J Gen Microbiol, 1975 Mar, 87(1), 141 - 9 The effect of culture age, chloramphenicol and B6 inhibitors on intra- and extracellular keto and amino acids of Escherichia coli B; Raunio RP et al.; Keto acids and free amino acids were assayed in the cells and the medium of Escherichia coli B growing in the presence of chloramphenicol, cycloserines, aminooxyacetate, and limiting nitrogen source . Under these growth-limiting conditions the cells accumulated ketoglutarate and 'ketovaline' but no other keto acids . In all experiments only ketoglutarate, pyruvate, and 'ketovaline' were found in the medium . Amino acids are released into the medium in the early phases of growth and the composition of the extracellular amino acids is similar to that of the amino acid pool . The concentrations of free amino acids were 10-3-10-4 times higher in the cell than in the medium . The internal pool composition is fixed under all growth-limiting conditions . In the presence of the drugs the cells release amino acids into the medium. Res Vet Sci, 1975 Mar, 18(2), 117 - 20 The immune response of hens to multiple Escherichia coli injections and transfer of immunoglobulins to the egg and hatched chick; Heller ED; Four spaced E coli antigen injections were given to laying hens in order to cause an extended period of antibody production . Antibody could be detected as early as four days after the first antigen injection reaching a peak at the 20th day . IgM was the first antibody to be detected . From the eighth day IgG could be detected replacing the IgM fraction gradually . Antibody to E coli produced by the hen could be detected in yolk of eggs and in chicks from the 15th day but not after the 87th day post treatment . Fluctuation or antibody transmitted by the individual hen was observed . Yolk and chick antibody titres were slightly lower than those found in the serum of the hens on the same day . Only IgG antibodies were found in yolks and serum of day-old chicks. Proc Soc Exp Biol Med, 1975 Mar, 148(3), 725 - 8 Immunosuppressive effects of experimental infection with Plasmodium gallinaceum; Weidanz WP et al.; Experimental infection of chickens with P . gallinaceum mardedly suppressed the splenic PFC response to SRBC . Suppression was most pronounced when birds were immunized at the time of peak parasitemia . The PFC response to E . coli LPS was of low magnitiude in both normal and infected chickens; however, it, too, was suppressed in infected birds, but not to the same degree as observed in response to SRBC . Cellular immunity as evidenced by allograft rejection was not influenced by infection. Proc Soc Exp Biol Med, 1975 Mar, 148(3), 675 - 8 Mechanisms of endotoxin tolerance . IX . Effect of exchange transfusion; Greisman SE et al.; Healthy New Zealand rabbits were injected iv with an LD-80 dose of E . coli endotoxin . Twenty minutes later, after removal of over 50% of the endotoxin by the RES, exchange transfusion was performed, accomplishing a rapid and sustained reduction in the level of endotoxemia simulating that seen in animals rendered highly tolerant by seven prior sublethal injections of toxin . Depite such reduction in endotoxemia, 96-hr mortality was only slightly, and not significantly reduced compared to sham exchanged controls (70 vs 83% respectively) . Additional control studies indicated that exchange tranfusion per se did not enhance endotoxin mortality . The findings directly support the concept that endotoxin tolerance is based primarily upon enhanced RES resistance to endotoxin toxicity rather than upon enhanced RES clearance of circulating endotoxin. Proc Natl Acad Sci U S A, 1975 Mar, 72(3), 921 - 5 Interaction of Escherichia coli dnaB and dnaC(D) gene products in vitro; Wickner S et al.; Purified E . coli dnaB and dnaC(D) gene products interact physically and functionally in vitro . This interaction was demonstrated as follows: (a) A complex of dnaB and dnaC(D) gene products was isolated by gel filtration; ATP specifically was required for isolation of the complex . (b) The DNA-independent ribonucleoside triphosphatase activity associated with dnaB gene product was inhibited by dnaC(D) gene product . (c) The dnaC(D) gene product was protected from inactivation by N-ethyl-maleimide by the combination of dnaB gene product and ATP; this protection required ATP specifically. Proc Natl Acad Sci U S A, 1975 Mar, 72(3), 911 - 5 Regulation of RNA polymerase synthesis in Escherichia coli: a mutant unable to synthesize the enzyme at 43 degrees; Oeschger MP et al.; We report the isolation of a mutant of E . coli in which the capacity to synthesize RNA polymerase (EC 2.7.7.6) (the beta and beta' subunits) is rapidly lost at 43 degrees . The mutation has no effect on the stability or activity of the polymerase itself . The mutation is recessive and is closely linked to the rif locus (the structural gene for the beta subunit) . Using strains carrying the mutation, we have shown that polymerase is present in excess in rapidly growing E . coli cells. Proc Natl Acad Sci U S A, 1975 Mar, 72(3), 829 - 33 Replicative intermediates of colicin E1 plasmid DNA in minicells; Oka A et al.; Pulse-labeled colicin E1 plasmid (Col E1) DNA in minicells was examined to characterize replicating molecules . Replication of Col E1 DNA principally occurred in covalently closed molecules and involved the synthesis of variable length single-stranded DNA fragments that were dissociable from the template DNA and ultimately incorporated into the completely replicated molecules . RNA was found to be associated with some of these newly synthesized fragments . Replicating molecules containing different size displacement loops were observed by electron microscopy. Proc Natl Acad Sci U S A, 1975 Mar, 72(3), 784 - 8 Nucleotide sequence of an RNA polymerase binding site at an early T7 promoter; Pribnow D; Escherichia coli RNA polymerase (EC 2.7.7.6), bound in a tight complex at an early T7 promoter, protects 41 to 43 base pairs of DNA from digestion by DNase . I . The protected DNA fragment contains both the binding site for RNA polymerase and the mRNA initiation point for the promoter . The sequence of the DNA fragment and the sequence of the mRNA that it codes for are presented here . A seven-base-pair sequence, apparently common to all promoters, is implicated in the formation of a tight binary complex with RNA polymerase. Proc Natl Acad Sci U S A, 1975 Mar, 72(3), 1112 - 6 Biogenesis of membrane lipids: mutants of Escherichia coli with temperature-sensitive phosphatidylserine decarboxylase; Hawrot E et al.; Phosphatidylserine decarboxylase catalyzes the last step in the pathway leading to phosphatidylethanolamine, the principal membrane lipid of E . coli . Mutants of E . coli have now been isolated in which this enzyme is theramolabile . The structural gene for phosphatidylserine decarboxylase (psd gene) is closely linked to the pur A locus at about 83 min on the standard map of the E . coli chromosome . When a mutant with thermolabile decarboxylase is incubated at 42 degrees, growth ceases, but only after a substantial fraction (20-40%) of the total phospholipid of the cell has been replaced by phosphatidylserine . Examination of such mutants with altered content of phospholipids may shed light on the role of specific phospholipids in membrane function. Proc Natl Acad Sci U S A, 1975 Mar, 72(3), 1068 - 71 Separation of transfer ribonucleic acid by sepharose chromatography using reverse salt gradients; Holmes WM et al.; The transfer ribonucleic acids of Escherichia coli bind to unsubstituted Sepharose in the presence of high concentrations of ammonium sulfate at pH 4.5 . Transfer RNA species are eluted individually from the Sepharose by a gradient from high to low concentrations of ammonium sulfate; leucine tRNA is fractionated into five isoaccepting species . The order of elution of these isoaccepting species differs from that seen with reverse phase chromatography . By means of only these two procedures, one isoaccepting species of leucine tRNA can be purified to apparent homogeneity . Isoaccepting tRNA species for 9 amino acids have been resolved . This established the general utility of this chromatographic system for the separation and purification of specific isoaccepting transfer RNAs. Proc Natl Acad Sci U S A, 1975 Mar, 72(3), 1059 - 62 N-formylmethionyl peptides as chemoattractants for leucocytes; Schiffmann E et al.; Leucocytes such as neutrophils are attracted by N-formylmethionine, but not by methionine . Di- and tripeptides containing formylmethionine are strong attractants for both neutrophils and macrophages, whereas the corresponding nonacylated compounds are not chemotactic . The formylated peptides may be related to an incompletely characterized chemotactic material normally produced by bacteria which attract the same animal cells. Proc Natl Acad Sci U S A, 1975 Mar, 72(3), 1050 - 4 Replication of colicin E1 plasmid DNA added to cell extracts; Tomizawa JI et al.; Closed-circular DNA of colicin E1 plasmid can undergo a round of semiconservative replication when added to an extract of Escherichia coli . Extracts of cells that do not carry the plasmid are able to perform complete replication of the plasmid . Replication requires de novo RNA synthesis but not protein synthesis. Nucleic Acids Res, 1975 Mar, 02(3), 327 - 45 On the statistical significance of primary structural features found in DNA-protein interaction sites; Dykes G et al.; Probabilities of occurrence for a number of the symmetries and other sequence regularities found in DNA-protein interaction site sequences have been calculated for segments of random DNA sequence . Results show that many of the symmetrical and repetitive features seen in these interaction sites are likely to have occured by chance . Other features are so unlikely to have occurred by chance that they are probably involved in the DNA-protein interaction processes. Nucleic Acids Res, 1975 Mar, 02(3), 303 - 18 Binding of lactose repressor to poly d(A-T) : OD AND CD melting of the complex; Clement R et al.; The binding of lactose repressor to poly d(A-T) at low ionic strength has been investigated by heat denaturation . The poly d(A-T) melting is monitored by optical density and the protein melting by circular dichroism . From the modification of the poly d(A-T) melting curve we estimate a maximum binding ratio of about one tetrameric repressor to about 20 basic pairs . The repressor melting can be interpreted as a global shift from a to b structure of about 25 residues per subunit . The melting curves of poly d(A-T) and repressor have not a shape easy to interpret; nevertheless both show a cooperative transition in the same temperature range where we can evaluate that about 3.8 aminoacid residues shift from a to b structure when 1 bases pair melt. Immunology, 1975 Mar, 28(3), 509 - 22 The inter-relationship of antigenic structure, thymus-independence and adjuvanticity . IV . A general model for B-cell induction; Waldmann H et al.; Polymerized flagellin and E . coli lipopolysaccharide both express adjuvanticity in vivo and in vitro for responses to hapten conjugated to sheep erythrocytes, and hapten conjugated to soluble thymus-dependent antigens . In the case of erythrocyte-bound hapten, adjuvanticity is expressed in the absence of thymus-derived cells (T cells) . However, in the case of responses to soluble thymus-dependent conjugates, carrier-specific T cells would appear to be necessary for adjuvanticity to be expressed . On the basis of these observations an hypothesis for the mechanism of B-cell induction and tolerance is proposed. Carbohydr Res, 1975 Mar, 40(1), 183 - 92 Human blood-group MN and precursor specificities: structural and biological aspects; Springer GF et al.; The human blood-group MM and NN antigens carry 2 to 4 immunodominant groupings per repeating subunit and differ only by one sialic acid residue per immunodominant group . This residue covers in the MM antigen the beta-D-galactopyranosyl group that is terminal in the N immunodominant structure and that, together with a terminal alpha-linked N-acetylneuraminic acid residue, is responsible for N specificity . M specificity was readily converted into N specificity by mild acid treatment . N structure is the immediate biochemical precursor of M structure, and M and N antigenic specificities are not determined by two allelic genes as believed hitherto . The NN antigen was inactivated by beta-D-galactosidase as well as by removal of N-acetylneuraminic acid . Some of the reactivities of the NN antigen, lost upon beta-D-galactosidase treatment, reappeared on subsequent partial N-acetylneuraminic acid removal . The structure uncovered by complete sialic acid depletion of MN antigens is the Thomsen-Friedenreich T antigen, the specificity of which is determined by beta-D-galactopyranosyl groups . Beta-D-Galactosidase treatment transformed the T antigen into one possessing Tnactivity . The significance of blood-group MN active substances extends to human breast cancer, where MN antigens were found in benign and malignant glands, but some of their precursors in cancerous tissue only. Can J Biochem, 1975 Mar, 53(3), 328 - 37 Chemical modification of ribosomes with dimethyl sulfate: a probe to the structural organization of ribosomal proteins and RNA; Moore G; Ribosomal proteins from {14-C}dimethyl sulfate treated with 30S and 50S subunits of Escherichia coli ribosomes were separated by two-dimensional polyacrylamide gel electrophoresis and the degree of methylation of each protein was determined . Comparison of the results from this relatively non-specific chemical modification procedure with results from the milerd lysine-specific reductive alkylation procedure reported previously (Moore, G . & Crichton, R.R . (1974) Biochem . J . 143, 607-612) has permitted a topographical classification of ribosomal proteins in terms of 'degree of exposure' in the 30S and 50S subunits . The reaction of dimethyl sulfate with ribosomal RNA, both in intact subunits and after isolation from the subunits, has indicated that approximately half of the RNA in 30S and 50S subunits is exposed on the surface of the ribonucleoprotein complexes, and that no large sections (extended sequences) of 16S RNA are concealed in the 30S subunit . It is proposed that modification of ribosomes with dimethyl sulfate is a potentially useful technique for probing exposed and hidden regions and also exposed single-stranded regions of RNA in ribosomes. Can J Biochem, 1975 Mar, 53(3), 262 - 8 Coupling of glycine and alanine transport to respiration in cells of Escherichia coli; Sprott GD et al.; Energy coupling for uptake of glycine and alanine in glycerol grown cells of Escherichia coli differs from that of the aromatic amino acids . Respiration and uptake of glycine and alanine show similar inactivations in cells exposed to high intensity violet light or to various concentrations of cyanide . In contrast,uptake of phenylalanine, tyrosine, and tryptophan is resistant to effects of light or cyanide . Anoxia largely inhibits uptake of glycine and alanine while that of the aromatic amino acids is only partially affected . Furthermore, ferricyanide (but not ferrocyanide) completely restores active uptake of aromatic amino acids under anoxic conditions but is without effect on glycine and alanine uptake . Adenosine 5'-triphosphate (ATP) concentration does not increase in anoxic cells exposed to ferricyanide, indicating that ATP cannot be responsible for this restoration . The data suggest that glycine and alanine represent amino acids whose transport shows a complete dependence on energy derived from respiration, while the energy for transport of the aromatic amino acids may be obtained from other sources Biophys J, 1975 Mar, 15(3), 253 - 61 Light-scattering study of the temperature dependence of Escherichia coli motility; Banks G et al.; Two light-scattering techniques are used to study the temperature dependence of translational and rotational motility in Escherichia coli . The method of number fluctuation spectroscopy is developed theoretically and experimentally to measure the translational swimming speed of a smooth swimming strain of E . coli . Interference fluctuation techniques are used to study the rotational component of the motion . The results demonstrate that the thrust remains proportional the the torque generated by the flagella throughout the range studied and also show that relative changes in translational swimming speed may be inferred from the dynamics of rotational motion. Urology, 1975 Mar, 05(3), 390 - 3 Complete duplication of urethra; Susan LP et al.; Complete duplication of the urethra with a single bladder is an exceptional finding . The anomalous urethral canal originates from the bladder separately, runs parallel and usually dorsal to the normally situated urethra, and opens on the dorsum of the penis . To our knowledge there are 41 reported cases of this anomaly . We present 2 cases of complete urethral duplication originating from a single bladder, one in a male patient and the other in a female . It is concluded that conservative therapy is the treatment of choice, and that surgery should be reserved for incontinent patients only . The complications of surgery have been emphasized. J Bacteriol, 1975 Mar, 121(3), 994 - 9 Regulation of ribosomal protein synthesis in Escherichia coli B/r; Dennis PP et al.; The differential synthesis rate of ribosomal protein (r-protein), alpha-r (synthesis rate of r-protein divided by synthesis rate of total protein), was measured during the cell division cycle . It was observed that alpha-r remained essentially constant and was not measurably affected by duplication of the r-protein gene cluster (i.e., str-spc region) during the process of chromosome replication . It was further observed that the rate of total protein synthesis and r-protein synthesis increased continuously and uniformly during the entire cell cycle . This gene dosage independence of the synthesis rate of r-protein was similar to that observed earlier for the synthesis of ribosomal ribonucleic acid (rRNA) . These observations indicate that the synthesis rates of the protein and RNA components of the ribosome are coordinately balanced during the entire cell division cycle and are not significantly perturbed by duplication of the r-protein or rRNA genes . Furthermore, this balanced synthesis insures that neither free rRNA nor free r-protein accumulate in appreciable amounts during balanced growth. J Bacteriol, 1975 Mar, 121(3), 923 - 32 Strain of Escherichia coli with a temperature-sensitive mutation affecting ribosomal ribonucleic acid accumulation; Frey T et al.; A mutant of Escherichia coli has been isolated that has a temperature-sensitive mutation that results in specific loss of ribosomal ribonucleic acid (RNA) synthesis and some reduction in messenger RNA synthesis . When the strain was grown in glucose medium at a restrictive temperature, RNA accumulation ceased, but both messenger RNA and protein synthesis continued for an extended time . Because carbon metabolism was slowed drastically when strain AA-157 was placed at the restrictive temperature, this phenotype can be compared with carbon depletion conditions present during diauxic lag . However, the phenotype of mutant AA-157 differs from shift-down conditions in that guanosine-3',5'-tetraphosphate levels are unaffected; therefore, a different site is affected . This mutant strain (AA-157) thus shows many characteristics similar to an aldolase mutant previously reported (Bock and Neidhardt, 1966) . However, the mutation occurred in a different position on the E . coli genetic map, and furthermore, aldolase was not temperature sensitive in strain AA-157 . In this paper we present a study of macromolecular biosynthesis in this mutant. J Bacteriol, 1975 Mar, 121(3), 892 - 900 Temperature-sensitive recA mutant of Escherichia coli K-12: deoxyribonucleic acid metabolism after ultraviolet irradiation; Hall JD et al.; A mutant of Escherichia coli K-12 temperature sensitive for genetic recombination was investigated and found to carry a mutation that could be cotransduced with cysC and hence could be in the recA gene . To determine whether recA+ can complement this mutation, matings were carried out at 35 and 40 C between Hfr donors that transfer recA+ or recA1 early and recipients carrying wild-type or mutant alleles . It was found that recA+ but not recA1 complements this mutation in zygotic temporary partial diploids . The mutant allele was accordingly designated recA44 . A transductant carrying recA44 behaved normally at low temperatures but more like recA- strains at high temperatures with respect to recombinant colony formation in Hfr matings, cell survival, and deoxyribonucleic acid (DNA) synthesis after ultraviolet irradiation, cellular DNA breakdown, and prophage induction when lysogenic for lambda . Alkaline sucrose sedimentation studies of DNA from recA44 cells showed that short DNA molecules synthesized immediately after ultraviolet irradiation increased in molecular weight during subsequent incubation at 32 C but not at 45 C . Hence, recA+ is required for this molecular weight increase . Cells exposed to ultraviolet light synthesized DNA that remained of low molecular weight during a 40-min incubation at 32 C . This material increased in molecular weight in recArut not in recA44 cells during subsequent incubation at 45 C . Thus, the availability of recA+ during the first 40 min at 32 C after irradiation did not obviate the need for recA+ in the subsequent phases of this post-replication repair process. J Bacteriol, 1975 Mar, 121(3), 883 - 91 Accumulation of the capacity of initiation of deoxyribonucleic acid replication in Escherichia coli; Evans IM et al.; Several temperature-sensitive initiation mutants of Escherichia coli were examined for the ability to initiate more than one round of replication after being held at nonpermissive temperature for approximately 1.5 generation equivalents . The capacity for initiation was measured by residual synthesis experiments and rate experiments under conditions where protein synthesis and ribonucleic acid synthesis were inhibited . Results of the rate and density transfer experiments suggest that the cells may initiate more than one round of replication in the absence of protein or ribonucleic acid synthesis . This contrasts with the results of the residual synthesis experiments which suggest that, under these conditions, only one round of synthesis is achieved . These findings suggest that the total amount of residual synthesis achieved in the presence of an inhibitor may be both a function of the number of initiation events which occur and the effect of the inhibitor of protein or ribonucleic acid synthesis on chain elongation. J Bacteriol, 1975 Mar, 121(3), 873 - 82 Model for the enchancement of lambde-gal integration into partially induced Mu-1 lysogens; Faelen M et al.; Temperate phage Mu-1, which is able to integrate at random in its host chromosome, is also able to mediate integration of other circular deoxyribonucleic acid, as a lambda-gal mutant unable to integrate by itself . After mixed infection with lambda-gal and Mucplus, galplus transductants are recovered that have the lambda-gal integrated in any circular permutation, sandwiched between two complete Mu genomes in the same orientation, the whole Mu-lambda-gal-Mu structure being found at any location in the bacterial chromosome . Here we show that such a lambda-gal can integrate in an induced Mu lysogen . In this case the lambda-gal is again in any circular permutation, between two Mu in the same orientation, but it is always located at the site of the original Mu prophage, and the two surrounding Mu have always the same genotype as the original Mu prophage . Active Mu replication functions are not essential for that process to occur . This suggests that bacterial replication may generate two Mu copies that in some way can regenerate a Mu attachment site that recombines with the lambda-gal . A model is presented that accounts for these observations, may be helpful for understanding some complex features of Mu development, and may possibly offer a basis for explaining spontaneous duplications. J Bacteriol, 1975 Mar, 121(3), 857 - 62 Integration of R plasmid Rts1 to the gal region of the Escherichia coli chromosome; Terawaki Y et al.; An R plasmid Rts1 was integrated into the gal region of the chromosome of Escherichia coli XA-7012 (galE) strain by the directed transposition technique . The integration of the Rts1 genome was confirmed mainly by conjugation studies and also by transduction experiments using phage P1 . As a result, it was found that the integrated genome contained genes responsible for kanamycin resistance, conjugal transferability, and for autonomous replication . As reported previously, Rts1 is temperature sensitive in replication and inhibits the growth of the host at nonpermissive temperature . However, although a plasmid derived from the integrated Rts1 genome still demonstrates temperature sensitivity upon transfer and high level of kanamycin resistance, this plasmid no longer displays temperature sensitivity in replication and the inhibitory effect on the host . These results indicate that the temperature sensitivity of replication of Rts1 and its inhibitory effect on the host cell are due to the presence of a gene or gene cluster on the Rts1 genome and that the gene(s) is clearly discriminated from the one responsible for the temperature sensitivity of transfer. J Bacteriol, 1975 Mar, 121(3), 753 - 8 Stability of Escherichia coli polysomes at high hydrostatic pressure; Pope DH et al.; The stability of Escherichia coli polysomes at increased hydrostatic pressure was investigated in actively growing cells, in which the initiation of transcription was blocked by rifampin . In these cells, {3-H}uridine incorporation into messenger ribonucleic acid and the subsequent degradation of the message (and therefore of polysomes) by ribonuclease could be observed . Evidence is presented that the activity of the RNases is unaffected by a pressure of 680 atm, that protein synthesis is completely inhibited at 680 atm but immediately resumes at the 1 atm rate on release of pressure, and that no degradation of messenger ribonucleic acid in polysomes occurs at 680 atm . The effects of pressure; puromycin, and chloramphenicol on polysomal degradation are discussed . These results indicate that, contrary to some previous reports, polysomes are probably stabilized by high pressures . Therefore, we consider that polysomal instability is not a factor in the inhibition of protein synthesis by high pressures. J Bacteriol, 1975 Mar, 121(3), 1158 - 65 Energy efficiency of intraperiplasmic growth of Bdellovibrio bacteriovorus; Rittenberg SC et al.; The Y-ATP (energy efficiency) of intraperiplasmic growth of Bdellovibrio bacteriovorus was determined from the distribution of radioactivity of the substrate organism ({U-14C}Escherichia coli) btween CO2 and bdellovibrio cells at the end of growth . A "best" Y-ATP value of 18.5 was obtained from single growth cycle experiments and an average value of 25.9 from multicycle experiments . Both values are much higher than the usual value of 10.5 for bacteria growing in rich media . The bases for the unusual energy efficiency for growth of B . bacteriovorus are discussed. J Bacteriol, 1975 Mar, 121(3), 1145 - 57 Incorporation of long-chain fatty acids of the substrate organism by Bdellovibrio bacteriovorus during intraperiplasmic growth; Kuenen JG et al.; Data are presented showing that a large proportion of the fatty acids of Bdellovibrio bacteriovorus grown intraperiplasmically are derived unaltered from the fatty acids of its substrate organism . Those fatty acids of the bdellovibrio not homologous with those of the substrate organism are derived mainly by metabolic alteration of preexisting fatty acids in the latter . De novo synthesis from acetate occurs only to a small extent . These characteristics of bdellovibrio physiology are in part responsible for its minimal energy expenditure for intraperiplasmic growth . The data presented also indicate that B . bacteriovorus is capable of hydrogenating unsaturated fatty acids, of beta-oxidation of fatty acids, and of regulating the proportion of saturated and unsaturated fatty acids in the lipids. J Bacteriol, 1975 Mar, 121(3), 1137 - 44 Utilization of nucleoside monophosphates per Se for intraperiplasmic growth of Bdellovibrio bacteriovorus; Rittenberg SC et al.; During growth of Bdellovibrio bacteriovorus on Escherichia coli, there was a marked preferential use of E . coli phosphorus over exogenous orthophosphate even though the latter permeated into the intraperiplasmic space where the bdellovibrio was growing . This preferential use occurred to an equal extent for lipid phosphorus and nucleic acid phosphorus . Exogenous thymidine-5'-monophosphate competed effectively with {3H}thymine residues of E . coli as a precursor for bdellovibrio deoxyribonucleic acid; exogenous thymidine competed less effectively and thymine and uridine not at all . A mixture of exogenous nucleoside-5'-monophosphates equilibrated effectively with E . coli phosphorus as a phosphorus source for B . bacteriovorus; the nucleotide phosphorus entered preferentially into bdellovibrio nucleic acids . A comparable mixture of exogenous nucleosides plus orthophosphate had only a small effect on utilization of E . coli phosphorus by B . bacteriovorus, as did orthophosphate alone . A mixture of exogenous deoxyriboside monophosphates equilibrium effectively with E . coli phosphorus as a phosphorus source for bdellovibrio growth; the phosphorus from this source entered preferentially into deoxyribonucleic acid . These data show that nucleoside monophosphates derived from the substrate organism are utilized directly for n-cleic acid biosynthesis by B . bacteriovorus growing intraperiplasmically . As a consequence, the phosphate ester bonds preexisting in the nucleic acids of the substrate organism are conserved by the bdellovibrio, presumably lessening its energy requirement for intraperiplasmic growth . The data also suggest, but do not prove, that the phosphate ester bonds of phospholipids are also conserved. J Bacteriol, 1975 Mar, 121(3), 1131 - 6 Effects of methotrexate on intraperiplasmic and axenic growth of Bdellovibrio bacteriovorus; Pritchard MA et al.; The intraperiplasmic growth rate and cell yield of wild-type Bdellovibrio bacteriovorus 109J, growing on Escherichia coli of normal composition as the substrate, were not markedly inhibited by 10-3 M methotrexate (4-amino-N10-methylpteroylglutamic acid) . In contrast, the growth rate and cell yield of the mutant 109Ja, growing axenically in 0.5% yeast extract +0.15% peptone, were strongly inhibited by 10-4 and 10-3 M methotrexate . Thymine, thymidine, and thymidine-5'-monophosphate, in increasing order of effectiveness, partially or completely reversed the inhibition . E . coli depleted of tetrahydrofolate and having an abnormally high protein/deoxyribonucleic acid (DNA) ratio was obtained by growing it in the presence of methotrexate . B . bacteriovourus grew at a normal rate on these depleted E . coli cells but with somewhat reduced cell yield . Mexthotrexate (10-3 M) inhibited intraperiplasmic growth of bdellovibrio on the depleted E . coli somewhat more than it inhibited growth on normal E . coli, but the effects were small compared with inhibition of axenic growth of the mutant . Total bdellovibrio DNA after growth on the depleted E . coli in the presence or absence of methotrexate exceeded the initial quanity of E . coli DNA present . Thymidine-5'-monophosphate (10-3 M) largely reversed the inhibition and increased the amount of net synthesis of DNA . The data are consistent with the prediction that intraperiplasmic growth of B . bacteriovorus should be insensitive to all metabolic inhibitors that act by specifically preventing synthesis of essential monomers . The data also indicate that B . bacteriovorus possesses thymidylate synthetase, thymidine phosphorylase, and thymidine kinase, and has the potential to carry out de novo DNA synthesis from non-DNA precursors during intraperiplasmic growth . The results also suggest that methionyl tRNAfMet is not required for initiation of protein synthesis by B . bacteriovorus. J Bacteriol, 1975 Mar, 121(3), 1117 - 21 Synthesis of nitrate reductase components in chlorate-resistant mutants of Escherichia coli; MacGregor CH; Specific antibody to purified nitrate reductase from Escherichia coli was used to identify enzyme components present in mutants which lack functional nitrate reductase . chlA and B mutants contained all three subunits present in the wild-type enzyme . Different peptides with a broad range of molecular weights could be precipitated from chlCmutants, and chlE mutants contained either slightly degraded enzyme subunits or no precipitable protein . No mutants produced significant amounts of cytoplasmic enzyme . The chlA and B loci are suggested to function in the synthesis and attachment of a molybdenum-containing factor . The chlC locus is suggested to be the structural gene for nitrate reductase subunit A and chlE is suggested to be involved in the synthesis of the cytochrome b1 apoprotein. J Bacteriol, 1975 Mar, 121(3), 1111 - 6 Anaerobic cytochrome b1 in Escherichia coli: association with and regulation of nitrate reductase; MacGregor CH; Nitrate reductase solubilized from the membrane of Escherichia coli by alkaline heat treatment was purified to homogeneity and used to prepare specific antibody . Nitrate reductase, precipitated by this antibody from Triton extracts of the membrane, contained a third subunit, not present in the purified enzyme used to prepare the antibody . This third subunit was identified as the cytochrome b1 apoprotein . This cytochrome is bound to nitrate reductase from wild-type E . coli in a ratio of 2 mol of cytochrome per mol of enzyme complex . In mutants unable to synthesize heme, this cytochrome b1 apoprotein is not bound to nitrate reductase . In these same mutants, the enzyme is overproduced and accumulates in the cytoplasm . The absence of cytochrome also affects the stability of the membrane-bound form of the enzyme. J Bacteriol, 1975 Mar, 121(3), 1102 - 10 Solubilization of Escherichia coli nitrate reductase by a membrane-bound protease; MacGregor CH; Nitrate reductase extracted from the membrane of Escherichia coli by alkaline heat treatment was purified to homogeneity and used to prepare specific antibody . Nitrate reductase, precipitated by this antibody from Triton extracts of the membrane, contained a third subunit not present in the purified enzyme used to prepare the antibody . Nitrate reductase precipitated by antibody from alkaline heat extracts was composed of peptide fragments of various sizes . These fragments were produced by a membrane-bound protease which was activated by alkaline pH and heat . It is the action of this protease that releases the enzyme from the membrane, as shown by the observations that protease inhibitors decreased the amount of solubilization of the enzyme, and the enzyme remaining in the membrane after heating showed much less proteolytic cleavage than that which was released. J Bacteriol, 1975 Mar, 121(3), 1092 - 101 Positive control in the D-serine deaminase system of Escherichia coli K-12; Bloom FR et al.; Two new types of D-serine deaminase (Dsdase)-negative mutants have been isolated and characterized . The first fails to synthesize a functional dsdC gene product as a result of dsdC- (regulator negative) mutations . The mutations lie in the dsdC region, are cis and trans recessive to dsdC+, and give rise to revertants of novel regulatory phenotype . The second class consists of Dsdase-negative lysogens in which the phenotype is the result of the integration of lambdac1857 Sam7 into the dsdC region . Lambda lysates derived from two of the Dsdase-negative lysogens can transduce the structural gene for Dsdase (dsdA) but not the dsdC region . The dsdC+ gene product had no repressor effect on constitutive synthesis in a strain containing a dsdO (initiator constitutive) and a dsdC- mutation . These and other findings indicate that control of Dsdase synthesis is strictly positive . The partial trans effect of the dsdC+ gene product on constitutive synthesis in dsdCc (regulator constitutive) strains can thus be explained by "subunit mixing" between active dsdCc subunits and dsdC+ subunits which are inactive in the absence of the inducer, D-serine . The order of genes in the dsd region is supN-dsdC-dsdP-dsdA-aroC. J Bacteriol, 1975 Mar, 121(3), 1078 - 84 Isolation and characterization of D-serine deaminase constitutive mutants by utilization of D-serine as sole carbon or nitrogen source; Bloom FR et al.; Mutants constitutive for D-serine deaminase (Dsdase) synthesis were isolated by utilizing D-serine as sole nitrogen or carbon source in the chemostat . This method generated only regulatory constitutive (dsdC) mutants . The altered dsdC gene product in these strains is apparently able to bind D-serine more efficiently than the wild-type dsdC+ gene product--a selective advantage . Constitutive synthesis of Dsdase in all of these dsdC mutants is extremely sensitive to catabolite repression, and catabolite repression is reversed by the addition of D-serine . Of the 15 mutants generated by this method, none are suppressible by supD, supE, or supF . Mutations to a low level of constitutivity (maximal specific activity of 9) occur much more frequently than mutations to a high level (maximal specific activity of 79) . High level constitutive synthesis of Dsdase results from the synthesis of an altered dsdC gene product--not from loss of ability to form the dsdC product . Dsdase synthesis is not regulated by the nitrogen supply in the medium, as nitrogen starvation does not result in the derepression of Dsdase synthesis. J Bacteriol, 1975 Mar, 121(3), 1074 - 7 Escherichia coli K-12 mutant forming a temperature-sensitive D-serine deaminase; McFall E; A single-site mutant of Escherichia coli K-12 able to grow in minimal medium in the presence of D-serine at 30 C but not at 42 C was isolated . The mutant forms a D-serine deaminase that is much more sensitive to thermal denaturation in vitro at temperatures above but not below 47 C than that of the wild type . No detectable enzyme is formed by the mutant at 42 C, however, and very little is formed at 37 C . The mutant enzyme is probably more sensitive to intracellular inactivation at high temperatures than the wild-type enzyme . The mutation lies in the dsdA region . The mutant also contains a dsdO mutation, which does not permit hyperinduction of D-serine deaminase synthesis. J Bacteriol, 1975 Mar, 121(3), 1047 - 55 Vinylglycolate resistance in Escherichia coli; Shaw L et al.; Escherichia coli K-12 vinylglycolate-resistant mutants have been isolated and characterized . Two of the mutants, JSH 150 and JSH 151, have been determined to be double mutants, lacking both membrane-bound L-and D-lactate dehydrogenases . The lactate transport system is intact in all strains; both radioactive lactate and vinylglycolate are actively taken up . Likewise, the phosphoenolypyruvate-dependent phosphotransferase system for hexose uptake is active . Vinylglycolate, previously shown to inhibit the phosphoenolpyruvate-dependent phosphotransferase system, has very little effect in the double mutants . The extent of vinylglycolate inhibition in other mutants seems directly related to the activity of the lactate dehydrogenases . This indicates that vinylglycolate is oxidized to 2-keto-3-butenoate before inactivating the phosphoenolpyruvate-dependent phosphotransferase system . These results were found in whole cells and confirmed in isolated membrane vesicles. J Bacteriol, 1975 Mar, 121(3), 1036 - 46 Mapping of the fabD locus for fatty acid biosynthesis in Escherichia coli; Semple KS et al.; fabD mutants of Escherichia coli contain a thermolabile malonyl-coenzyme A-acyl carrier protein transacylase which causes defective fatty acid synthesis and temperature-sensitive growth . By conjugation and P1 transduction the fabD locus has now been mapped at min 24, between pyrC and purB and close to cat . The order of sites is tentatively given as pyrC, cat, fabD, and purB, though the orientation of cat and fabD could be reversed . The possible relationship of fabD with another mutation lying in this region and also affecting acid synthesis is discussed . In the course of these studies we also confirmed the location of the fabA gene, determined that poaA lies between fabA and pyrC, and inadvertently found that the pyr mutation in strain AT3143 is probably pyrF and not pyrC. J Bacteriol, 1975 Mar, 121(3), 1007 - 13 F deoxyribonucleic acid superinfected into phenocopies of donor strains; Saitoh T et al.; When F+ donor cells of Escherichia coli are conjugated with F-, F+, or Hfr recipients under the conditions of phenocopy mating, the male recipients are found capable of accepting the F episome as effectively as the F- recipients . The F deoxyribonucleic acid (DNA) superinfected into the male recipients is converted to the covalently closed, circular duplex form, as in the F- recipients . It is also found that the synthesis of the strand complementary to the transferred single strand and its subsequent conversion to the covalently closed, circular duplex occur effectively in male recipients as well as in female recipients . Under these mating conditions, F-ilv+ episome superinfected to F+ and Hfr cells is diluted out during growth, whereas F-ilv+ transferred into F-cells is replicated and established in almost all progeny cells . These results suggest that the incompatibility of the F episome is not due to the reduction in the rate of the conversion of transferred single-straned F DNA to covalently closed, circular duplex, but, rather, to an inhibition of further replication of the covalently closed, circular F DNA. Infect Immun, 1975 Mar, 11(3), 493 - 6 Characterization of Escherichia coli obtained from newborn calves with diarrhea; Myers LL; A total of 373 isolates of Escherichia coli were obtained from one or more calves with diarrhea in 155 herds during the 1974 calving season in Montana . Sixty-seven (18 percent) of the isolates representing 59 of the 155 herds were found to be enterotoxigenic as indicated by their ability to cause distention of the calf ligated intestinal segment . The 67 isolates of enterotoxigenic E . coli (ETEC) were placed in one of six different antigenic groups based upon agglutination of formolized whole cells in KO antiserum . Eighty-seven percent (58 of 67) of the ETEC had antigen 1, 2, or 3, whereas only 2.3 per cent (7 of 310) of the non-ETEC (NETEC) had antigen 1, 2, or 3 . This antigen numbering system was used for convenience and is not related to any established typing system . Antigens 1, 2, and 3 do not belong to any of the O groups 1 to 157 or K groups 1 to 93 of the International Schema . Colony color or morphology of ETEC and NETEC grown on Tergitol-7 agar with triphenyltetrazolium chloride added could not be used as an indicator of enterotoxigenicity, although there was a tendency for E . coli with the smooth and mucoid colony type to be enterotoxigenic whereas rough colonies were seldom enterotoxigenic . Among ETEC isolates with antigen 1, 2, or 3, there was good correlation between colony type and antigen number . All 13 ETEC isolates with antigen 1 had the smooth colony type, 17 of 19 ETEC isolates with antigen 2 had the smooth, mucoid colony type, and all 25 isolates of ETEC with antigen 3 had the intermediate colony type . Conversely, of the 310 isolates of NETEC, none had antigen 1 and the smooth colony type; none had antigen 2 and the smooth and mucoid colony type; and only one isolate of NETEC had antigen 3 and the intermediate colony type . Sixteen of the 67 isolates of ETEC (24 percent) were motile . Fifteen of the 16 motile isolates of ETEC had the intermediate colony type, and none of the ETEC with smooth or smooth and mucoid colonies were motile. Infect Immun, 1975 Mar, 11(3), 441 - 4 Immunosuppression by hydroxystilbamidine isethionate, a lysosome-stabilizing, anti-proteolytic, antifungal drug; Folds JD et al.; Hydroxystilbamidine (HSB) is a potent suppressor of the plaque-forming cell response of mice injected with heterologous erythrocytes . HSB, given in varying doses and injection schedules, suppressed both the primary and secondary immune responses to bovine serum albumin . Apparently the effect is not simply a toxic effect on spleen cells, because there was no appreciable difference in cell numbers between control and HSB-treated mice . The effect of HSB was most apparent in the early phase of the immune respone. Appl Microbiol, 1975 Mar, 29(3), 430 - 1 Prophage induction of Escherichia coli (lambda) by N-nitrosamines; Thomson JA et al.; Carcinogenic N-nitrosamines were tested for their ability to induce lambda in a lysogenic strain of Escherichia coli K-12 (58-161 F+ Dimethylnitrosamine, di-n-propylnitrosamine, methyl-n-propylnitrosamine, and N-nitrosopiperidine were shown to be inducers of prophage. Surg Gynecol Obstet, 1975 Mar, 140(3), 371 - 6 Cholecystokinin cholangiography and analysis of duodenal bile in the investigation of pain in the right upper quadrant of the abdomen without gallstones; Freeman JB et al.; Thirty-one patients with recurrent symptoms of the biliary tract and repeated normal oral cholecystograms were studied by a combination of cholecystokinin cholangiography and biliary drainage . Ten patients had reduplication of their symptons because of dyskinetic contractions or obstruction of the cystic duct, and seven patients had delayed gallbladder emptying without pain due to hypokinetic contractions . Five patients had abnormal duodenal bile characterized by supersaturation and the presence of crystals or bacteria . Based upon these studies, 22 patients had cholecystectomy and 20 were cured, while two showed improvement . There were no therapeutic failures . Cholecystokinin cholangiography capably detects the presence of neuromuscular disease of the gallbladder wall, whereas the oral cholecystogram tests for mucosal function or the presence of filling defects . An additional group of patients who have cholesterosis, cholecystitis, or cholelithiasis missed by the oral cholecystogram will not be diagnosed by cholecystokinin cholangiography unless the duodenal bile is also examined. Surg Gynecol Obstet, 1975 Mar, 140(3), 365 - 7 Septic complications following endoscopic retrograde cholangiopancreatography; Davis JL et al.; In four patients, septic complications developed following endoscopic retrograde cholangiopancreatography which required surgical intervention . The pathogenesis involves stasis in the pancreatic or biliary tree, but the source of infection is unclear . Prompt recognition and early surgical intervention should decrease the seriousness of these complications. J Immunol, 1975 Mar, 114(3), 950 - 7 Receptor sites for antigen-antibody complexes on cells derived from solid tumors: detection by means of antibody sensitized sheep erythrocytes labeled with technetium-99m; Wood GW et al.; Surface receptor sites for the Fc portion of antigen-antibody complexes were demonstrated on cells derived from three methylcholanthrene-induced fibrosarcomas, one of strain C3H and two of strain BALB/c origin, two spontaneously occurring malignant melanomas (B16 in strain C57BL/6 and Harding-Passey in strain BALB/c mice), a Moloney sarcoma virus-induced tumor of strain BALB/c origin and the Walker 256 carcinosarcoma of Holtzman rats . Primary cell cultures derived from these tumors adsorbed technetium-99m labeled, antibody-sensitized sheep erythrocytes (99mTc EA) as determined either by visual scoring of adherence or radioisotopic quantitation . Depending upon the tumor tested, from 20% to greater than 95% of the target cells absorbed 99mTc EA . All cells lost their reactivity after 1 or 2 passages in vitro, but this was regained after a single passage in vivo . Indicator erythrocytes coated with F(ab')2 fragments of the sensitizing sheep erythrocytes (SRBC) antiserum did not adhere thereby demonstrating that the hemadsorption required an intact Fc portion of the antibody molecule . Adherence of 99mTc EA was blocked by soluble immune complexes prepared with ovalbumin and rabbit antibody directed against it and Escherichia coli 055:B5 lipopolysaccharide and mouse antibody directed against it . Normal rabbit or mouse serum, immune serum, or antigen alone did not block adherence of 99mTc EA thereby demonstrating that the receptors had greater affinity for immune complexes than for either antigen or antibody alone . The existence of membrane receptors on tumor-derived cells which react with the Fc portion of antigen-antibody complexes may provide an explanation for the mechanism by which immune complexes are capable of blocking cell-mediated tumor cell destruction irrespective of whether the receptors are on the tumor cells themselves or on admixed lymphocytes and macrophages. Invest Urol, 1975 Mar, 12(5), 337 - 45 Experimental lipid A-induced nephritis in the dog; Westenfelder M et al.; The effect of radioactive lipid A, obtained from Escherichia coli, on the kidney of adult dogs and puppies was investigated . Injection of lipid A into the temporarily occluded renal pelvis of adult dogs caused abacterial interstitial nephritis in all animals tested . The intensity and duration of the kidney response coorelated well with the lipid A dose administered . Lipid A was demonstrated autoradiographically in the renal cortex, extra- and intracellularly . Radioactivity was still present after 10 weeks in 16 of 20 examined dogs.In the remaining four dogs the interstitial nephritis had subsided . Thirteen of 14 puppies in which the immunologic defense mechanisms had not yet developed showed no histologic reaction in the kidney after the same procedure as in the adult dogs . Lipid A appeared in the renal parenchyma through the blood stream rather than through the retrograde route . Lipid A antibody titers could be detected only in adult dogs, never in puppies . Lipid A apparently provoked an immunologic process in the kidney damaged by the operative manipulations, thus resulting in an abacterial interstitial nephritis . The possibility that chronic abacterial pyelonephritis can be induced clinically through lipid A and thus may have a pathogenesis similar to abacterial interstitial nephritis is discussed. Proc Natl Acad Sci U S A, 1975 Mar, 72(3), 864 - 8 Sequence of 71 nucleotides at the 3'-end of tobacco mosaic virus RNA; Guilley H et al.; The sequence of the first 71 nucleotides from the 3'-OH end of tobacco mosaic virus RNA has been determined . After total T1 ribonuclease digestion of the viral RNA, the oligonucleotide C-C-C-A-OH, which originates from the 3'-OH terminus of the RNA, may be readily detected by electrophoresis at pH 2.5 or pH 3.0; it is the only oligonucleotide that migrates toward the cathode at these pHs . This property has been used to screen the purified products of partial T1 ribonuclease digestion of tobacco mosaic virus RNA for the fragment originating from the 3'-end of the native molecule . The sequence of nucleotides in the 3'-terminal fragment, identified in this manner, was determined by radiochemical methods . The fragment contained 71 nucleotides; no abnormal bases could be detected . Although it has been reported that the 3'-end of tobacco mosaic virus RNA is a substrate for aminoacylation by the histidyl-tRNA synthetase of yeast or Escherichia coli, we were unable to fold the sequence into the cloverleaf structure characteristic of tRNAs. Antibiotiki, 1975 Mar, 20(3), 243 - 8 {Actinomycin DO inhibition of RNA biosynthesis and its interaction with DNA preparations}; Devan ML et al.; Actinomycin D0 differing from actinomycin D in replacement of I residue of sarcosine by glycine in one of the peptide chains was found to have a lower inhibitory effect on RNA synthesis in various biological systems . On interaction of E . coli with DNA actinomycin D0 formed a complex with DNA having the binding constant K=1.6 with 10-5 which was 100 times lower than the binding constant of actinomycin D . Therefore, actinomycin D0 formed a complex with DNA which was 100 times less stable than actinomycin D . Actinomycins D0, D and II having different conformations in buffer on interaction with DNA formed complexes having similar conformations . The conformational change from free actinomycin to bound actinomycin was found difficult in case of actinomycin D0 because of its slow rate of complex formation with DNA as compared to that of other actinomycins complex formation with DNA. Biophys J, 1975 Mar, 15(3), 223 - 32 Viscoelastic characterization of single-stranded DNA from Escherichia coli; Uhlenhopp EL et al.; Single-stranded DNA released from E . coli wild type and mutant cells by alkaline-EDTA-detergent was analyzed using the recently developed biophysical technique of viscoelastometry . Under the lysis conditions used, it was possible to detect single strands of molecular weight approximately 2 times 10-9 daltons . Little difference was detected in the size of single-stranded DNA from log phase vs . stationary phase cultures, or from cells treated with chloramphenicol to allow completion of replicating chromosomes . The largest single strands from ligase overproducing, endonuclease minus, and pol A1 mutants were likewise of approximately the same size as wild type, but were present in smaller yields . The reduction in single-strand molecular weight as a result of heating intact cells was investigated as a function of time and temperature . Heating at 37 degrees C for up to 20 min produced no additional single-strand breaks, but temperatures from 45 to 65 degrees introduced breaks . Solutions maintained at pH 12.5 were not stable indefinitely, and the relative viscosity of such solutions was found to decrease over a period of several hours. Am J Surg, 1975 Mar, 129(3), 266 - 8 Emergency subclavian vein catheterization and intravenous hyperalimentation; Merk EA et al.; One hundred consecutive subclavian catheter insertions were performed by the surgical house staff of Martland Hospital, Newark, New Jersey, over a ten month period . The only complications were three punctures of the subclavian artery and one systemic infection . The following conclusions were drawn from these data . Maintaining a closed intravenous system with minimal manipulation of the catheter is the most important factor in avoiding infectious complication . Neither the routine use of irrigation of the catheter with amphotericin B nor insertion of the catheter under strict aseptic conditions is necessary to minimize infectious complications . The morbidity related to insertion of the catheter can be kept to a minimum if the catheters are inserted by experienced personnel. Appl Microbiol, 1975 Mar, 29(3), 405 - 13 Two-dimensional polyacylamide gel electrophoresis of envelope proteins of Escherichia coli; Johnson WC et al.; A method of separating envelope proteins by two-dimensional polyacrylamide gel electrophoresis is described . Escherichia coli envelopes (inner and outer membranes) were prepared by French pressing and washed by repeated centrifugation . Membrane proteins were solubilized with guanidine thiocyanate and were dialyzed against urea prior to two-dimensional electrophoretic analysis . The slab gel apparatus and conditions were similar to the technique developed by Metz and Bogorad (1974) for the separation of ribosomal proteins . This separation occurs in 8 M urea for the first dimension and in 0.2% sodium dodecyl sulfate for the second dimension . The technique separates about 70 different membrane proteins in a highly reproducible fashion according to both intrinsic charge and molecular weight . Some examples of alterations in the membrane protein pattern are demonstrated . These alterations are caused by a mutation affecting a sugar transport system and by growth in the presence of D-fucose, inducer of the transport system . A further example of membrane protein changes introduced by growth at the nonpermissive temperature of a temperature-sensitive cell division mutant is shown . Finally, it is demonstrated that the major outer membrane component of Escherichia coli K-12 contains more than four proteins of similar molecular weight. Ann Sclavo, 1975 Mar-Apr, 17(2), 140 - 5 {1st experiences on the protective effect of Limulus polyphemus amebocyte lysate in the endotoxic shock in the rat}; Fumarola D et al.; The injection in the peritoneum of Limulus amebocyte lysate at the same time of inoculation of lethal dose of E . coli LPS is able to protect the rats against endotoxin lethality. Biomedicine, 1975 Mar, 22(2), 122 - 7 The effect of prednisone on the non-stimulated and the stimulated NBT test; Wantzin GL; The non-stimulated and stimulated nitroblue tetrazolium (NBT) dye test was studied in five normal adults in sequence, viz . before and at intervals after prednisone was given as one large dose . A negative effect of prednisone on the NBT response was seen . This applied to both the non-stimulated and stimulated NBT test . For the latter five different toxins were used . At the same time a pronounced neutrophil granulocytosis was seen . The depression of the NBT test is present both in the percentage of NBT-stained neutrophils and when expressed in absolute counts of NBT-strained neutrophils. J Bioenerg, 1975 Mar, 7(1), 17 - 38 Conversion of Escherichia coli cell-produced metabolic energy into electric form; Griniuviene B et al.; The formation of membrane potential in energized E . coli cells has been investigated by means of ionic penetrants . The fluxes of anions and cations in opposite directions have been observed: anions moved out and cations moved into the cells . The energy-linked uptake of cations was stoichiometrically coupled with the outflow of H+ ions from the cells . The value of a membrane potential in the energized cells calculated from a distribution of permanent cations was in the range of -140 mV (inside minus) . The uptake of penetrating cations by deenergized cells has been observed following the non-enzymatic generation of a membrane potential . The influx of synthetic and natural (lactose) penetrants collapsed the non-enzymatic membrane potential . The effect of lactose was sensitive to N-ethyl maleimide . These results are in favour of the conception that in the energized E . coli cells an energy-linked H+-pump generates a membrane potential which is a driving force for the transport of synthetic and some natural penetrants. Cell Differ, 1975 Mar, 3(6), 335 - 45 In situ studies on the effect of acid extraction on the DNA template activity of mature avian erythrocytes; Klass MR; Exogenous E . coli RNA polymerase was used to determine the in situ DNA template activity of ethanol/acetone fixed avian erythrocytes . No RNA polymerase-catalyzed incorporation of 3H-UTP was detected in mature avian erythrocytes while simultaneously fixed avian lymphocytes did exhibit incorporation of 3H-UTP . Nuclei of mature erythrocytes which were subjected to treatments known to remove histones showed dramatic increases in RNA polymerase-catalyzed incorporation of 3H-UTP . The chromatin of treated cells was presumed to be more accessible to RNA polymerase as determined by the increase in RNA polymerase-catalyzed incorporation of 3H-UTP . Incubation of acid-treated nuclei in poly-L-lysine prior to incubation with RNA polymerase failed to inhibit the incorporation of 3H-UTP . Possible mechanisms for the inactivation of avian erythrocyte nuclei are discussed. J Biochem (Tokyo), 1975 Mar, 77(3), 567 - 73 Effect of cyclic nucleotides on the cyanide-insensitive respiration of polymorphonuclear leucocytes; Nakagawara A et al.; The effects of N6,O2'-dibutyryladenosine 3',5'-monophosphate (Bu2-cyclic AMP) and N2,O2'-dibutyrylguanosine 3',5'-monophosphate (Bu2-cyclic GMP) on the cyanide-insensitive respiration of guinea pig peritoneal exudate polymorphonuclear leucocytes were studied . Bu2-cyclic AMP inhibited the respiration induced both by phagocytosis of E . coli and by the interaction with trypsin-digested rat liver microsomes . The addition of theophylline gave rise to an inhibitory pattern similar to that with Bu2-cyclic AMP against both the respirations induced . On the other hand, Bu2-cyclic GMP did not affect the respiration induced by phagocytosis whereas it inhibited the respiration induced by the addition of myristic acid was inhibited by Bu2-cyclic AMP, which was similar to that with E . coli . The respiration induced by methylene blue was inhibited neither by Bu2-cyclic AMP nor by Bu2-cyclic GMP . These observations suggest that the cyanide-insensitive respiration of polymorphonuclear leucocytes may be classified into at least three types from the inhibitory pattern of cyclic AMP and cyclic GMP. Antibiotiki, 1975 Mar, 20(3), 239 - 43 {Determination of penicillinase and acylase by chromatography on a thin layer of sorbent in the case of their joint formation by different strains}; Tochenaia NP et al.; The products of penicillinase and acylase hydrolysis of benzylpenicillin were studied with a method of sorbent thin-layer chromatography . The method provided qualitative determination and differentiation of penicillinase and acylase activity in cultures of E . coli capable of simultaneous production of both enzymes . It was shown that when the cultures of E . coli were grown under conditions optimal for acylase production, the amounts of penicillinase were insignificant. Proc Natl Acad Sci U S A, 1975 Mar, 72(3), 1171 - 4 Stimulation of DNA polymerase by factors isolated from Novikoff hepatoma; Probst GS et al.; Extracts of Novikoff hepatoma cells contain factors capable of stimulating in vitro DNA synthesis several fold . The activity can be resolved into three separate protein peaks on DEAE-Sephadex . Two of these, factors II and III, have been purified and partially characterized . Both factors increase the initial rate of DNA synthesis and allow synthesis to proceed much longer . If either factor is added after synthesis by the DNA polymerase has reached a plateau, resumption of synthesis occurs . The factors appear to have different modes of action or sites of action since they show an additive effect even when a single one is used at saturating conditions . These factors are present in normal rat liver but at a concentration less than 5% of that found in the tumor cells . When tested with several highly purified DNA polymerases (DNA nucleotidyltransferase, EC 2.7.7.7), the factors show a much greater stimulation of homologous, non-mitochondrial enzymes (rat liver nuclear-, rat liver cytoplasmic-, or Novikoff-DNA polymerases) when compared with rat liver or calf liver mitochondrial-, Escherichia coli I-, or sea urchin nuclear-DNA polymerases . The mechanism of action of these factors is not known at present . No enzymatic activity has been associated with factor III . Highly purified, but not homogeneous, preparations of factor II contain low levels of endonuclease; it has not been established whether endonuclease is a contaminant or is responsible for the stimulating activity. J Bacteriol, 1975 Mar, 121(3), 907 - 16 Kinectics of beta-galactosidase synthesis in Escherichia coli at 5 C; Anderson WA; The defect in protein synthesis that is observed in Escherichia coli after transfer to low temperature was studied . For the enzyme beta-galactosidase, the elongation reactions of transcription and translation can take place slowly but normally at 5 C . The time necessary to complete the coupled synthesis of the beta-galactosidase messenger ribonucleic acid and polypeptide chain was found to be about 80 min at 5 C . From this result and from the known length of the beta-galactosidase monomer, it is possible to calculate that at 5 C one amino acid is added to the growing polypeptide chain every 4 s . The initiation of transcription of the beta-galactosidase messenger is inhibited after transfer to 5 C . This fact alone, however, cannot account for all of the phenomena observed at 5 C, because a given amount of messenger yields less enzyme at 5 C than it does at 37 C . Furthermore, in cells induced for short periods at 37 C, the capacity to synthesize beta-galactosidase after transfer to 5 C was found to accumulate linearily with the square of the time of induction . Two alternative models could account for these data . If all ribosomes that initiate translation at 37 C yield complete beta-galactosidase polypeptide chains at 5 C, then an inhibition of translation initiation after transfer to 5 C must be invoked to explain the results . If, on the other hand, a substantial portion of the ribosomes that initiate translation at 37 C do not yield complete beta-galactosidase polypeptides at 5 C, then intracistronic polarity could account for the data, and there is no need to invoke an inhibition of translation initiation at 5 C. J Bacteriol, 1975 Mar, 121(3), 1203 - 7 Effect of tsl mutations in decreasing radiation sensitivity of a recA- strain of Escherichia coli K-12; Mount DW et al.; It has been shown previously that the radiation sensitivity of lexA- strains of Escherichia coli K-12 can be suppressed by thermosensitive mutations (designated tsl) that are closely linked to the lexA locus and are thought to be intragenic suppressors of lexA mutations (Mount et al., 1973) . When a recA mutation is crossed into a suppressed tsl- strain, the extreme radiation sensitivity usually conferred by a recA mutation is decreased, but there is no detectable change in genetic recombination deficiency . Increased resistance to UV in the tsl-reA-strains depends upon ability to synthesize active uvrA+ product. J Bacteriol, 1975 Mar, 121(3), 1085 - 91 Isolation and characterization of catabolite-resistant mutants in the D-serine deaminase system of Escherichia coli K-12; Bloom FR; Two classes of D-serine deaminase (Dsdase)-specific secondary mutants of Escherichia coli K-12 were isolated from a Dsdase low constitutive nonhyperinducible mutant as types which could grow in the presence of both D-serine and glucose . These strains contain cis dominant, nonsuppressible mutations in the dsdO (operator-initiator) region . In the first class of mutants (e.g., FB4010), Dsdase synthesis is completely insensitive to catabolite repression, and synthesis occurs at a high constitutive rate in the absence of cyclic adenosine 5'-monophosphate . In the second class (e.g., FB4005), Dsdase synthesis is partially insensitive to catabolite repression, and catabolite repression is reversed by the addition of cyclic adenosine 5'-monophosphate . Dsdase synthesis in strain FB4005 is partially independent of the cyclic adenosine 5'-monophosphate binding protein, as constitutive synthesis is reduced only 65% (relative to the cap+ strain) in strains unable to synthesize the cyclic adenosine 5'-monophosphate binding protein . Surprisingly, the constitutive rate of Dsdase synthesis is fourfold higher in all mutants of both classes than in the parent, indicating a close interrelationship between the sites of response to induction and catabolite repression. Infect Immun, 1975 Mar, 11(3), 429 - 35 Escherichia coli enterotoxin: stimulation of adenylate cyclase in broken-cell preparations; Dorner F et al.; The enterotoxin from cell-free filtrates of the enteropathogenic Escherichia coli strain P-263 was found to stimulate adenylate cyclase activity in broken-cell preparations from myocardial tissue . Particulate and detergent-solubilized fractions from cat heart were incubated with enterotoxin and assayed for adenylate cyclase activity . Adenylate cyclase activity was stimulated by enterotoxin; the extent of stimulation was proportional to the concentration of enterotoxin . The data demonstrate that stimulation of enterotoxin-sensitive adenylate cyclase in this system provides a sensitive in vitro assay, either as an accurate measure of enterotoxin concentration or as an assay for antitoxic titers in sera . A parallel comparison showed that stimulation of fluid production in rabbit intestinal loops by enterotoxin was less sensitive. Clin Chem, 1975 Mar, 21(3), 420 - 4 Mechanized enzymatic determination of triglycerides in serum; Bucolo G et al.; A procedure for enzymatic determination of serum triglycerides {Clin . Chem . 19, 476 (1973)} has been adapted for use in continuous-flow analysis (Technicon AutoAnalyzer) . A very simple manifold is used; serum is incubated at 37 degrees C with the lipase and alpha-chymotrypsin in potassium phosphate buffer (0.1 mol/liter, pH 7, containing 1.50 g of bovine serum albumin per liter) . The liberated glycerol is dialyzed against the complete glycerol reagent . The change in absorbance at 340 nm resulting from oxidation of NADH is proportional to the dialyzed glycerol . The same manifold can be used to determine preformed glycerol if the hydrolyzing enzymes are omitted . The hydrolysis is complete, as shown by the use of equivalent glycerol standards . No prior treatment of the samples is necessary . Assays are run at 60 per hour in the AutoAnalyzer l, 80 per hour in the AutoAnalyzer ll . Results with both instruments for 150 samples correlated well with those obtained by the same enzymatic manual method and by the AutoAnalyzer fluorometric procedure. Ann N Y Acad Sci, 1975 Feb 28, 249, 505 - 22 The nature and function of T-cell antigens; Schlesinger M et al.; T-lymphocytes differ antigenically from B-lymphocytes . In the present study attempts were made to determine the role of surface antigens of T-cells, in their migration in vivo and in their response to mitogens . Exposure of thymus cells to anti-H2 sera inhibits migration to the lymph nodes (LN) to a greater extent than to the spleen . Fab fragments of H-2 antisera had only a slight effect on lymphocyte migration, inhibiting the LN-seeking stream only slightly more than the spleen-seeking stream . The interaction of Con-A with carbohydrates on the cell-surface of lymphocytes inhibits preferentially their localization in LN . Studies on the migration of lymphocytes that had localized either in the LN or spleens of primary host indicate that Con-A does not eliminate LN-seeking cells, but rather inhibits their active localization in LN . The subpopulation of lymphocytes, in both thymus and spleen that responds to Con-A was found to possess a higher H-2 antigenicity and a lower Ly and theta-antigenicity than the cells responding to PHA . Spleen cells responding to low concentrations of PHA had a relatively high H-2 antigenicity, whereas thos responding to high concentrations of PHA had a low H-2 antigenicity . Exposure of thymus cells to H-2 antiserum alone markedly inhibited their response to Con-A . Similar treatment of spleen cells had only a weak inhibitory effect. Ann N Y Acad Sci, 1975 Feb 28, 249, 290 - 9 Antigenic and functional evidence for the in vitro inductive activity of thymopoietin (thymin) on thymocyte precursors; Basch RS et al.; Thymopoietin, a polypeptide hormone isolated from bovine thymus, induced in vitro the differentiation of prothymocytes to cells with the antigenic and functional characteristics of intrathymic thymocytes . These changes included the acquisition of the differentiation antigens TL and Thy-1 (theta) and the ability to respond to the mitogen Con-A . Thymopoietin appears to be highly speicfic in inducing the prothymocyte to be highly specific in inducing the prothymocyte to thymocyte differentiation and does not affect the pluripotential stem cell measured by the colony forming assay (CFU-S), the erythropoietin-sensitive cell or B-cells . Experiments are in progress to determine whether additional hormonal inductive signals are required to complete the differentiation of an immunologically competent T-cell. Ann N Y Acad Sci, 1975 Feb 28, 249, 154 - 65 Examination of lymphocyte membranes of athymic "nude" mice by scanning electron microscopy; Thurman GB et al.; Recent reports 1-3 have proposed that T-lymphocytes (thymus derived) could be distinguished from B-lymphocytes (thymus independent) by examining their features under the scanning electron microscope . T-cells were designated as having relatively smooth surfaced cells, whereas B-cells had "hairy" surfaces with many microvilli . We have examined this hypothesis in congenitally athymic "nude" mice, animals lacking T-cells,4 and have not been able to confirm these reports . We have found that lymphocytes from nude (nu/nu) mice are indistinguishable from lymphocytes obtained from normal littermates (NLM) and CBA/J mice . We have found that in all the murine lymphoid tissue examined, including nude, the complete gamut of cell surface types, ranging from smooth to "hairy" are present . Our studies indicate that the proposed T- and B-cell classification based upon human surface morphology under the scanning electron microscope is questionable in a mouse model . It is presently unclear whether the smooth lymphocytes seen in nude mice are immature B-cells, pre-T-cells, or another unidentified population of cells. Ann N Y Acad Sci, 1975 Feb 28, 249, 145 - 53 Thymosin-induced differentiation of murine thymocytes in allogeneic mixed lymphocyte cultures; Cohen GH et al.; Calf thymosin is shown to enhance the one-way MLR of CBA thymocytes cultured with allogeneic mitomycin-C- treated C57BL/J6 spleen cells . Thymosin does not enhance the one-way MLR of CBA thymocytes cultured with syngeneic mitomucin-C-treated spleen cells . Based on this finding we present a relatively simple, rapid and quantitative in vitro microculture hioassay for inducers of T-cell differentiation and propose that thymosin treatment, when accompanied by antigen presentation, induces the two-step maturational sequence of pre-T yields T1 yields T2. Biochim Biophys Acta, 1975 Feb 27, 379(2), 418 - 25 The purification and properties of superoxide dismutase from a blue-green alga; Misra HP et al.; Soluble extracts of Plectonema boryanum have been shown to contain a single, electrophoretically distinct, superoxide dismutase . The enzyme has been isolated and has been found to be an iron-containing enzyme similar to that described from the periplasm of Escherichia coli . It contains 1 Fe3+/mole of enzyme . The molecular weight was approximately 36 500, and the enzyme appeared to be composed of two subunits of equal size joined by non-covalent interactions . ESR data are presented, as are the results of amino acid analysis. J Biol Chem, 1975 Feb 25, 250(4), 1218 - 22 Chemical modification of amino acid residues associated with the delta-4-3-ketosteroid-dependent photoinactivation of delta-5-3-ketosteroid isomerase; Martyr RJ et al.; We have studied the accumulation of dibenzyldimethyl-ammonium ion (DDA+) by respiring membrane vesicles of Escherichia coli, as an index of the generation of an electrical gradient during respiration . Nonrespiring vesicles accumulated DDA+ when K+ efflux was induced by valinomycin or monactin . By various criteria this was shown to be the exchange of one cation for another, independent of metabolism and coupled entirely by electrical forces . Uptake of DDA+ by respiring vesicles was inhibited by ionophores that translocate electrical charge and by reagents that block the respiratory chain . Oxamate and p-chloromercuribenzoate inhibited accumulation of DDA+ but did not dissipate a preformed pool; the reason appears to be that these reagents are less inhibitory to transport after lactate oxidation has begun than they are in resting vesicles . Uptake does not appear to involve a biological carrier, but requires trace amounts of a lipid-soluble anion such as tetraphenylboron, which has a catalytic role in DDA+ translocation . Respiring K+ vesicles accumulated substantially less DDA+ than did Na+ vesicles . Na+ was expelled from the vesicles concurrently with DDA+ uptake, whereas Rb+ and K+ were not . Thus, DDA+ uptake may be limited in the latter case by the availability of anionic groups . This explanation was supported by the finding that the addition of nigericin doubled the capacity of K+ vesicles to take up DDA+, presumably by providing a route for K+ to exit in exchange for H+ . Parallel experiments on the valinomycin-dependent accumulation of Rb+ by respiring vesicles indicate that this process is analogous to the uptake of DDA+ . Ionophores that elicit electrogenic K+ movement also induced respiration-linked transport . Proton-conducting ionophores and several inhibitors of respiration blocked Rb+ uptake and dissipated a preformed gradient . Preincubation of the vesicles with oxamate or p-chloromrecuribenzoate inhibited Rb+ uptake, but their addition to respiring vesicles again did not cause efflux . Rb+ and DDA+ be, but their addition to respiring vesicles again did not cause efflux . Rb+ and DDA+ compete for uptake when present simultaneously . We conclude that the accumulation of both DDA+ and Rb+ occurs in response to an electrical gradient, vesicle interior negative, produced by respiration. Biochemistry, 1975 Feb 25, 14(4), 817 - 24 Stepwise enzymatic oligoribonucleotide synthesis including modified nucleotides; Walker GC et al.; A method has been developed for the routine synthesis of 2'(3')-o-monoacyl ribonucleoside 5'-diphosphates for stepwise synthesis of oligoribonucleotides with Escherichia coli polynucleotide phosphorylase . The use of triethyl orthoisovalerate allows the facile preparation of 2'(3')-o-isovaleryl-UDP, -CDP, -ADP, -GDP, -IDP, -EPLISON-APD, eplison-CDP, and N6-isopentenyl-ADP . The synthesis of N6-isopentenyl-ADP from ADP by N1-alkylation and the Dimroth rearrangement to N6 is reported . The effects of several factors including the nature of the divalent cation, pH, SALT CONCENTRATION, AND TIME ON THE EFFICIENCY OF THE POLYNUCLEOTIDE PHPSPHORYLASE CATALYZED SINGLE ADDITIONS OF THE 2'(3')-O-ISOVALERYL RIBONUCLEOSIDE 5'-DIPHOSPHATES TO AN OLIGORIBONUCLEOTIDE PRIMER ARE REPORTED . The syntheses of many tetranucleoside triphosphates and two pentanucleoside tetraphosphates in yields of 20-75 per cent are reported . The 2'(3')-o-isovaleryl derivatives of IDP, eplison-ADP, eplison-CDP, and N6-isopentenyl-ADP were all accepted by polynucleotide phosphorylase as substrates for the monoaddition reaction . The extension of the method to include the syntheses of oligoribonucleotides containing modified nucleosides offers a means of studying the role s of these modification by the use of relatively simple model compounds. Biochemistry, 1975 Feb 25, 14(4), 782 - 9 DNA-protein interactions of the rat liver non-histone chromosomal protein; Sevall JS et al.; Native rat liver NHC protein-DNA interactions have been investigated by use of a nitrocellulose filter assay sensitive in detection of protein-DNA complexes . Optimal conditions for DNA-protein interactions occurs at low ionic strength conditions (110 mM phosphate buffer) . A fraction of NHC proteins was enriched 25-fold by their affinity for rat DNA immobilized on cellulose columns under these conditions . At higher ionic strength (260 mM-0.04M phosphate buffer and 0.15 M sodium chloride), this fraction binds approximately sevenfold less to rat DNA but with a substantial increase in stability of the complexes . Equilibrium competition experiments indicate that at the higher ionic strength there is a considerable DNA sequence specificity of the rat DNA binding NHC protein . Since rat DNA contains three components as defined by their reassociation kinetics: single copy DNA (C0t1/2pure = 1.6 times 103); middle repetitive DNA (C0t1?1PURE = 1.1); and highly repetitive (C0t1/2pure smaller than 0.02) . The two former were isolated and employed in the DNA binding assays . At the high ionic strength criterion, the rat DNA binding NHC proteins showed a substantial preference for a subset of middle repetitive DNA sequences . This suggests a preferential interaction between a class of NHC proteins and a class of middle repetitive DNA sequences. J Biol Chem, 1975 Feb 25, 250(4), 1556 - 62 Purification and properties of a soluble factor required for the deoxyribonucleic acid-directed in vitro synthesis of beta-galactosidase; Kung H et al.; The DNA-directed in vitro synthesis of beta-galactosidase has been investigated in a system dependent on Escherichia coli ribosomes, a salt wash of the ribosomes, and a supernatant fraction . Fractionation of the supernatant has made it possible to obtain dependencies on RNA polymerase and another protein factor for beta-galactosidase synthesis . The other factor (called L factor) cannot be replaced by a variety of proteins known to be required for transcription and translation . It has been purified to homogeneity and has a molecular weight of approximately 65,000 . Although it is required for the in vitro synthesis of beta-galactosidase, it has no effect on total DNA-dependent amino acid incorporation under the conditions of the incubation . However, total RNA synthesis is depressed by the addition of L factor in a manner similar to what is observed with rho factor could not replace L factor in beta-galactosidase synthesis. J Biol Chem, 1975 Feb 25, 250(4), 1451 - 9 Partial purification and properties of an endoribonuclease isolated from human KB cells; Bothwell AL et al.; With the use of a precursor to Escherichia coli tRNA-Tyr as a substrate, we have detected and partially purified a novel endoribonuclease from the cytoplasm of human KB tissue culture cells . This activity, which we have called RNase NU, cleaves the tRNA precursor at two sites in that part of the molecule which is not included in the mature tRNA sequence and which is normally degraded in vivo . In keeping with this observation, we have found that, of a variety of substrates tested, only those which are unstable in vivo are attacked by RNase NU . RNase NU can be purified from the 0.2 M NH4Cl wash of ribosomes followed by ammonium sulfate fractionation and DEAE-Sephadex chromatography . RNase NU cleaves RNA to create 3'-phosphate-terminated oligonucleotides . It has a pH optimum near 8.0, requires either a monovalent cation (NH4+ is most efficient) or Ca-2+ for optimal activity, and is inhibited by 0.1 M PO4-3- . In the course of purifying RNase NU we have detected and studied the intracellular distribution of other ribonuclease activities in human KB cells. J Biol Chem, 1975 Feb 25, 250(4), 1405 - 12 Accumulation of lipid-soluble ions and of rubidium as indicators of the electrical potential in membrane vesicles of Escherichia coli; Altendorf K et al.; We have studied the accumulation of dibenzyldimethyl-ammonium ion (DDA+) by respiring membrane vesicles of Escherichia coli, as an index of the generation of an electrical gradient during respiration . Nonrespiring vesicles accumulated DDA+ when K+ efflux was induced by valinomycin or monactin . By various criteria this was shown to be the exchange of one cation for another, independent of metabolism and coupled entirely by electrical forces . Uptake of DDA+ by respiring vesicles was inhibited by ionophores that translocate electrical charge and by reagents that block the respiratory chain . Oxamate and p-chloromercuribenzoate inhibited accumulation of DDA+ but did not dissipate a preformed pool; the reason appears to be that these reagents are less inhibitory to transport after lactate oxidation has begun than they are in resting vesicles . Uptake does not appear to involve a biological carrier, but requires trace amounts of a lipid-soluble anion such as tetraphenylboron, which has a catalytic role in DDA+ translocation . Respiring K+ vesicles accumulated substantially less DDA+ than did Na+ vesicles . Na+ was expelled from the vesicles concurrently with DDA+ uptake, whereas Rb+ and K+ were not . Thus, DDA+ uptake, whereas Rb+ and K+ were not . Thus, DDA+ uptake may be limited in the latter case by the availability of anionic groups . This explanation was supported by the finding that the addition of nigericin doubled the capacity of K+ vesicles to take up DDA+, presumably by providing a route for K+ to exit in exchange for H+ . Parallel experiments on the valinomycin-dependent accumulation of Rb+ by respiring vesicles indicate that this process is analogous to the uptake of DDA+ . Ionophores that elicit electrogenic K+ movement also induced respiration-linked transport . Proton-conducting ionophores and several inhibitors of respiration block Rb+ uptake and dissipated a preformed gradient . Preincubation of the vesicles with oxamate or p-chloromercuribenzoate inhibited Rb+ uptake, but their addition to respiring vesicles again did not cause efflux . Rb+ and DDA+ complete for uptake when present simultaneously . We conclude that the accumulation of both DDA+ and Rb+ occurs in response to an electrical gradient, vesicle interior negative, produced by respiration. J Biol Chem, 1975 Feb 25, 250(4), 1371 - 5 Photoinactivation of the beta-galactoside transport system in Escherichia coli membrane vesicles with 2-nitro-4-azidophenyl-1-thio-beta-D-galactopyranoside; Rudnick G et al.; 2-Nitro-4-azidophenyl-1-thio-beta-D-galactopyranoside (azidophenylgalactoside) is a competitive inhibitor of lactose transport in membrane vesicles isolated from Escherichia coli ML 308-225, exhibiting an apparent Ki of 75 muM . The initial rate and steady state level of {3H}azidophenylgalactoside accumulation are markedly stimulated by the addition of D-lactate to vesicles containing the lac transport system, and kinetic studies reveal an apparent Km of 75 muM . Membrane vesicles devoid of the lac transport system do not take up significant amounts of azidophenylgalactoside in the presence or absence of D-lactate . When exposed to visible light in the presence of D-lactate, azidophenylgalactoside irreversibly inactivates the lac transport system . Strikingly, photolytic inactivation is not observed in the absence of D-lactate . Kinetic studies of the inactivation process yield a KD of 77 muM . Since lactose protects against inactivation and azidophenylgalactoside does not inactivate amino acid transport, it is apparent that these effects are specific for the lac transport system . The results are consistent with the proposal that the lac carrier protein is inaccessible to substrate in the absence of energy coupling. J Biol Chem, 1975 Feb 25, 250(4), 1361 - 70 Energy-dependent binding of dansylgalactosides to the beta-galactoside carrier protein; Schuldiner S et al.; Fluorescent beta-galactosides (1-(N-dansyl)amino-beta-D-galactopyranoside (DG0), 2'-(N-dansyl)aminoethyl-beta-D-thiogalactopyranoside (DG2), 2'-(N-dansyl)aminoethyl-beta-D-galactopyranoside (oxy-DG2), and 6'-(N-dansyl)aminohexyl-beta-D-thiogalactopyranoside (DG6)) competitively inhibit lactose transport by membrane vesicles from Escherichia coli ML 309-225, but are not actively transported . An increase in the fluorescence of these dansylgalactosides is observed upon addition of D-lactate, imposition of a membrane diffusion potential (positive outside), or dilution-induced, carrier-mediated lactose efflux . The increase is not observed with 2'-(N-dansyl)aminoethyl-beta-D-thioglucopyranoside nor with membrane vesicles lacking the beta-galactoside transport system . Moreover, the D-lactate-induced fluorescence increase is blocked or rapidly reversed by addition of beta-galactosides, sulfhydryl reagents, inhibitors of D-lactate oxidation, or uncoupling agents . The fluorescence increase exhibits an emission maximum at 500 nm and excitation maxima at 345 nm and at 292 nm . The latter excitation maximum is absent unless D-lactate is added, indicating that the bound dansylgalactoside molecules are excited by energy transfer from the membrane proteins . Titration of vesicles with dansylgalactosides in the presence of D-lactate demonstrates that the lac carrier protein constitutes 3 to 4% oof the total membrane protein, and that the affinity of the carrier for substrate is directly related to the length of the alkyl chain between the galactosidic and the dansyl moieties of the dansylgalactosides . In addition, there is excellent agreement between the affinity constants of the various dansylgalactosides as determined by fluorimetric titration and their apparent Kis for lactose transport (KDs and/or apparent Kis are approximately 550, 3o, 40, and 5 muM FOR DG0, DG2, oxy-DG2, and DG6, respectively) . Polarization of fluorescence measurements with DG2 and DG6 demonstrate a dramatic increase in polarization on addition of D-lactate which is reversed by addition of lactose or anaerobiosis . These findings provide strong evidence for the contention that the fluorescence changes observed on "energization" of the membrane are due to binding of the dansylgalactosides per se, rather than binding followed by transfer into the hydrophobic interior of the membrane J Biol Chem, 1975 Feb 25, 250(4), 1600 - 2 Isolation of the soluble substrate recognition component of the dicarboxylate transport system of Escherichia coli; Lo TC et al.; A soluble, periplasmic protein was isolated from Escherichia coli cells by chromatography on columns of aspartate-coupled Sepharose 4B . This protein has a molecular weight of about 15,000 and, as judged by competition experiments, binds the three dicarboxylic acids, succinate, malate, and fumarate and, addition, the monocarboxylic acid D-lactate . The periplasmic protein seems to be missing from some mutants of E . coli (designated cbt) which are incapable of transporting succinate in whole cells. J Biol Chem, 1975 Feb 25, 250(4), 1340 - 7 Reconstitution of Escherichia coli thioredoxin from complementing peptide fragments obtained by cleavage at methionine-37 or arginine-73; Slaby I et al.; Thioredoxin from Escherichia coli (a small hydrogen transport protein containing 108 amino acid residues and having in its oxidized form a single disulfide bond) was acylated with citraconic anhydride . Citraconylation of all amino groups resulted in total loss of enzymatic activity with thioredoxin reductase and immunoprecipitin activity with antithioredoxin antibodies; both these activities were fully restored after deblocking of the citraconylated protein by acid treatment . Large enzymatically inactive peptide fragments of thioredoxin were prepared by selective cleavage at Arg-73 and Met-37, respectively, and tested for their antigenic activity with antibodies against native thioredoxin . Thioredoxin-T-(1-73) and thioredoxin-T-(74-108) were separated by Sephadex G-50 chromatography in 50% acetic acid of a deblocked trypsin digest of citraconylated thioredoxin . Thioredoxin-T-(1-73) afforded about 25% of the corresponding immunoprecipitate of native thioredoxin without significant inhibition at antigen excess . Thioredoxin-T-(74-108) gave no immunoprecipitate but was a potent inhibitor of the reaction of thioredoxin and antithioredoxin as measured by turbidity formation . A major antigenic determinant of thioredoxin was present in the COOH-terminal sequence of the molecule . An equimolar mixture of thioredoxin-T-(1-73) and thioredoxin-T-(74-108) showed full immunoprecipitation activity with antithioredoxin and significant enzymatic activity with thioredoxin reductase . Gel chromatography experiments at pH 8 with the peptide mixture showed a main symmetrical peak with elution volume and amino acid composition identical with native thioredoxin . The results strongly suggested reconstitution of these two fragments to a complex, thioredoxin-T', with a conformation similar to native thioredoxin . Reconstitution of a thioredoxin-like structure was also obtained from a mixture of the overlapping fragments thioredoxin-T-(1-73) and thioredoxin-C-(38-108), although these peptides represented more than the 108 amino acid residues of the protein . Previous results showed reconstitution of thioredoxin-C-(1-37) and thioredoxin-C-(38-108) to a complex called thioredoxin-C' (Holmgren, A . (1972) Fed . Eur . Biochem . Soc . Lett . 24, 351-354) . Together with the present results, this shows that three different combinations of two larger peptide fragments obtained by cleavage at two permissible sites gives reconstitution of thioredoxin . In each case, at least one of the component peptides showed strong immunochemical activity with antibodies to native thioredoxin, Netion from a tetrameric to a dimeri J Biol Chem, 1975 Feb 25, 250(4), 1242 - 50 Aspartokinase I-homoserine dehydrogenase I of Escherichia coli K12 (lambda) . Activation by monovalent cations and an analysis of the effect of the adenosine triphosphate-magnesium ion complex on this activation process; Ogilvie JW et al.; The dehydrogenase activity of the aspartokinase I-homoserine dehydrogenase I complex isolated from Escherichia coli K12 is subject to a cooperative activation by K+ or Rb+, which is characterized by a Hill coefficient of approximately 2 . Ionic strength has little effect on the Hill coefficient for this activation process; however, high ionic strength appears to increase the enzyme's affinity for K+ and decrease its affinity for Rb+ . The Vmax of the K+-activated dehydrogenase is greater than that of the Rb+-activated dehydrogenase . The results of a study of the competition between K+ and Rb+ in the activation process suggest the presence of an activated species containing both K+ and Rb+ . The cooperative activation by K+ is antagonized by Na+ via a process that is noncooperative with respect to Na+ . The MgATP-2- complex, a substrate for the kinase activity of aspartokinase I-homoserine dehydrogenase I, has a marked effect on the K+ activation of the dehydrogenase activity . Kinetic studies of this effect of MgATP-2- on the K+ requirement of the dehydrogenase at pH 8.9 indicate that: (a) activation by a monovalent cation is essential in the presence as well as in the absence of MgATP-2-; (b) the concentration of K+ required to activate fully the dehydrogenase is reduced in the presence of MgATP-2-; (c) activation of the dehydrogenase by K+ is noncooperative in the presence of MgATP-2-; and (d) the maximum velocity for the dehydrogenase catalyzed oxidation of homoserine is greater in the presence of MgATP-2- than in its absence . Based on these results, a simple model consistent with these data is proposed . Destruction of the kinase activity and the threonine sensitivity of the aspartokinase-homoserine dehydrogenase complex by treatment with 5,5'-dithiobis(2-nitrobenzoic acid) or by incubation at pH 9 also converts the K+ activation of the dehydrogenase from a cooperative to a noncooperative process . Marked protection of the enzyme against loss of threonine sensitivity at pH 9 is afforded by MgATP-2- plus K+ and homoserine . The apparent molecular radius of the enzyme complex as determined by gel filtration at pH 8.85 in the presence of threonine or MgATP-2- plus K+ and homoserine is dependent on the enzyme concentration . The observed apparent molecular radii of 70 A at high enzyme concentrations and 61 A at low enzyme concentrations are consistent with the enzyme's undergoing a concentration-dependent dissociation from a tetrameric to a dimeri J Biol Chem, 1975 Feb 25, 250(4), 1212 - 7 Purification and properties of stringent factor; Block R et al.; The stringent factor from Escherichia coli is the product of the relA locus . It is the enzyme that catalyzes the synthesis of pppGpp and ppGpp eliciting a pyrophosphate transfer from ATP to the 3'--OH of GTP (or GDP) . This protein is responsible for the synthesis of pppGpp and ppGpp in stringent strains in response to an amino acid starvation . In vitro it catalyzes the synthesis of these guanosine compounds in either a ribosome-dependent reaction that requires a particular conformation of the ribosome i.e . the presence of an uncharged tRNA recognizing a codon in the acceptor (A) site of the ribosome or in a ribosome-independent reaction at temperatures under 30 degrees in the presence of only buffer, salts, and substrates . Here we report the purification of the stringent factor to near homogeneity . It is a monomeric protein with a molecular weight of 75,000 . The properties of the ribosome-independent reaction are studied and it is shown that the presence of certain acidic proteins, such as the 50 S ribosomal proteins L7 and L12 or casein, or 20% methanol or both stimulates the reaction by creating an environment that together with the low temperature further stabilizes the stringent factor. Biochim Biophys Acta, 1975 Feb 24, 383(1), 106 - 16 Alteration of regulation of arginine biosynthesis in Escherichia coli W by mutation to rifampin resistance; Wozny ME et al.; The synthesis of acetylornithine delta-transaminase is induced by arginine and by rifampin in an arginine-inducible mutant of Escherichia coli W . A mutant of the arginine-inducible strain was isolated which is resistant to rifampin . The mutation which has brought about rifampin resistance has altered RNA polymerase and has simultaneously altered the regulation of arginine biosynthesis . Three of the enzymes of arginine biosynthesis, acetylornithinase, ornithine transcarbamylase, and argininosuccinase, show a greater rate of derepression and a 2--12-fold higher level of enzyme activity in the rifampin-resistant mutant than in the parent arginine-inducible strain . Acetylornithine delta-transaminase is no longer inducible by rifampin alone, and the level of inducibility by arginine plus rifampin has been reduced by 70% . The results indicate that RNA polymerase is involved in regulation of arginine biosynthesis in E . coli W. Eur J Biochem, 1975 Feb 21, 51(2), 483 - 93 The effects of overloading in density-gradient centrifugation; Steensgaard J et al.; The effects of overloading of the sample zone in density gradient centrifugation have been studied by use of a three-component shelf-lavered sample in which the total protein concentration was increased by addition of different amounts of albumin . It is found that overloading of the gradient gives rise to particle movements which are not predictable from the Svedberg equation . The two typical effects of overloading are dislocation of the zone mass centres and changes in the zone shapes . It is found that the magnitude of the calculated sedimentation coefficients increases nearly linearly with increasing sample load . The changes in zone shapes are found to depend on the specific load and two different patterns may be distinguished . The zone of the sample component which causes the overloading is defined as primarily overloaded and the others as secondarily overloaded . In primarily overloaded zones the original Gaussian shape is lost, while in secondarily overloaded zones the Gaussian zone shape is maintained, although a zone broadening is seen . Extreme high loads are found to be able to divide single zones . As a whole these experiments show that evidence for a non-overloaded set of experimental conditions must be provided, when density gradient centrifugation is used for determination of sedimentation coefficients . For preparative gradient centrifugations the power of resolution will decrease with increasing sample load . A simple method to detect overloading in density gradient centrifugations is described. Eur J Biochem, 1975 Feb 21, 51(2), 459 - 65 Ribosomal RNA genes in the amoebal and plasmodial forms of the slime mould Physarum polycephalum; Hall L et al.; 1 . The degree of homology between ribosomal RNA isolated from microplasmodia and amoebae of the slime mould Physarum polycephalum has been determined by competive hybridisation of the RNA from the two sources to homologous DNA in solution . The extent of competition was measured both by hybridisation to saturation and by following the kinetics of hybrid formation . In each case competition was found to be 100%, indicating that the ribosomal RNAs from the two, quite different vegetative forms of the organism exhibit a high degree of homology and are probably transcribed from the same genes . 2 . The relationship between the amount of nuclear DNA that codes for ribosomal RNA (rDNA) and ploidy has been investigated in three strains of P . polycephalum which exhibit a 1:2:5 variation in the amount of DNA per nucleus . Ribosomal RNA saturation values were determined by hybridisation to DNA isolated from prophase nuclei of plasmodia . The proportion of rDNA was found to be constant at 0.16--0.18% of the total genome in the three strains. Eur J Biochem, 1975 Feb 21, 51(2), 369 - 76 Stimultaneous purification of Escherichia coli termination factor rho, RNAase III and RNAase H; Darlix JL; This communication describes a method for the stimultaneous purification of Escherichia coli termination factor rho, RNAase III and RNAase H, which is rapid, reproducible and high in yield . Depending on how cells are grown 0.5 to 1 mg of RNAase III, 1 to 2 mg of RNAase H and 1 to 2 mg of rho are obtained from 100 g wet cells . RNAase III and rho are pure proteins, and RNAase H 80% pure . In addition it is shown that pure RNAase III degrades only RNA . RNA duplexes, and is responsible for the sizing of early T7 mRNA . The active form of RNAase III is composed of two identical subunits having a molecular weight of 23 500 . Native RNAase H which specifically hydrolyses the RNA moiety of an RNA . DNA hybrid, is a single polypeptide chain with a molecular weight of 21 000 The amino acid composition of termination factor rho is also reported. Med Klin, 1975 Feb 21, 70(8), 328 - 31 {Balkan-nephropathy, a particular form of interstitial nephritis (author's transl)}; Glawtschew; Because of epidemiological, clinical, pathomorphological, and etiological criteria the Balkan-nephropathy is suggested to be a particular form of chronic interstitial nephritis with super-imposed pyelonephritis in about 30 p.c . of the patients . A basic scheme illustrates the origin and the development of the endemia as well as etiology and clinical course of the disease . Another scheme shows pathogenesis and pathomorphogenesis of the nephritis . This analysis about the characteristics of the endemic Balkan-nephropathy allows for the clarification of the triad: endemic occurrance, familial susceptibility, and mosaik like morbidity . The following important aspects of the disease are given: rarely occuring hypertension, facultative leukuria, and bacteriuria, smooth nephrocirrhosis . Prophylactic and therapeutic prospects are given. Eur J Biochem, 1975 Feb 21, 51(2), 567 - 71 The mechanism of action of methionyl-tRNA synthetase from Escherichia coli . Inhibition by adenosine and 8-aminoadenosine of the amino-acid activation reaction; Blanquet S et al.; Adenosine and 8-aminoadenosine, both competitive inhibitors of ATP-Mg2+ in the ATP-PPi exchange reaction catalyzed by methionyl-tRNA synthetase, are used to investigate the active center for methionyl-adenylate formation . Resolution of the kinetics parameters of the reaction indicates that methionine markedly enhances the affinity of the nucleosides for the enzyme, providing evidence for coupling between the sites for amino acid and the nucleoside moiety of ATP . Furthermore, occupation of both of these sites is a prerequisite for binding of pyrophosphate . Introduction of an amino group in position 8 of the adenine ring strongly increases the affinity constants for the nucleoside and for pyrophosphate in the coupled reactions described above. Biochim Biophys Acta, 1975 Feb 20, 380(2), 344 - 54 13C-enriched phosphatidylethanolamines from Escherichia coli; Birdsall NJ et al.; Escherichia coli have been grown using {1-13C}acetate and {2-13C}acetate as the sole carbon source . The 13C NMR spectra of the whole cells and spheroplasts can be readily obtained but give limited information . The 13C NMR spectra of t-e isolated 13C-enriched phosphatidylethanolamines are assigned and analysed to give the biosynthetic pathway for acetate incorporation . This method provides a ready source of 13C-enriched phospholipids. Biochim Biophys Acta, 1975 Feb 19, 377(2), 271 - 81 A cyclic adenosine 3',5'-monophosphate-dependent histone kinase from pig brain . Purification and some properties of the enzyme; Nesterova MV et al.; A cyclic adenosine 3',5'-monophosphate-dependent histone kinase (ATP: protein phosphotransferase, EC 2.7.1.37) was isolated from pig brain . The enzyme has been purified 1140-fold; it is homogeneous on polyacrylamide gel electrophoresis and gel filtration . The estimated molecular weight of the enzyme is 120 000 . Histone kinase dissociates into a catalytic subunit and a regulatory one (molecular weights 40 000 and 90 000, respectively) . The catalytic subunit has been obtained in homogeneous state as evidenced by sodium dodecylsulphate-polyacrylamide gel electrophoresis . At all purification steps, enzymatic activity is stimulated 5-fold by cyclic AMP . An apparent Km value for cyclic AMP is about 3.3 - 10- minus 7 M . In the presence of cyclic AMP(5 - 10- minus 6 M), the Km value for ATP and F1 histone were 1.2 - 10- minus five and 3 - 10- minus 5 M, respectively . Optimum pH value for histone kinase is 6.5, its isoelectric point is situated at pH 4.6 . The purified enzyme displays high specificity for the lysine-rich and moderately lysine-rich histones F1, F2a2 and F2b . Arginine-rich histones and other known protein substrates for cyclic AMP-dependent protein kinases (casein, Escherichia coli RNA polymerase, etc.) are extremely poor substrates for this enzyme. Biochim Biophys Acta, 1975 Feb 19, 377(2), 217 - 28 Glutamate dehydrogenase from Escherichia coli: induction, purification and properties of the enzyme; Veronese FM et al.; When Escherichia coli was grown in a minimum medium with glucose as sole carbon source and a proper level of ammonia, NADP+ specific glutamate dehydrogenase (L-glutamate: NADP+ oxidoreductase (deaminating), ED 1.4.1.4) was induced . The enzyme was solubilized by French press treatment and purified to homogeneity by (NH4)2SO4 fractionation, heat treatment followed by DEAE-cellulose, hydroxylapatite and Bio-Gel chromatography with an overall yield of 30% . The enzyme proved to be heat stable and relatively resistant to protein denaturants . The optimum of enzymic activity for the reductive amination is at pH 8 and at pH 9 for the oxidative deamination . The activity is affected by adenine nucleotides . The molecular weight (about 250 000 for the native form and 46 000 for the inactive subunit) and amino acid composition, suggest strict similarities with the NADP+ enzyme from fungal origin. Lancet, 1975 Feb 15, 1(7903), 358 - 61 Controlled trial of therapy in covert bacteriuria of childhood; Savage DC et al.; Sixty-three girls with covert bacteriuria were included in a controlled trial of therapy . Recurrent infection in the treated group was common and was not significantly different from the rate of persistent infection in the untreated control group . Two children in each group developed clinical pyelonephritis; the others have remained healthy and all of them have a normal rate of growth . 2 years after diagnosis three of the thirty-four children in the control group and one of twenty-six children in the treated group have radiological evidence of new scars of pyelonephritis . These changes were relatively minor and in both groups of children renal growth was similar to that in normal children . It is suggested that for most of these children therapy is not essential, and that when renal changes occur they are of little or no significance . Prescriptive screening for cobert bacteriuria of childhood cannot be recommended at present. Biochim Biophys Acta, 1975 Feb 13, 381(2), 257 - 68 Regulation of the amount and of the activity of phosphofructokinases and pyruvate kinases in Escherichia coli; Kotlarz D et al.; Two isozymes of fructose-6-phosphate kinase and two isozymes of pyruvate kinase have been detected in Escherichia coli under a wide variety of growth conditions . Their kinetic behavior has been characteriized with respect to different effectors and substrates . The conclusions reached on one hand by Malcovati and Kornberg (Biochim . Biophys . Acta (1969) 178, 420-423), on the other hand by Fraenkel, Kotlarz and Buc (J . Biol . Chem . (1973) 248, 4865-4866) have been found to be true in aerobiosis as well as in anaerobiosis . The biosynthesis of the four proteins is sensitive to the nature of the carbon sources as well as to the shift from aerobic to anaerobic conditions . Kinetics of depression after a shift to anaerobiosis have been followed and found to be of the order of the doubling time. Biochemistry, 1975 Feb 11, 14(3), 599 - 607 Multiple forms of phosphodeoxyribomutase from Escherichia coli . Physical and chemical characterization; Leer JC et al.; Phosphodeoxyribomutase from Escherichia coli has been purified to homogeneity . Chromatography on DEAE-Sephadex revealed three peaks of enzyme activity, designated form I, form II, and form III . Form III could be further separated into form III-1 and form III-2 by polyacrylamide gel electrophoresis . The four different molecular forms of the enzyme thus isolated were shown not to be products of the column chromatography per se . The amino acid composition as well as the N-terminal amino acid were found to be identical for the different forms . Molecular weight determinations demonstrated that all four forms of the enzyme consist of a single polypeptide chain with a molecular weight of 45,000 plus or minus 1000 . Measurements of partial specific volumes, sedimentation coefficients, and absorption coefficients for form I and form II did not reveal any differences . It is concluded that the multiple forms of phosphodeoxyribomutase are caused by modifications of the polypeptide chain . Evidence is presented that form II is formed in vitro from form I by deamidation . It is probable that this deamidation occurs in vivo also . The different forms displayed only minor changes with respect to KM for substrate and cofactor . Greater differences seem to exist among the four enzyme forms with respect to VM and to cobalt binding. Biochemistry, 1975 Feb 11, 14(3), 514 - 20 Recognition of the 3' terminus of 2'-O-aminoacyl transfer ribonucleic acid by the acceptor site of ribosomal peptidyltransferase; Ringer D et al.; The interaction of the 3' terminus of 2'- and 3'-O-aminoacyl-tRNA with the peptidyltransferase A site of Escherichia coli ribosomes has been studied using the following aminoacyl oligonucleotides as models of the 3' terminus of AA-tRNA: C-A-Phe, C-A(2'Phe)H, C-A(2'H)Phe, C-A(2'Phe)Me, C-A(2'Me)Phe, C-A(2'Gly)H, and C-A(2'H)Gly . The transfer of Ac-(14C)Phe from the Ac-(14C)Phe-tRNA-oly(U)-70S ribosome complex to puromycin (10-4 and 10-5 M) was inhibited by C-A-Phe, C-A(2'Phe)H, C-A(2'H)Phe, C-A(2'Gly)H, and C-A(2'H)Gly . Kinetic analysis of the inhibition of Ac(14C)Phe-puromycin formation by C-A(2'Phe)H failed to show simple competitive inhibition . Binding of C-A-C-C-A-(14C)Phe to 70S ribosomes in the presence of an excess of deacylated tRNA was also inhibited by C-A-Phe, C-A(2'Phe)H, C-A(2'H)Phe, C-A(2'Phe)Me, and C-A(2'Me)Phe . It appears that the acceptor site of peptidyltransferase can recognize the 3' terminus of either 2'- or 3'-O-AA-tRNA, with preference for the 2' isomer . It therefore follows that 2'-O-AA-tRNA amy be bound to ribosomes prior to peptide bone formation and that 3'-O-AA-tRNA, which is used exclusively by peptidyltransferase as an acceptor, is supplied by 2' leads to 3' transacylation occuring at the peptidyltransferase A site. Biochemistry, 1975 Feb 11, 14(3), 478 - 84 Apparent high degree of asymmetry of protein arrangement in the Escherichia coli outer cell envelope membrane; Haller I et al.; Ghosts from Escherichia coli have been oxidized with CuSO4-o-phenanthroline or ferricyanide-ferrocene . Upon oxidation they became resistant to boiling dodecyl sulfate . The resulting rod-shaped "oxidation containers" apparently held together by disulfide bridges, are practically pure protein . They are soluble in dodecyl sulfate when reduced and they contain a set of about 30 different polypeptide chains . The four major ghost membrane proteins are not represented among the "oxidation proteins." Comparison of data obtained from digestion of ghosts with trypsin or particle-bound trypsin showed that most of the "oxidation proteins" appear to be located at the outer surface of