual sequence analysis of the intact protein, the fragments produced by cyanogen bromide cleavage, and peptides derived from the largest cyanogen bromide fragment by digestion with trypsin, Staphylococcus aureus V8 proteus, chymotrypsin, and Lysobacter enzymogenes alpha-lytic protease. The complete sequence comprises 78 residues in a single polypeptide chain of molecular weight 8444. No evidence of heterogeneity was obtained, indicating that all subunits of the native enzyme are identical. Comparison of the sequence with that of all known dihydrofolate reductases shows no significant sequence homology.


Identification of two different RNase H activities associated with yeast RNA polymerase A. Two ribonuclease H activities have been found in yeast RNA polymerase A. The nuclease activities comigrated with subunits A49 (Mr = 49,000) and A40 (Mr = 40,000), after electrophoresis in a sodium dodecyl sulfate polyacrylamide gel containing [32P](rG)n . (dC)n as substrate. Both activities were also found, among other nucleases, in a high salt chromatin extract. Several lines of evidence suggest that the chromatin RNase H of 49,000 daltons (RNase H49) is the same protein as subunit A49. They co-migrate on sodium dodecyl sulfate-gel electrophoresis, have the same chromatographic properties, and dissociate simultaneously from RNA polymerase A. Fractions containing RNase H49 stimulate RNA synthesis by RNA polymerase A* lacking A49 and A34.5 subunits. Finally, limited proteolysis of the protein band having RNase H49 activity yields the characteristic fingerprint of the A49 subunit. This subunit, therefore, exists in two states: bound to chromatin and associated with RNA polymerase A. On the other hand, it is not yet clear whether the RNase H activity of 40,000 daltons, associated with RNA polymerase A, is due to the A40 subunit or whether it represents a trace contamination by a very active nuclease tightly bound to the enzyme.


Evidence for a second histidine at the active site of succinyl-CoA synthetase from Escherichia coli. Ethoxyformic anhydride was used to demonstrate the existence of a second important histidine in succinyl-CoA synthetase from Escherichia coli. Differential labeling of the enzyme by [3H]ethoxyformic anhydride gave a stoichiometry of one important histidine per alpha beta catalytic unit. Data are presented suggesting that this residue and an important thiol group on the beta subunit (Collier, G., and Nishimura, J.S. (1978) J. Biol. Chem. 253, 4938-4943) interact with each other during catalysis. A mechanism of action involving these 2 residues is proposed for one of the partial reactions catalyzed by succinyl-CoA synthetase.


Purification and properties of the sn-glycerol 3-phosphate-binding protein of Escherichia coli. A binding protein for sn-glycerol 3-phosphate was isolated from the cell envelope of Escherichia coli by the cold osmotic shock procedure. The protein was purified to homogeneity. It has a molecular weight of 45,000 and binds sn-glycerol 3-phosphate with a KD of 0.2 microM. The protein is monomeric and has L-leucine as NH2-terminal amino acid. The intrinsic fluorescence of the protein is altered upon binding of substrate. At an excitation of 285 nm, the emission maximum at 340 nm is quenched and shifted to 330 nm. Binding of sn-glycerol 3-phosphate is reversible and no chemical alteration occurs with the substrate. The appearance of the binding protein in the periplasm is the result of a mutation that renders the cells constitutive for sn-glycerol 3-phosphate transport. Simultaneously, two other proteins appear in the periplasm. These proteins were also purified. They do not bind sn-glycerol 3-phosphate and do not cross-react with antibodies against the pure binding protein.


Role of secretory component, a secreted glycoprotein, in the specific uptake of IgA dimer by epithelial cells. The binding of secretory component (SC) to epithelial cells and its role in the specific uptake of immunoglobulin A (IgA) dimer has been studied in rabbit mammary gland and liver. SC, Mr approximately 80,000, secreted by epithelial cells of the mammary gland was found associated with the cell surface of mammary cells in intact tissue. Dispersed mammary cells and plasma membrane-enriched fractions obtained from mammary glands of midpregnant rabbits bound 125I-labeled SC in a saturable time- and temperature-dependent process. The association rate followed a second order reversible reaction (k+1 approximately equal to 2.7 x 10(6) M-1 min-1 at 4 degrees C) and equilibrium was reached in about 4 h at 4 degrees C. The dissociation rate for membranes was first order (k-1 approximately equal to 1.7 x 10(-2) min-1 at 4 degrees C), whereas displacement from cells was incomplete. The apparent affinity constant was similar for membranes and cells (Ka approximately equal to 5 x 10(8) M-1) with one class of binding sites. The number of binding sites varied from one animal to another (260 to 7,000 sites/mammary cell) in relation to endogenous occupancy by SC, which was assessed by immunocytochemistry and complement-mediated cytotoxicity. Rabbit liver and heart membranes did not bind SC, and serum proteins present in rabbit milk failed to interact with mammary cells or membranes. Mammary membranes or cells and liver membranes bound 125I-labeled IgA dimer in a saturable, reversible time- and temperature-dependent process. Association and dissociation rate constants at 4 degrees C (k+1 approximately equal to 5 x 10(6) M-1 min-1 and k-1 approximately equal to 5 x 10(-3) min-1, respectively) and the apparent affinity constant (Ka approximately equal to 10(9) M-1) were similar for liver and mammary membranes; these parameters differed, however, from those reported for free SC-IgA dimer interaction. The binding capacity of membranes for IgA dimer was directly related to the amount of free SC bound to membranes. Interaction of IgA dimer with mammary or liver membranes or cells was abrogated by excess of free SC and was prevented by preincubation of membranes or cells with Fab antibody fragments directed against SC. These data indicate that the first step in the translocation process of polymeric immunoglobulins across epithelia consists of binding of SC to the surface of epithelial cells which in turn acts as a receptor for the specific uptake of IgA dimer.


RNase E, an RNA processing enzyme from Escherichia coli. An activity, RNase E, was purified about 100-fold from Escherichia coli cells, it can process p5 rRNA from a 9 S RNA molecule which accumulates in a mutant of E. coli defective in the maturation of 5 S rRNA. The enzyme requires Na+, K+, or NH4+, and Mg2+ or Mn2+. The molecular weight of the enzyme is about 70,000 and its pH optimum is 7.6 to 8.0. Its temperature optimum is around 30 degrees C, and it can be irreversibly inactivated at 50 degrees C. It has a very high degree of specificity but the reaction can be inhibited by nonspecific RNAs. We interpret its mode of action in producing p5 RNA as being accomplished in two steps, 9 S RNA is first processed to 7 S and 4 S, and subsequently 7 S is further processed to p5.


Blocking of the initiation of protein biosynthesis by a pentanucleotide complementary to the 3' end of Escherichia coli 16 S rRNA. Hydrogen bonding between the 3' terminus of 16 S rRNA (... C-A-C-C-U-C-C-U-U-A-OH3) and complementary sequences within the initiator region of mRNA may be a crucial event in the specific initiation of protein biosynthesis (Shine, J., and Dalgarno, L. (1974) Proc. Natl. Acad. Sci. U. S. A. 71, 1342-1346; Steitz, J. A., and Jakes, K. (1975) Proc. Natl. Acad. Sci. U. S. A. 72, 4734-4738). Using equilibrium dialysis, we have studied the binding of G-A-dG-dG-U (which is complementary to the 3' end of 16 S rRNA and which has been synthesized enzymatically) to initiation factor-free Escherichia coli ribosomes. We have also investigated the effects of the pentanucleotide on initiation reactions in E. coli ribosomes. G-A-dG-dG-U has a specific binding site on the 30 S ribosome with an association constant of 2 x 10(6) M-1 at 0 degrees C. G-A-dG-dG-U inhibits the R17 mRNA-dependent binding of fMet-tRNA by about 70%, both with 70 S ribosomes and 30 S subunits. In contrast, the A-U-G-dependent initiation reaction and the poly(U)-dependent Phe-tRNA binding was not affected by the pentanucleotide with both ribosomal species.


Spectroscopic analysis of the interaction of Escherichia coli DNA-dependent RNA polymerase with T7 DNA and synthetic polynucleotides. We have studied the circular dichroism and ultraviolet difference spectra of T7 bacteriophage DNA and various  synthetic polynucleotides upon addition of Escherichia coli RNA polymerase. When RNA polymerase binds nonspecifically to T7 DNA, the CD spectrum shows a decrease in the maximum at 272 but no detectable changes in other regions of the spectrum. This CD change can be compared with those associated with known conformational changes in DNA. Nonspecific binding to RNA polymerase leads to an increase in the winding angle, theta, in T7 DNA. The CD and UV difference spectra for poly[d(A-T)] at 4 degrees C show similar effects. At 25 degrees C, binding of RNA polymerase to poly[d(A-T)] leads to hyperchromicity at 263 nm and to significant changes in CD. These effects are consistent with an opening of the double helix, i.e. melting of a short region of the DNA. The hyperchromicity observed at 263 nm for poly[d(A-T)] is used to determine the number of base pairs disrupted in the binding of RNA polymerase holoenzyme. The melting effect involves about 10 base pairs/RNA polymerase molecule. Changes in the CD of poly(dT) and poly(dA) on binding to RNA polymerase suggest an unstacking of the bases with a change in the backbone conformation. This is further confirmed by the UV difference spectra. We also show direct evidence for differences in the template binding site between holo- and core enzyme, presumably induced by the sigma subunit. By titration of the enzyme with poly(dT) the physical site size of RNA polymerase on single-stranded DNA is approximately equal to 30 bases for both holo- and core enzyme. Titration of poly[d(A-T)] with polymerase places the figure at approximately equal to 28 base pairs for double-stranded DNA.


Potentiometric analysis of Escherichia coli cytochromes in the optical absorbance range of 500 nm to 700 nm. The oxidation-reduction potentials of Escherichia coli cytochromes have been studied by a recently described technique for automated electrodic potentiometry (Hendler, R.W., Songco, D., and Clem, T.R. (1977) Anal. Chem. 49, 1908-1913; Hendler, R.W. (1977) Anal. Chem. 49, 1914-1918), where entire spectra are recorded at a series of solution potentials. New techniques for resolution of the spectra versus voltage data have been applied. The results indicate that a 1-electron transport chain conducts electrons from substrate to cytochrome d, which is the cytochrome oxidase. Cytochrome d contains several components which appear to increase electron transfer first to a 2-electron stage and then to a 4-electron stage for the final reduction of a molecule of oxygen to 2 molecules of water.


Substrate binding closes the cleft between the domains of yeast phosphoglycerate kinase. Using small angle x-ray scattering from solutions of yeast phosphoglycerate kinase, we have measured the radius of gyration of the enzyme both in the presence and in the abscence of ligands. We find that the radius of gyration decreases by 1.09 +/- 0.34 A upon binding both substrates MgATP and 3-phosphoglycerate to form the ternary complex. Smaller decreases, at the limit of the precision of the measurement, were found for the separate binding of MgATP (0.30 +/- 0.50 A). Using computer modeling, it has been estimated that a substrate-induced cleft closure in phosphoglycerate kinase resulting from one lobe rotating 8-14 degrees relative to the other lobe lobe is consistent with this observed change in radius of gyration. We suggest, therefore, that the conformational change that results in the smaller radius of gyration for the ternary complex is a hinge motion of the two lobes which produces a closing of the cleft between the two lobes. The apparent similarity of the ligand-induced change in phosphoglycerate kinase to the cleft closure in hexokinase suggests that this kind of conformational change may prove to be a rather general kinase phenomenon (Bennett, W.S., and Steitz T.A. (1978) Proc. Natl. Acad. Sci. U.S.A. 75, 4848-4852; Anderson, C.M., Zucker, F.H., and Steitz, T.A. (1979) Science 204, 375-380).


Mechanisms of action of histidinol dehydrogenase and UDP-Glc dehydrogenase. Evidence that the half-reactions proceed on separate subunits. Histidinol dehydrogenase and UDP-Glc dehydrogenase catalyze 4-electron dehydrogenations that convert primary alcohol groups to the corresponding acids. Both reactions proceed in two distinct steps involving the oxidation of the primary alcohol to a bound form of the intermediate aldehyde, followed by oxidation of this to the corresponding acid. The enzymes have subunit structure, the former is made up of two subunits and the latter of six (beef liver enzyme). Evidence is presented that the two half-reactions proceed independently of the overall reaction. Histidinol dehydrogenase preparations that approach total dissociation into subunits also approach total inhibition of the overall reaction, while the second half reaction is completely unaffected and 50% of the first half-reaction survives. Further, the fraction of overall activity surviving in partially dissociated preparations follows the weight fraction of residual dimer. UDP-Glc dehydrogenase behaves in an analogous fashion. These data are interpreted on the basis that both enzymes function by carrying out first oxidation step at a site on one subunit and then pass the intermediate to a vicinal site on the adjacent subunit, where the reaction is completed.


Regulation of 22Na+ transport by calcium in an established kidney epithelial cell line. The role of calcium in regulating the Na+ channel in an established kidney epithelial cell line has been examined. Extracellular calcium was inhibitory to Na+ uptake, and a Dixon plot of the initial Na+ uptake rate in the presence of Ca2+ was nonlinear, suggesting a mixed pattern of inhibition. Similar patterns of inhibition were also observed for other divalent cations, including Ba2+, Mg2+, and Mn2+. In contrast elevated concentrations of intracellular calcium resulted in a stimulation of Na+ entry. This intracellular effect was specific to calcium, with Mg2+ and Mn2+ appearing much less effective. Lineweaver-Burk plots of Na+ influx in calcium-loaded and unloaded cells were linear, suggesting that under both conditions a single system transported Na+. Although Na+ entry was stimulated by intracellular Ca2+, the cells did not exhibit other counter transport phenomena reported with cell types in which a Na+/Ca2+ exchange system is operative. Thus, the results indicate that calcium acts as an allosteric regulator of Na+ transport by the Na+ channel.


Specificity of phospholipases in methylcholanthrene-transformed mouse fibroblasts activated by bradykinin, thrombin, serum, and ionophore A23187. Methylcholanthrene-transformed mouse fibroblasts synthesize prostaglandins in response to bradykinin, thrombin, serum, and the ionophore A23187. These agents activate phospholipases, thereby releasing fatty acids from phospholipids. To examine the phospholipid specificity of the phospholipases activated by bradykinin, thrombin, serum, and A23187, cells were labeled with [14C]arachidonic acid and stimulated with these agents in the presence of delipidated bovine serum albumin. Phospholipid classes were resolved by two-dimensional chromatography on silica gel-coated paper. Only phosphatidylinositol and phosphatidylcholine lost radioactivity upon stimulation. To characterize the fatty acid specificity of the phospholipases, cells were incubated with 14C-labeled stearic, oleic, linoleic, eicosatrienoic, or arachidonic acid and then exposed to the stimuli. Bradykinin, thrombin, and serum caused specific release of radioactivity into the medium only from cells labeled with arachidonic acid or eicosatrienoic acid, whereas A23187 caused release from cells labeled with any one of the five fatty acids. We conclude that bradykinin, thrombin, and serum activate phospholipases that specifically hydrolyze arachidonyl and eicosatrienoyl phosphatidylinositol and phosphatidylcholine, whereas A23187 is less specific activator of phospholipases.


Deoxyribonucleic acid polymerase of bacteriophage T7. Purification and properties of the phage-encoded subunit, the gene 5 protein. DNA polymerase of bacteriophage T7 is composed of two subunits, the gene 5 protein of the phage and the host-specified thioredoxin. The gene 5 protein has been purified 7400-fold to homogeneity from bacteriophage T7-infected Escherichia coli 7400 trxA cells that lack thioredoxin. The purification procedure has been monitored by using a complementation assay in which thioredoxin interacts with the gene 5 protein to form an active DNA polymerase. The purified gene 5 protein is a single polypeptide having a molecular weight of 87,000. The gene 5 protein itself has only 1 to 2% of the polymerase activity of T7 DNA polymerase. However, T7 DNA polymerase can be reconstituted by the addition of homogeneous thioredoxin to the gene 5 protein. Optimal reconstitution is obtained when the molar ratio of thioredoxin/gene 5 protein is 150. Under these conditions, the gene 5 protein attains approximately 80% of the activity of an equal amount of T7 DNA polymerase. The apparent Km for thioredoxin in the reaction to restore DNA polymerase activity is 2.8 x 10(-8) M. The enzymatic properties of the reconstituted enzyme are indistinguishable from those of T7 DNA polymerase synthesized in vivo; the reconstituted polymerase interacts with T7 gene 4 protein to catalyze DNA synthesis on duplex DNA templates.


T7-induced DNA polymerase. Characterization of associated exonuclease activities and resolution into biologically active subunits. Bacteriophage T7-induced DNA polymerase has been isolated by a procedure suitable for large scale use and which yields near homogeneous enzyme. In addition to previously described DNA polymerase activity and 3' to 5' exonucleolytic activity on single stranded DNA (Grippo, P., and Richardson, C. C. (1971) J. Biol. Chem. 246, 6867-6873), the enzyme also possesses a highly active exonuclease which hydrolyzes duplex substrates with 3' to 5' directionality. The native polymerase has been dissociated using 6 M guanidine HCl and resolved into biologically active subunits: T7 gene 5 protein and Escherichia coli thioredoxin. The phage-specified subunit obtained by this procedure is deficient in DNA polymerase and double strand exonuclease activities, with deficiencies in these activities being apparent at the level of a single turnover. However, it possesses near normal levels of a single strand hydrolytic activity which is identical to that associated with the native polymerase with respect to substrate specificity and suppression of hydrolysis by low levels of deoxyribonucleoside 5'-triphosphates. Thioredoxin forms a molecular complex with the T7 gene 5 protein, and addition of the host protein restores restores DNA polymerase and double strand exonuclease activities to near normal levels.


Sequence analysis of two yeast mitochondrial DNA fragments containing the genes for tRNA Ser UCR and tRNA Phe UUY. Two restriction enzyme fragments containing yeast mitochondrial tRNA genes have been characterized by DNA sequence analysis. One of these fragments is 320 base pairs long and contains a tRNA Ser gene. The corresponding tRNA SER was isolated from yeast mitochondria and its nucleotide sequence also was determined. This mitochondrial tRNA is 90 nucleotides in length, has a G + C content of 38%, and has UGA as the anticodon. A portion of a 680-base-pair DNA fragment containing a tRNA Phe gene was also sequenced. The portion of this gene which codes for the mature tRNA is 75 base pairs in length, has a G + C content of 33%, and contains the anticodon GAA. Neither gene contains an intervening sequence or codes for the 3' CCA terminus. Both are surrounded by regions of more than 90% A + T. The significance of these sequences is discussed.


Primary structure and properties of an inactive mutant aspartate transcarbamoylase. A mutant form of Escherichia coli aspartate transcarbamoylase (ATCase) which lacks catalytic activity has been purified and characterized (Wall, K.A., Flatgaard, J.E., Gibbons, I., and Schachman, H.K. (1979) J. Biol. Chem 254, 11910-11916). Peptide mapping of the mutant and wild type catalytic chains followed by the determination of the amino acid sequence of the one altered peptide in the mutant indicated that a glycyl residue was replaced by aspartic acid. This substitution is located at position 125 in the tentative sequence kindly provided by W. Konigsberg (personal communication). The mutant protein has an overall secondary structure similar to that of the wild type as indicated by circular dichroism spectroscopy. However, marked changes in the reactivity of several amino acid residues were demonstrated. Lysyl residue 84 which in the wild type subunits reacts specifically with pyridoxal 5'-phosphate is only slightly reactive in the mutant even though the peptide containing that residue was not altered in amino acid composition. Another residue, cysteinyl 46, which is thought to be in the active site, is much more reactive toward p-hydroxymercuribenzoate in the mutant subunit than in the wild type protein. Finally, tyrosyl residue 213, which according to recent crystallographic studies is not near the active site and which exhibits an unusually low pK (9.1) in the wild type catalytic subunits, appears to have its pK shifted to 10.5 or higher as a result of the mutation. The evidence indicates that the substitution of an aspartyl for a glycyl residue at a region of the amino acid sequence remote from those residues in the active site causes sufficient modification of the tertiary structure to cause the loss of enzyme activity and to affect the reactivity of other residues in the protein. Moreover, the quaternary structure of the intact enzyme is altered as well since the subunit interactions are greatly weakened.


Synthesis and properties of fluorescent nucleotide substrates for DNA-dependent RNA polymerases. A new class of fluorescent nucleotide analogs which contain the fluorophore 1-aminonaphthalene-5-sulfonate attached via a gamma-phosphoamidate bond has been synthesized. Both the purine and pyrimidine analogs have fluorescence emission maxima at 460 nm. Cleavage of the alpha-beta-phosphoryl bond produces change in both the absorption and fluorescence emission spectra. The fluorescence of the pyrimidine analogs is quenched; cleavage of the alpha-beta-phosphoryl bond of the UTP analog produces about a 14-fold increase in fluorescence intensity at 500 nm. Under the same conditions the fluorescence of the CTP analog increases about 8-fold, whereas the fluorescence of the purine analogs shows only a slight change. These derivatives are good substrates for Escherichia coli RNA polymerase with only slightly increased Km values and with Vmax values about 50 to 70% that of the normal nucleotides. They are used less efficiently by wheat germ RNA polymerase II. The ATP analog can be used by E. coli RNA polymerase to initiate RNA chains.


Primary structure of yeast proteinase B inhibitor 2. The complete amino acid sequence of yeast proteinase B inhibitor 2 (IB2) was determined to be H3N+-Thr-Lys-Asn-Phe-Ile-Val-Thr-Leu-Lys-Lys-Asn-Thr-Pro-Asp-Val-Glu-Ala-Lys-Lys-Phe-Leu-Asp-Ser-Val-His-His-Ala-Gly-Gly-Ser-Ile-Leu-His-Glu-Phe-Asp-Ile-Ile-Lys-Gly-Tyr-Thr-Ile-Lys-Val-Pro-Asp-Val-Leu-His-Leu-Asn-Lys-Leu-Lys-Glu-Lys-His-Asn-Asp-Val-Ile-Glu-Asn-Val-Glu-Asp-Lys-Glu-Val-His-Thr-Asn-COO-. Elucidation of the primary structure was enabled by automated Edman degradation and COOH-terminal hydrolysis with carboxypeptidases A (bovine pancreas and Y (yeast). IB2 is the first proteinase inhibitor to be sequenced that possesses a structure devoid of disulfide bridges.


Isolation of yeast tRNALeu genes. DNA sequence of a cloned tRNALeu3 gene. A library of cloned yeast DNA fragments generated by digestion of yeast DNA with the restriction endonuclease Bam HI has been screened by colony hybridization to total yeast [32P]tRNA. Four hundred colonies carrying yeast tRNA genes were isolated. By hybridization to 125I-tRNALeu3, we have isolated from this collection 14 colonies carrying fragments containing yeast tRNALeu genes. The size of the yeast Bam HI inserts ranged from 2.45 x 10(6) to 14 x 10(6) daltons. One of these fragments was mapped in detail by restriction endonuclease digestion and hybridization to 125I-tRNALeu3. The presence of a tRNALeu3 gene was confirmed by DNA sequence. The results indicate that the tRNALeu3 coding region is not co-linear with the tRNALeu3. An intervening tract of 33 base pairs interrupts the coding sequences 1 base pair past the anticodon coding region. The putative structure of a tRNALeu3 precursor is deduced in which the anticodon base pairs with residues from the intervening sequence.


Transcription, processing, and mapping of mitochondrial RNA from grande and petite yeast. Mitochondrial RNA (mtRNA) from petite yeast strains was analyzed by electrophoresis in agarose-urea, acrylamide-urea, and agarose-methyl mercuric hydroxide gels, and by transfer to diazobenzyloxy-methyl paper and hybridization to labeled mitochondrial DNA (mtDNA). Petites contain numerous mitochondrial transcripts, including processed species like 21 S and 14 S rRNA. Petite transcripts were found to fall into three classes: 1) bands that comigrate with grande mtRNA species; 2) "group-specific" new bands found in multiple strains and coinciding with specific regions of the mitochondrial genome; and 3) "strain-specific" new bands found only in individual petite strains. A deletion map was constructed in which we used the presence or absence of the first two types of mtRNA bands in specific strains, and the restriction endonuclease map of these strains. This map confirmed the localization of 21 S and 14 S rRNA, which were mapped previously by hybridization, and also localized more than 20 additional mtRNA species. The mtRNA species were grouped in regions of the genome in a fashion that strongly suggests that many of them are precursors to fully processed mtRNA species. Hybridization experiments with grande mtRNA and cloned mtDNA fragments have shown the same kind of transcript grouping. Other hybridization experiments have demonstrated two apparent precursors to 21 S rRNA (3700 nucleotides) measuring 5500 and 4500 nucleotides. Processed tRNAs are found only in petites that contain a specific region of the genome near the P (paromomycin resistance) locus. When this region is absent, processed tRNAs are not detected, even for tRNA genes quite distant from the P locus. Since this phenotype is expressed in petites that lack mitochondrial protein synthesis, and since it maps to a specific location in the mitochondrial genome, there appears to be a mtRNA species which has a role in processing of mitochondrial tRNA.


Ca2+-dependent protein phosphorylation of purely cholinergic Torpedo synaptosomes. Preincubation of intact, purely cholinergic Torpedo synaptosomes with [32P]Pi results in the incorporation of 32P into about 10 specific proteins. Depolarizing the Torpedo synaptosomes by a high K+ buffer or treatment with the Ca2+ ionophore A23187 result in Ca2+ uptake, in acetylcholine (ACh) release, and in a marked increase of 32P incorporation into a specific protein band with an apparent subunit molecular weight of 100,000 (band alpha). The kinetics of synaptosomal 45Ca2+ uptake, of 32P incorporation into band alpha, and of ACh release is similar and reach maximal values about 45 s after the synaptosomes have been treated. Sr2+ and Ba2+ can replace Ca2+ in evoking both K+ depolarization-dependent ACh release and 32P incorporation into band alpha. The effectiveness of these ions (Ca2+ greater than Sr2+ greater than Ba2+) is similar in both cases. The data presented suggest that Ca2+ accumulation by Torpedo synaptosomes leads to an increase in the phosphorylation of a specific protein and to ACh release. This phosphoprotein may be involved in the regulation of presynaptic processes which underly ACh release.


dnaG (primase)-dependent origins of DNA replication. Nucleotide sequences of the negative strand initiation sites of bacteriophages St-1, phi K, and alpha 3. The simplest known origins of DNA replication occur in the single-stranded bacteriophages. In one set of phages, negative strand synthesis is initiated by a single protein, the product of the Escherichia coli replication gene dnaG. Evidently, in these phages--G4, St-1, phi K, and alpha 3--the origin for negative strand synthesis consists of a nucleic acid element capable of direct recognition by the dnaG priming protein. We have located and sequenced the origins of negative strand synthesis in St-1, phi K, and alpha 3, and compared them with the origin sequence previously determined for G4. In each case, the point at which the negative strand is initiated can be identified at the nucleotide level. The data lead to the following conclusions: 1. In all four phages, the negative strand initiation site occurs within an intercistronic region of approximately 135 bases. While in G4, the origin lies between genes specifying the viral coat proteins F and G, the origin is shifted in St-1, phi K, and alpha 3 to a position between coat protein genes G and H. 2. Extensive nucleotide conservation exists at the negative strand origin, but does not extend into the adjacent coding regions. The conserved origin DNA occurs in two regions, 42 and 45 bases long, which are separated by 13 bases of divergent sequence. 3. Correlated with the two stretches of conserved nucleotide sequence are two regions of potential secondary structure. The start point of negative strand synthesis lies just prior to one of these hairpins. Similarities in both primary sequence and secondary structure can be found between the negative strand origins of G4, St-1, phi K, and alpha 3 and the general origin regions of bacteriophage lambda and of E. coli.


Bacteriophage fd gene II-protein. II. Specific cleavage and relaxation of supercoiled RF from filamentous phages. Bacteriophage fd gene II-protein was characterized as an endonuclease which specifically nicked supercoiled replicative form (RF) of filamentous phages in the viral strand. No other supercoiled DNAs tested were attacked by the enzyme, nor were doubly closed fd RF in the relaxed state nor phage fd single strands. Maximal activity was found at pH 8.5 and 80 mM KCl using fd RFI of physiological superhelicity. Mg2+, but no other cofactor, was required for the cleavage reaction. A sealing activity was found to be associated with the enzyme. At a higher concentration of Mg2+ up to 40% of the reaction products were found as doubly closed relaxed fd RF. The protein was not found to be tightly attached to the cleaved strand.


The treatment of congenital vertical talus. Operative correction of congenital vertical talus in nine feet in six patients is described. The operation consists of the comprehensive lengthening of the tendons in the foot and full peritalar release without excision of the navicular; it is performed through a dorsal transverse incision.


Wear of high-density polyethylene on bone and cartilage. Wear of high-density polyethylene on bone and cartilage has resulted in a large volume of plastic particles being shed into the two knees and two hips studied. The giant-cell foreign-body reaction of the synovium may not be sufficient to cope with the amount of debris presented and the destruction of the endosteal bone in one hip, caused by the wear particles and movement of the prosthesis, has made revision impossible. Articulation of high-density polyethylene against bone or cartilage either by design or by the failure of alignment of the component must be avoided.


Hodgkin's disease in childhood.

Hodgkin's disease in childhood. The histopathologic study of 103 patients with Hodgkin's disease including 5 cases who had staging laparotomy during the last 10 years were reviewed. The following conclusions were drawn: 1. No significant sex difference among subtypes were noted. 2. Associated glomerulopathy, nephrotic syndromes, and amyloidosis were occasionally found. 3. In childhood lymphomas excluding leukemia are the most frequent malignacies. Among the lymphomas, Hodgkin's disease predominates. 4. The most frequent subtype both in the original biopsy and after staging laparotomy was mixed cellularity. The nodular sclerosis type was the rarest. The subtypes generally did not change in the subsequent biopsies and in laparatomy done up to 6 months later. 5. It is most frequently noted in cervical area. Males are affected more than females. The peak incidence was in the first decade of age group. A brief review of the literature and discussion on the comparison of data from other countries is given.


Involvement of histone H1 in the organization of the nucleosome and of the salt-dependent superstructures of chromatin. We describe the results of a systematic study, using electron microscopy, of the effects of ionic strength on the morphology of chromatin and of H1-depleted chromatin. With increasing ionic strength, chromatin folds up progressively from a filament of nucleosomes at approximately 1 mM monovalent salt through some intermediate higher-order helical structures (Thoma, F., and T. Koller, 1977, Cell 12:101-107) with a fairly constant pitch but increasing numbers of nucleosomes per turn, until finally at 60 mM (or else in approximately 0.3 mM Mg++) a thick fiber of 250 A diameter is formed, corresponding to a structurally well-organized but not perfectly regular superhelix or solenoid of pitch approximately 110 A as described by Finch and Klug (1976, Proc. Natl. Acad. Sci. U.S.A. 73:1897-1901). The numbers of nucleosomes per turn of the helical structures agree well with those which can be calculated from the light-scattering data of Campbell et al. (1978, Nucleic Acids Res. 5:1571-1580). H1-depleted chromatin also condenses with increasing ionic strength but not so densely as chromatin and not into a definite structure with a well-defined fiber direction. At very low ionic strengths, nucleosomes are present in chromatin but not in H1-depleted chromatin which has the form of an unravelled filament. At somewhat higher ionic strengths (greater than 5 mM triethanolamine chloride), nucleosomes are visible in both types of specimen but the fine details are different. In chromatin containing H1, the DNA enters and leaves the nucleosome on the same side but in chromatin depleted of H1 the entrance and exit points are much more random and more or less on opposite sides of the nucleosome. We conclude that H1 stabilizes the nucleosome and is located in the region of the exit and entry points of the DNA. This result is correlated with biochemical and x-ray crystallographic results on the internal structure of the nucleosome core to give a picture of a nucleosome in which H1 is bound to the unique region on a complete two-turn, 166 base pair particle (Fig. 15). In the formation of higher-order structures, these regions on neighboring nucleosomes come closer together so that an H1 polymer may be formed in the center of the superhelical structures.


Rapid isolation of Legionella pneumophila from seeded donor blood. The laboratory isolation of Legionella pneumophila from seeded donor blood, using the lysis-centrifugation technique, is described. Time to pure culture isolate was 3 to 4 days.


Detection of Candida antigen in sera of patients with candidiasis by an enzyme-linked immunosorbent assay-inhibition technique. A total of 37 serum samples from 27 cancer patients were tested by an enzyme-linked immunosorbent assay-inhibition technique for the detection of Candida antigen. In 20 randomly chosen sera from patients without clinical evidence of candidiasis and in 10 sera from patients proven by autopsy not to have candidiasis, the inhibition ranged up to 17%; in contrast, inhibition ranged from 22 to 56% in all seven patients proven by autopsy to have systemic candidiasis, indicating the presence of Candida antigen in the sera of these patients. This technique appears promising in diagnosing disseminated candidiasis in cancer patients.


API and Minitek systems in identification of clinical isolates of anaerobic gram-negative bacilli and Clostridium species. A comparison of the API and Minitek methods of biochemical testing was made on a variety of anaerobic bacteria. Although API and Minitek results were not compared to more standardized or conventional procedures of identification, multiple repeat testing of the two systems was done on routine clinical isolates and known organisms to determine (i) whether the reactions were reliably consistent, (ii) the ease of reading the two systems with respect to the frequency of questionable results, and (iii) the percentage of routine clinical isolates for which each system yielded an identification. The Minitek system gave a much lower incidence of difficult to interpret reactions. The two systems were comparable in terms of reproducibility and capability of yielding an identification of the anaerobic gram-negative bacilli and Clostridium species, but were unsatisfactory for routine use on most of the other anaerobic bacteria isolated.


Serotyping of Serratia marcescens: current status of seven recently described flagellar (H) antigens. The slightly revised, current scheme of 20 flagellar (H) antigens of Serratia marcesens was examined. The seven new H antigens were demonstrated to be antigenically distinct as determined with Le Minor's H-immobilization test. The H-immobilization antibodies of rabbit anti-H immune sera proved resistant to treatment with 2-mercaptoethanol and dithiothreitol, respectively. On the other hand, dual absorptions of rabbit anti-H immune sera with killed cells of Staphylococcus aureus strain Cowan I, i.e., protein A, failed to reduce significantly H-immobilization titers of rabbit sera, although human immunoglobulins G and M were bound by protein A. It was tentatively concluded that the 2-mercaptoethanol- and dithiothreitol-refractory H-immobilizing rabbit antibodies belonged to the immunoglublin M class. H-antigen (phase) variation was not demonstrable in several extramural, clinical isolates of S. marcescens for which this phenomenon had been claimed. Rather, four of these six isolates were found to consist of cell populations of two distinct serotypes, as also borne out by bacteriocin typing; the flagellar H-antigens of the remaining two isolates were stable, with minor, hterologous H-antigen cross-reactivity.


Primary plate identification of group A beta-hemolytic streptococci utilizing a two-disk technique. A two-disk system is described which allows primary plate identification of group A beta-hemolytic streptococci. Group A beta-hemolytic streptococci could be visualized on primary throat culture plates by using trimethoprim-sulfamethoxazole to inhibit normal flora. In the heavily inoculated area of Trypticase soy agar plates containing 5% sheep blood, a 25-microgram/ml trimethoprim-sulfamethoxazole disk was placed contiguous to a 0.04-U bacitracin disk. A total of 259 throat specimens were examined with this two-disk system. The swabs from these throat specimens were incubated in Todd-Hewitt broth. The bacterial pellet from the broths was stained by fluorescent antibody as a control. Of the cultures that were determined to be positive on the plates, 75% could be read unequivocally after overnight incubation, whereas the remaining 25% required subculture. The plates recovered 91% of the cultures which were considered as true positives by the broth-fluorescent-antibody technique. This method provided a significant savings in time compared with standard plate methods and in cost of materials compared with broth-fluorescent-antibody methods. This technique is particularly valuable for producing rapid results in laboratories where fluorescence microscopy would not be cost-effective.


Characteristics of noncultivable adenoviruses associated with diarrhea in infants: a new subgroup of human adenoviruses. Virus particles morphologically resembling adenovirus were found in fecal specimens from infants and were examined for cultivability with standard cell culture techniques and for characteristics of human adenoviruses. Specimens from 13 of 15 infants could not be cultivated in cell cultures. The two adenoviruses that were cultivated, types 1 and 31, reacted in the expected manner in all tests. Counterimmunoelectrophoresis with group-specific anti-hexon serum confirmed that the observed particles in the 15 specimens were human adenoviruses. The buoyant density in sucrose of five of the noncultivable adenoviruses in original stool suspensions averaged 1.335 g/cm(3) and that of the two cultivable ones averaged 1.332 g/cm(3); both groups had typical adenovirus morphology by electron microscopy. Treatment of the specimens and of a variety of tissue culture cells with proteolytic and other enzymes did not improve cultivability. Examination of partially purified virus by immunoelectron microscopy did not reveal evidence of immunoglobulin A, G, or M coating on the particles, an indication that coproantibody inhibition was not the cause of noncultivability. Fluorescent-antibody studies with an antihexon conjugate and counterimmunoelectrophoresis studies of serially passaged noncultivable viruses indicated that the viruses are infecting cells but are not undergoing effective replication. Antisera to three of the noncultivable viruses demonstrated homologous reactions in counterimmunoelectrophoresis with the respective immunizing antigens but showed only low levels of hemagglutination-inhibiting and neutralizing activity to a few of the known human adenoviruses. We concluded that the noncultivable viruses in these infant diarrhea cases were indeed human adenoviruses, were not defective particles, were not bound to coproantibody, were infectious but incapable of effective relication in conventional cell cultures, were serologically related to types 11, 17, 32, and 33, and should be considered a new, distinct subgroup.


Antigen requirements, sensitivity, and specificity of enzyme immunoassays for measles and rubella viral antibodies. Enzyme immunoassay (EIA) systems for measles virus and rubella virus were studied from the standpoints of requirements for suitable viral antigens and control antigens, and the sensitivity and specificity of the tests for detecting antibody elicited by past infection (determination of immunity status), and for serodiagnosis of currenet infections. Crude or semipurified measles virus antigens were satisfactory for EIA, but antigens derived by pelleting virus from infected cell culture fluids were slightly more specific in their reactivity than were antigens produced from lysates of infected cells. However, reliable rubella EIA antigens could be produced only from infected cell culture fluids, and they required density gradient purification to render them suitably specific. Even with gradient-purified rubella antigens, it was necessary to use antigen prepared in an identical fashion from uninfected cell culture fluids as a control on the specificity of reactions obtained with test sera. With appropriate viral antigens and control antigens, both measles and rubella EIA systems were highly sensitive and specific for determination of immunity status and for serodiagnosis of current infections. Antibody was detectable earlier in the course of infection by EIA than by hemagglutination inhibition or complement fixation, but this did not limit the diagnostic value of the test, since titer increases demonstrable by EIA were usually greater than those detectable by hemagglutination inhibition or complement fixation tests.


Presumptive identification of group A, B, and D streptococci on agar plate media. Several presumptive tests were evaluated for their effectiveness in differentiating streptococci. When the tests were combined into a battery and the resulting reactions were interpreted as patterns, the overall presumptive identification rate was at least 97%. We used the hemolytic reaction, susceptibility to bacitracin and sulfamethoxazole plus trimethoprim (1.25 micrograms plus 23.75 micrograms), and standard CAMP reactions on sheep blood Trypticase soy agar, and bile-esculin and 6.5% NaCl agar tolerance tests with incubation in candle extinction jars. Subsequently, 98.9% of the group A; 95.3% of the group B; 100% of the beta-hemolytic non-group A, B, or D; 92.3% of group D enterococcal; 100% of the group D non-enterococcal; and 92.8% of the viridans streptococci were presumptively identified. We then used the hemolytic reactions, susceptibility of bacitracin and sulfamethoxazole-plus-trimethoprim disks, CAMP disk reactions on sheep blood Trypticase soy agar and bile-esculin and 6.5% NaCl agar tolerance tests with incubation in normal atmosphere. Subsequently, 98.1% of the group A; 98.6% of the group B; 99.2% of the beta hemolytic non-group A, B, or D; 97.5% of the group D entercoccal; 97.6% of the group D non-entercoccal; and 92.4% of the viridans strains were presumptively identified.


Isolation and characterization of a polyene-resistant variant of Candida tropicalis. An atypical variant of Candida tropicalis was recovered from multiple specimens from a patient who had been a recipient of a bone marrow transplant. This yeast variant showed atypical morphology on corn meal agar distinguishable from typical isolates of C. tropicalis by the production of clusters of blastospores. Isolates of the variant produced acid, but no gas, from maltose and sucrose in fermentation tests. Isolates from blood, pleural fluid, respiratory secretions, and stool specimens were susceptible to amphotericin B and nystatin in an agar dilution system. However, eight isolates of the variant C. tropicalis recovered over a period of 4 weeks from the patient's urine after amphotericin B therapy were found to be resistant to amphotericin B and nystatin. The isolate recovered after 7 days of therapy had minimal inhibitory concentrations of 100 micrograms of amphotericin B and 20 micrograms of nystatin per ml, whereas the seven isolates recovered subsequently had minimal inhibitory concentrations of greater than 500 micrograms of amphotericin B and 50 micrograms of nystatin per ml. The resistant isolates concomitantly lost the capacity to utilize amino acids that susceptible isolates could utilize. Ultraviolet absorption spectra of nonsaponifiable fractions of whole cells showed that resistant isolates lacked ergosterol, which susceptible isolates contained.


Rapid diagnosis of influenza A infection by direct immunofluorescence of nasopharyngeal aspirates in adults. The efficacy for direct immunofluorescence of a commercial conjugate for influenza A virus prepared against whole A/Udorn (H3NS) virus was studied. The conjugate was specific for influenza A virus, but its sensitivity varied depending upon the strain of influenza A tested. Nasopharyngeal aspirates collected from 25 patients during an outbreak of influenza were examined for viral antigen with the conjugates and inoculated onto monkey kidney (MK) cells for virus isolation. Fifteen patients had isolates for influenza A/USSR/90/77 (H1N1); nasopharyngeal secretions were fluorescent antibody positive in 12. Fluorescent antibody was copositive with culture in 11/15 patients (73.3%) and conegative in 9/10 (90%). The one fluorescent antibody-positive, culture-negative patient had negative serology for influenza A and the fluorescent antibody result was considered to be a false positive. At a 1:10 dilution, the conjugate stained nasopharyngeal and MK cells infected with A/USSR (H1N1) 2 to 3+, whereas cells infected with H3N2 virus stained 4+. A conjugate made specifically against the ribonucleoprotein antigen, which is universal to all influenza A strains, may improve the sensitivity of the direct immunofluorescent test.


Isolation of Mycoplasma bovis from a patient with systemic illness. Mycoplasma bovis was cultured from the sputum of a patient with lobar pneumonia, psychosis, and probable myocarditis, nephritis, and hemolytic anemia. Although we cannot be certain that this species of mycoplasma was the etiological agent of the patient's acute illness, this case report is of interest because, to the best of our knowledge, it represents the first isolation of M. bovis from a human source.


Efficacy of cold enrichment techniques for recovery of Yersinia enterocolitica from human stools. Stool specimens from children with gastroenteritis and their household contacts were cultured for Yersinia enterocolitica by direct plating onto routine laboratory media. These stools were also inoculated into phosphate-buffered saline and subcultured to the same media after 1 day or 3 weeks of incubation at 4 degrees C. Y. enterocolitica was isolated from 174 index cases and 34 household contacts. One hundred eighty-one isolates were of serotype O:3, and the remaining 21 belonged to other serotypes. Eighty-one percent (147/181) of O:3 isolates were recovered by direct plating, and 6.1% (11/181) and 13% (23/181) were recovered by 1-day and 3-week cold enrichment, respectively. For other serotypes, 26% (7/27), 0%, and 74% (20/27) were isolated by direct plating, 1-day cold enrichment, and 3-week cold enrichment, respectively. The efficacy of the cold enrichment for the patients were still symptomatic, 94 and 6% of Y. enterocolitica were identified by direct plating and cold enrichment, respectively. Isolation rates were 66% by direct plating and 34% by cold enrichment when stools were obtained from asymptomatic carriers or from those convalescing from Y. enterocolitica gastroenteritis. These results indicate that the cold enrichment methods increase the sensitivity of Y. entercolitica culture methods considerably in convalescent and asymptomatic subjects but only minimally in patients with diarrhea caused by serotype O:3.


Metabolism of arachidonic acid in ionophore-stimulated neutrophils. Esterification of a hydroxylated metabolite into phospholipids. [14C]Arachidonic acid incubated with human neutrophils was esterified into phospholipids and triglycerides. Stimulation of these labeled neutrophils with ionophore A23187 (2 microM) results in release of [14C]arachidonate from phospholipid and its metabolism to prostaglandin E2 and 5-hydroxy-6,8,11,14-eicosatetraenoic acid (5-HETE), a lipoxygenase product. The released arachidonate is also metabolized to a polar lipid of unknown composition here disignated compound A. 5-HETE was found to be released into the medium and then taken up again by the cells. To determine its metabolic fate, [14C]5-HETE was prepared biosynthetically, purified, and incubated with stimulated, unlabeled neutrophils. Most of the radioactivity entered the cells and was esterified into phospholipids and triglycerides. The radiolabeled complex lipids were saponified, and the released fatty acids cochromatographed with authentic 5-HETE. The esterification of 5-HETE, a hydroxylated fatty acid, into membrane phospholipids may be an example of a more generalized mechanism for altering membrane characteristics.


Effects of neomycin alone and in combination with cholestyramine on serum cholesterol and fecal steroids in hypercholesterolemic subjects. Effects of neomycin were studied on serum cholesterol and fecal steroids in hypercholesterolemic patients during a short treatment period (4 wk) and a long treatment period (16 mo), using small (1.5 g/d) and large (up to 6 g/d) doses alone and in combination with cholestyramine. In the short-term low-dose study the decrease in serum cholesterol by 21% was associated with a proportionate increase in fecal cholesterol elimination as neutral sterols through impaired cholesterol absorption. Serum cholesterol remained low and fecal steroid excretion remained elevated in the long-term neomycin study. Increasing the dosage from 1.5 to 6 g/d at the end of the 16-mo period brought about a further slight decrease in serum cholesterol and a small further increase in fecal neutral and acidic steroids. The increases in fecal bile acids and fat but not in neutral sterols were positively correlated with the increases in the neomycin dosage. Thus, large neomycin doses can also cause bile acid malabsorption. In another series of patients, a decrease (25%) in serum cholesterol by cholestyramine was associated with a proportional increase in the fecal elimination of cholesterol (2.5-fold) as bile acids. The inclusion of neomycin in cholestyramine therapy further increased fecal steroid output (solely as neutral sterols) by only about one-fifth of that due to cholestyramine, but further decreased serum cholesterol almost to the same extent (-17%) as cholestyramine alone. The overall decrease was 38%, no side effects occurred, and the patients found combination therapy convenient. Neomycin decreased serum cholesterol in different studies by 10+/-2, 17+/-4, and 12+/-4% per 100 mg/d of the increment in fecal steroids, the respective decrease for cholestyramine being only 2.2+/-0.5%. Thus, neomycin effectively reduced serum cholesterol by a relatively small increase in cholesterol elimination (via cholesterol malabsorption) compared with cholestyramine-induced bile acid malabsorption.


A renal biopsy study in toxaemia of pregnancy. Renal biopsy specimens from 11 women with severe pre-eclamptic toxaemia were examined by light and electron microscopy linked with immunofluorescence and immuno-electron microscopy. The part played by the mesangium in causing capillary loop thickening is stressed, and the progress of this 'strangulation' is illustrated. In contrast to the findings of most previous authors, IgM was demonstrated by direct immunofluorescence in all biopsy specimens, and its presence and site within the glomerulus were shown by immuno-electron microscopy in three cases.


Biochemical identification of clinically important yeasts. Four commercially available kits for the speciation of yeasts were tested against 50 clinical isolates that had been identified by conventional methods. On biochemical grounds alone, the four systems varied from 71% to 100% in their efficiency in identifying Candida albicans. Yeasts other than C. albicans were identified with an efficiency varying from 24% to 83%. Conclusions are drawn on the value of these systems to the routine laboratory.


Threshold responses in formaldehyde-sensitive subjects. Patch tests with three low concentrations of formaldehyde were applied continuously in formaldehyde-sensitive subjects for 1 wk. The closed patch test method produced a response to 30 parts per million (ppm) in some subjects by 120 continuous hours of testing. Thirteen subjects then sprayed a 30-ppm formaldehyde solution in an axilla on a double-blind, controlled, 2-wk-use test. Their responses to 30 ppm aqueous formaldehyde indicate that levels below this concentration should be tolerated by sensitive subjects if repeatedly applied to normal skin. Popular formaldehyde-releasing preservatives can be above or below this threshold-eliciting response.


Cutaneous plasmacytomas. A review and presentation of an unusual case. Recent reviews separate four types of plasma cell tumor: multiple myeloma, extramedullary plasmacytoma (without multiple myeloma), solitary myeloma of bone, and plasma cell leukemia. Cutaneous plasma cell tumors may arise from lymphatic or vascular spread of tumor (metastatic cutaneous plasmacytoma) or by direct extension from bone lesions. The former, metastatic cutaneous plasmacytomas, are quite rare. Specific malignant plasmacyte cutaneous tumors can also be seen in extramedullary plasmacytoma, solitary myeloma of bone, and plasma cell leukemia. We present a patient with multiple myeloma and lymphedema of the right arm, who developed a pathologic fracture of the right humerus and subsequently developed numerous metastatic cutaneous plasmacytomas localized to the lymphedematous arm. Direct immunofluorescence of frozen sections and enzymatically released cells from tumor nodule failed to reveal cell-associated immunoglobulins.


Trace element deficiencies and fertility in ruminants: a review. Various minerals (copper, cobalt, selenium, manganese, iodine, zinc, and iron) can influence reproductive performance of ruminants. Reproductive failure may be induced by deficiencies of single or combined trace elements and by imbalances. This review is focused on maladjustments of trace elements leading to impaired breeding performance. Opinion is diverse as to the existence of various reproductive disturbances from either a severe copper depletion or a marginal dietary copper deficiency. Field experience suggests that administration of cobalt to ruminants on cobalt-deficient diets improves their impaired breeding performance. Selenium infertility in ewes is more prevalent in some areas and in some seasons, but the actual cause of this malady and the continuing role of additional factors are unknown. Manganese is necessary for normal fertility in ruminants, and feeding low-manganese rations depresses conception rates. Lack of iodine impairs thyroid activity and also ovarian function. Reproductive failure in the female and in spermatogenesis are manifestations of zinc deficiency. Despite forages rich in iron, low availability in certain instances could affect adversely ruminant reproduction. Knowledge of biochemical dysfunctions from trace element deficiencies is essential to determine the role which trace elements play in fertility of ruminant animals.


Microstructures in non-precious alloys near the procelain-metal interaction zone. The chemistry of microstructures near the interfacial reaction zones of four non-precious alloys and two different brands of dental porcelain has been analyzed. It was found that most non-precious dental alloys contain second phases which can alter elemental concentration profiles. Recommendations for assessing interfacial reaction zone chemistry in dental non-precious alloys are offered.


Antibodies to tooth allografts in H-2 matched mice. Teeth were transplanted among mice which represented the best match possible between unrelated humans. Sera were examined for their ability to kill donor cells with the help of complement, to trigger complement-independent, cell-mediated cytolysis, and to agglutinate RBC. All responses were weak, but rejection correlated best with antibody-dependent cytolysis.


Valproic acid: a new antiepileptic drug with potential side effects of dental concern. Although the exact mechanism by which valproic acid acts on hemostasis is unknown, an association between the agent and defective blood clotting and spontaneous hemorrhage is evident. This side effect should be considered in the management of dental patients taking the drug for whom treatment involves surgery of the soft tissues.


Abutment stabilization using a nonresilient gingival bar connector. A preformed or cast metal bar can be used to stabilize abutment teeth for removable partial dentures. In addition to splinting, the bar provides a method of indirect retention and aids in preserving the underlying alveolar bone.


Early active motion following a beveled technique of flexor tendon repair: report on fifty cases. In an effort to overcome restrictive adhesions following flexor tendon repair, a technique involving beveling of the tendon ends and fine compressive suturing was used in 50 patients (110 tendons). Free early active motion was allowed. There were 70% good-to-excellent results, with a 10% rupture rate.


H-2 unrestricted cytotoxic T cell activity against antigens controlled by genes in the QA/TLA region. The specificity of H-2 unrestricted cytotoxic T cells was analyzed in secondary CML responses. A/J strain effector cells, sensitized against A.Tlab lymphoid cells, lysed target cells from strains with differing H-2 haplotypes but all sharing Qa-1b/Tlab alleles; whereas, target cells from strains with Qa-1a/Tlaa were not. When B6.Tlaa animals were in vivo-primed and challenged in vitro with B6 stimulator cells, no cytotoxic effector cell activity was generated. However, if B6.Tlaa animals were primed in vivo with A.BY cells and then rechallenged in vitro with either A.BY or B6 stimulator cells, cytotoxic effector cells were generated that lysed target cells from strains with Qa-1b/Tlab alleles. This suggests that factors in addition to Qa/Tla may play a role in the generation of anti-Qa/Tla effector cell activity. It was also noted that targets from strains with Qa-1a/Tlaa alleles were killed, although to a much lesser extent than the Qa-1b/Tlab targets. SWR anti-DBA/1 efffector cells strongly lysed target cells frrom strains with Qa-1b/Tlab, lysed Qa-1a/Tlaa targets to a lesser extent, and produced no cytotoxic effect on B6.Tlaa target cells. These data suggest that in addition to a CML target antigen associated with Qa-1b/Tlab, there may be an additional specificity recognized by cytotoxic T cells controlled by a gene outside of Qa-1b/Tlab.


[Treatment of diabetic retinopathy by laser photocoagulation (author's transl)]. Recent advances in our knowledge concerning spontaneous changes in diabetic retinopathy, and improved classification of clinical forms due to fluorescein angiography, have established precise indications for the use of the laser for retinal photocoagulation in diabetic retinopathy. Controlled, randomized, multicentre studies have conclusively demonstrated the value of the techniques used and results obtained: indirect photocoagulation of the ischaemic zones (pan-retinal photocoagulation) can now effectively preserve the future vision of patients with preproliferating of proliferating retinopathy. The value of photocoagulation in the fight against diabetic oedematous maculopathy appears to be less conclusive, though early elective therapy for juxtafoveolar microvascular lesions appears to be a significant advance.


Experimental malaria in the CBA/N mouse. CBA/N mice carry an X-linked, recessive gene, which results in the absence of a B cell subset, and is expressed primarily as an inability to respond to a certain class of thymus-independent antigens. We have examined the responses of these mice to the malaria parasite Plasmodium yoelii and found that primary infections induced by this parasite are more severe and last longer in mice with X-linked defect than in normal controls. The decreased resistance of the defective mice is associated with a striking deficiency in their IgM antibody response. After recovery from a primary infection, defective mice resist reinfection with the homologous parasite as well as normal mice. Although as resistant as normal controls, B cells from defective mice transfer considerably less immunity to naive recipients than B cells from normal animals. Hence, two modes of thymus-dependent protective immunity may contribute to the host response to P. yoelii. Control of an acute primary infection appears to involve a thymus-dependent antibody response that CBA/N mice are deficient in. Resistance to reinfection may be mediated primarily by a different mechanism.


Serum opsonic activity in rodent malaria: functional and immunochemical characteristics in vitro. The functional and immunochemical characteristics of serum opsonic activity in rodent malaria were examined in the present study. Schizont- and late trophozoite-enriched populations of Plasmodium berghei-infected red blood cells (IRBC) were isolated on a Ficoll density-gradient and used in an in vitro phagocytosis system composed of serum and monolayer cultures of rat peritoneal macrophages. Hyperimmune serum augmented the phagocytosis of IRBC to a greater degree than did nonimmune serum. When either IRBC or macrophages were pre-incubated with serum, the phagocytosis-promoting factors acted on the IRBC rather than on the macrophages in a manner characteristic of serum opsonins. The opsonic activity was specific for IRBC since noninfected red blood cells were rarely phagocytized and were unable to absorb opsonic activity from serum. The opsonic activity of both hyperimmune and nonimmune sera was heat stable, and unaffected by agents known to inactivate or inhibit complement (cobra venom factor and ethylenediaminetetraacetic acid). Finally, the opsonic activity was identified in preparations of purified IgG isolated from both hyperimmune and nonimmune sera.


Functional characterization of mouse lymphocyte subpopulations identified by their natural binding of bacteria. I. Identification of the Ig-secreting cell subpopulation. Three mouse B cell subpopulations (B1, B2 and B3) can be identified by their natural binding of bacteria. To determine whether these subpopulations have unique functions, we assayed the number of anti-SRBC-secreting cells and the number of Ig-secreting cells in unseparated populations and in populations in which the B2 and B3 cells were removed by immobilized monolayers of Escherichia coli-2, a bacteria that binds B2 and B3 cells. Essentially all of the plaque forming cells present in the unseparated population were found in the B1-enriched population, suggesting that most of the antibody-secreting and Ig-secreting cells were in the B1 subpopulation. To show conclusively that the anti-SRBC-secreting cells resided in the B1 subpopulation, the Jerne plaque assay was performed on slides by using lymphocytes prelabeled with various bacteria and the cells that gave rise to the plaques were directly examined. Essentially all of the secreting cells were labeled with Corynebacterium xerosis, which binds to the B1 and B2 cells, whereas very few of the secreting cells were labelled with Arizona hinshawii, which binds to the B2 cells, or with Escherichia coli-2, which binds to the B2 and B3 cells. Thus, the B1 subpopulation contained essentially all of the antibody-secreting cells, which indicates that the B cell subpopulations identified by bacteria are functionally different.


Identification and separation of Thy-1 positive mouse spleen cells active in natural cytotoxicity and antibody-dependent cell-mediated cytotoxicity. The expression of the Thy-1 antigen on mouse spleen cells responsible for NK activity and ADCC was investigated by using a monoclonal IgM anti-Thy-1.2 antibody. Both C-mediated cytotoxicity and the fluorescence-activated cell sorter were used to fractionate cells. The effector cells were found to be heterogeneous in their expression of Thy-1. Effector cells from nude BALB/c mice were predominantly Thy-1 positive; some of the NK cells in CBA spleens appeared to be Thy-1 positive, but at least one-third of the lytic activity was due to Thy-1 negative cells. The effects of treatments on NK cytotoxicity and ADCC were very similar, supporting the hypothesis that the same cells mediate both activities.


Human cell membrane components dominant in T cell lineage: identification and characterization of human TL-like antigens. Cell membrane components that contain beta 2-microglobulin were purified from cells of a human T cell-type leukemia cell line, HPB-ALL. They contained membrane components that have the same molecular size and the same subunit structure as HLA(A,B,C) antigens but are separable from the typical beta 2-microglobulin-containing cell membrane components, i.e., the HLA (A,B,C) antigens, by xenoantibody reagents. A sensitive radioimmunoassay was constructed for detection of the T cell membrane components. The assay revealed that the cell membrane components are expressed exclusively on cells of T cell-type leukemia cell lines among the human lymphoid cell lines tested, predominantly in thymus, among the human organs and tissues tested. They were not present on cells of human B cell-type cell lines or on cells of nonlymphoid organs and tissues. No alloantibodies directed to the T cell membrane components, the putative human homologues of mouse TL antigens, were found in any of the human tissue typing sera tested.


Detection of M-components by an easy immunofixation procedure: comparison with agarose gel electrophoresis and classical immunoelectrophoresis. Commercial anti-gamma-alpha-mu and anti-kappa-lambda (mixture of commercial anti-kappa and anti-lambda) were used for immunofixation after agarose gel electrophoresis of 100 serum samples diluted 1 : 5. This simple screening method detected M-components in 39 sera. M-components were detected in 33 sera by agarose gel electrophoresis, and in 30 sera by classical immunoelectrophoresis. More than one band was detected in 15 sera by the screening method, in 3 sera by agarose gel electrophoresis, and in 2 sera by immunoelectrophoresis. The screening method was superior to the combined use of agarose gel electrophoresis and classical immunoelectrophoresis for detection of M-components, and easier to perform and interpret.


Cryosectioning of undecalcified tissues for immunofluorescence. The present report describes a procedure for preparing 4--6 micrometers cryostat sections of undecalcified fresh frozen tissue which contain hard tissue, for immunofluorescence. The apparatus used is a cryomicrotome originally designed for cutting sections for whole body autoradiography. To obtain cryostat sections suitable for tissue immunofluorescence the standard procedure was modified with respect to the hardness and edges of the microtome knife, the temperature of the cryostat and the carboxymethyl cellulose concentration of the embedding material.


Direct immunofluorescence studies in alopecia areata and male pattern alopecia. Direct immunofluroescence studies were performed on hairy and alopecic areas of scalp in patients with alopecia areata, alopecia totalis and male pattern alopecia. Abnormal deposits of C3 and occasionally of IgG and IgM were found in 92% of 12 patients with alopecia areata and in 21% of patients with male pattern alopecia. No abnormalities were seen in 4 patients with alopecia totalis. In both alopecia areata and male pattern alopecia, the deposits were most common along the basement zone of the inferior segment of hair follicles and occurred with equal frequency in alopecic and normal scalp. These observations suggest that immune factors may play a role in the pathogenesis of alopecia areata.


Immunofluorescence demonstration of type IV collagen and a noncollagenous glycoprotein in thickened vascular basal membranes in protoporphyria. Specific rabbit antibodies to type IV collagen isolated from a basement membrane producing mouse tumor were used in indirect immunofluorescence tests to study the thickened vascular basement membrane in skin biopsies from patients with erythropoietic porphyria (EPP) and from protoporphyric mice. In addition, rabbit antibodies to a noncollagenous, basement membrane specific glycoprotein also derived from the mouse tumor were tested. It was shown that normal as well as altered vascular basement membranes in both the human and the murine skin specimens react with the anti-type IV collagen and the antiglycoprotein antibodies. A particular strong reaction in the diseases skin indicated that formation of new vascular basement membrane layers involved deposition of the major structural proteins which also constitute normal basement membrane matrices.


Quantitative estimation of leishmanial antibody titers by enzyme-linked immunosorbent assay. The response to immunization against Leishmania braziliensis in rabbits was followed by measurement of antibody by enzyme-linked immunosorbent assay (ELISA), passive hemagglutination, complement fixation, and countercurrent immunoelectrophoresis. Passive hemagglutination and complement-fixation titers were interpreted according to standard methods, whereas ELISA exact titers were derived by the equation Y = a-b log X, which describes the straight line that results when absorbances are plotted against test serum dilutions. Peak titer was measured at 32 +/- 1 days after initial injection, irrespective of the assay method. After seven days, the reciprocal antibody titer was 1,130 by ELISA, 0 by passive hemagglutination, and 8 by complement fixation. Precipitin bands were shown by countercurrent immunoelectrophoresis only at peak titer. With sera from hamsters experimentally infected with Leishmania, 94% showed leishmanial antibody by ELISA, compared with 92% by complement fixation and 65% by passive hemagglutination. When 31 samples of sera from human patients with cutaneous leishmaniasis were tested only by ELISA, 23 were positive for leishmanial antibodies.


Endemic syphilis: passive transfer of resistance with serum and cells in hamsters. Serum or spleen cells from hamsters immune to infection with Treponema pallidum strain Bosnia A conferred partial protection against syphilitic infection by the same strain on recipient hamsters. Cutaneous lesions did not develop, and in the lymph nodes the average weight and number of treponemes detected were significantly lower than in normal and control hamsters. Treatment of the immune spleen cells with antithymocyte serum and complement abolished their ability to transfer resistance. This is the first direct evidence that thymus-derived cells are involved in resistance to syphilitic infection.


Scanning electron microscope observations of microvascular anastomosis in the rat carotid artery. Microvascular anastomosis is gaining increasing significance in the field of replantation and reconstructive surgery. Although traumatic interference is minimized by microsurgery, thrombotic occlusions remain the major reason for clinical failure. With the improvement of suturing technique studies were directed towards the causes of failure using light and scanning electron microscopy. Our study was focussed on the latter aspect. The left common carotid artery of 55 albino  rats was transsected and re-anastomosed by the eccentric biangulation technique. The suture-lines were observed after various postsurgical intervals, ranging from 12 hours up to 14 months. Twelve hours after the anastomosis had been performed, the most striking alterations of the endothelium were not found at the suture line, as was expected, but in those parts of the vessels, which were caught by the clamps. The injury leads to sloughing of the endothelium in extensive areas. The exposed subendothelial layer of connective tissue is covered with aggregates of thrombocytes. The suture itself does not lead to thrombus formation. After 14 days, healing is almost complete with total regeneration of the endothelium. From the 21st postoperative day on, the inner lining can no longer be differentiated from that found in experimental animals after 14 months.


Late results following treatment of naevus flammeus and facial burns with split thickness skin grafts, with special reference to post-operative colour changes. Two cases of extensive split thickness skin transplant to the face are presented. It was noted that the usual brownish pigmentation of grafted skin did not occur in these cases. Continued exposure in the open or to sunlight during work has been implicated as a causative factor.


A quality of care evaluation measure for emergency medical service systems. This paper presents the development and validation of an emergency medical service (EMS) systems quality of care evaluation measure. The System Input Severity Measure consists of a set of single and multiple injury survival rates that would be expected to occur in an EMS system classified as providing "baseline" advanced life support services. These expected survival rates were developed by a nationwide panel of emergency medicine experts through the use of the Delphi group opinion surveying technique. Validation of the System Input Severity Measure was a twofold process. First, reliability of the results of the Delphi surveying process was validated by comparing the resultant expected survival rates with Illinois Trauma Registry data. Second, the applicability of the measure was validated using data collected in EMS systems generally recognized to be providing exceptional (either superior or inferior) emergency patient care. It was recognized that the development of a comprehensive, large-scale System Input Severity Measure through the use of the Delphi technique alone is impractical. Consequently, a functional relationship between single and multiple injury survival rates was also developed. It appeared that when employed in conjunction with data developed from the Delphi technique, such an approach would make practicable the development of a comprehensive System Input Severity Measure.


Isolation of haemin-requiring mutants of Escherichia coli K12. Fifty-five haemin-requiring mutants were isolated from haemin-permeable mutants. According to their growth responses to haem precursors and their patterns of porphyrin accumulation, the 55 mutants fell into three groups which were judged to have defects in 5-aminolaevulinate dehydratase, ferrochelatase, and uroporphyrinogen III cosynthase or uroporphyrinogen decarboxylase. In mutants of the group deficient in 5-aminolaevulinate dehydratase, the mutations were adjacent to lac, and evidence is presented that the mutations were in hemB and were commonly deletions extending into proC.


Mutants of Escherichia coli K12 permeable to haemin. Mutants of Escherichia coli which require 5-aminolaevulinic acid (5-ALA), the first intermediate of haem biosynthesis, do not respond to haemin and porphyrins. The probable explanation of the lack of response is that E. coli may be impermeable to haemin and porphyrins. Mutants are described which responded to haemin and porphyrins as well as to 5-ALA. Indirect evidence is presented that the mutants were permeable to haemin. The mutants showed other phenotypic changes, and resembled some mutants which are known to have changes in the cell envelope.


Denaturation map of the ColE1-Km plasmid pCR11. The denaturation map of EcoRI-digested pCR11, a ColE1-Km plasmid, is described. The 2.0 kilobase ColE1-derived segment contains an adenine+thymine rich site in the colicin immunity gene region. In the 7.2 kilobase kanamycin resistance region, the transposon Tn903 consists of an adenine+thymine rich 0.98 kilobase kan gene region flanked by a guanine+cytosine rich 1.09 kilobase inverted duplication.


Taxonomy of Clostridium tetani and related species. Clostridium tetani and its related species C. tetanomorphum, C. cochlearium and C. lentoputrescens were examined for DNA-DNA homology and biochemical properties. Two distinctly different groups were included under the name of C. tetanomorphum: one was identical with C. cochlearium and the name C. tetanomorphum was applied to the other group with some amendment of biochemical properties. Comparison of the type strain of C. lentoputrescens with wild strains obtained from horse faeces indicated that the name C. lentoputrescens should be abolished as a later synonym of C. cochlearium. Liquefaction of gelatin and spore shape, which have been used as the important criteria for differentiation of C. tetani-related species, were genetically insignificant.


Interference of azide with cysteine biosynthesis in Salmonella typhimurium. The growth inhibition of Salmonella typhimurium aziA mutants by sodium azide is reversed by cystine and related compounds. NADPH-sulphite reductase (hydrogen-sulphide:NADP+ oxidoreductase; EC 1.8.1.2), an enzyme of cysteine biosynthesis, is inhibited in cell extracts by sodium azide. AziB mutants which are able to grow in the presence of the inhibitor without cystine were isolated. About half of them were mapped in the cysK gene and have only residual activity of its product, O-acetylserine sulphydrylase A [O-acetyl-L-serine acetate-lyase (adding hydrogen-sulphide); EC 4.2.99.8]. Sensitivity of wild type and aziA mutants to azide was also reversed by a constitutive mutation in cysB, the regulatory gene of cysteine biosynthesis. CysK and cysB mutants showed cross-resistance to azide and 1,2,4-triazole. It is suggested that the resistance of these mutants to azide is due to an increased activity of NADPH-sulphite reductase.


Characterization of plasmids from antibiotic-resistant Shigella isolates by agarose gell electrophoresis. Gel electrophoresis of DNA from 95 clinical isolates of Shigella sonnei and Shigella flexneri resistant to antibiotics revealed a heterogeneous plasmid population. Most of the plasmids were smaller than 6 megadaltons (Mdal). Six S. sonnei isolates with the most common antibiotic resistance pattern were characterized. They had two plasmids in common: one was a self-transmissible Fi+ plasmid of 46 Mdal encoding resistance to streptomycin and sulphafurazole. In addition, several cryptic plasmids ranging in size from 1.0 to 24.5 Mdal were present. Mobilization of the 5.5 Mdal SuSm plasmid and a 1.0 Mdal cryptic plasmid was demonstrated with all six S. sonnei isolates during conjugation. This mobilization was mediated by the 46 Mdal self-transmissible Fi+ R plasmid and also by a 24.5 Mdal Fi- plasmid carrying no known drug resistance determinants.


Effects of sheep pineal fractions on the activity of male rat hypothalami in vitro. High molecular weight substances could be isolated from sheep pineals with the "Bensinger" extraction method, followed by ultrafiltration of the waterlayer through different diaflomembranes. Two of the pineal fractions, XM100R and PM30R, stimulate the gonadotropin releasing activity of the medial basal hypothalamus (MBH). In experiments in which comparable pineal fractions were incubated without MBH and without pituitary and injected in immature mice no effect was detectable. All experiments in which a similar amount of rat cerebral cortex was used for incubation with pineal fractions did not show any activity.


Production of experimental brain abscess in the rat. Experimental evaluation of brain abscess has been inhibited by the lack of a simple and reproducible model in small animals. A stereotaxic headholder and slow infusion of 1 microliter of saline, containing a known number of bacteria, were used to produce brain abscess consistently in the rat. The natural history of the brain abscess produced by this technique closely simulated that found in the human clinical situation.