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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 the ghost membrane which is derived from the outer cell envelope membrane . One of the major ghost membrane proteins, II, is partially digested by trypsin, and it is shown that its trypsin sensitive part is also exposed only at the outer surface of the ghost membrane . Native cells could be oxidized only with low yields of "oxidation containers." However, cell envelopes prepared without detergents or chelating agents, as well as cells depleted of phospholipid or treated with sucrose-Triton X-100, are completely accessible to oxidation . In each case, the same set of proteins as that present in "oxidation containers" from ghosts was found to be covalently linked . Treatment of cells with trypsin caused the loss of about five "oxidation proteins" and a complete loss of oxidizability of the ghosts derived from these cells . It therefore appears that arrangement and localization of the "oxidation proteins" are not greatly different in cells and in ghosts, i.e., that these proteins are also situated asymmetrically at the outer cell envelope membrane.

Biochemistry, 1975 Feb 11, 14(3), 468 - 77
Methylation of the ribosomal proteins in Escherichia coli . Nature and stoichiometry of the methylated amino acids in 50S ribosomal proteins; Chang CN et al.; Methylated ribosomal proteins from Escherichia coli 50S subunit are localized by growing cells in a medium containing (1-14C)methionine and (3H-methyl)-methionine and comparing the 3H/14C ratio for each of the 50S ribosomal proteins . The following proteins are methylated: L11, L1, L3, L5, L7, L8, L9, L12, L18, and L33 . The nature and stoichiometry of the methylated amino acid(s) in each of the methylated proteins are determined . Protein L11 is the most heavily methylated of all the 50S subunit proteins . This protein has previously been implicated in the peptidyl transferase reaction during protein synthesis (K . H . Nierhaus and V . Montejo (1973), Proc . Nat . Acad . Sci . U . S . 47, 1588-1602) . Three proteins (L1, L3, and L5) have intermediate levels of methylation and contain about 0.4-0.6 methyl groups each per molecule of protein . Five other proteins (L7, L8, L9, L12, and L18) are also methylated to a slight extent (-0.1 methyl group/molecule of protein) . One unknown methylated neutral amino acid was detected in protein L11 and at least one and possibly two other unidentified methylated amino acids appeared to be present in protein L33.

Biochim Biophys Acta, 1975 Feb 10, 378(3), 363 - 77
Characterization of the genome of Phycomyces blakesleeanus; Dusenbery RL; DNA of Phycomyces blakesleeanus was extracted from whole mycelia and from nuclear and mitochondrial organelle fractions obtained from sporangiophores . DNA from all three sources exhibits one symmetrical band at p equals 1.688 g/ml in CsCl buoyant density gradients . Reassociation data are consistent with kinetic division of the DNA into three components: very rapidly renaturing (fraction I), rapidly reassociating (fraction II) and slowly reassociating (fraction III) base sequences . These components comprise approximately 10%, 20% and 64% of total cell DNA . Kinetic fractions were prepared from total cell DNA and reassociated separately . The corrected rate constant is 11.3 M-1-S-1 for the rapidly reassociating component and 0.055 M-1-S-1 for the slowly reassociating component . Based on these data and the data from unfractionated total cell DNA, the genome size of Phycomyces is approximately 1.9-10-10 daltons, 6.7 times that of Escherichia coli.

J Biol Chem, 1975 Feb 10, 250(3), 813 - 4
Assembly of ribosomal proteins L7, L10, L11, and L12, on the 50 S subunit of Escherichia coli; Highland JH et al.; We have determined the in vitro assembly sequence of ribosomal proteins L7, L10, L11, and L12 on Escherichia coli 50 S subunits by reconstitution experiments with the use of various ribosomal core particles and split protein fractions produced by treatment of 50 S subunits with 1 m NH4Cl and 50% ethanol . Proteins L7, L10, L11, and L12 were removed by a two-step treatment, first at 0 degrees, then at 37 degrees . Small amounts of proteins L1, L5, and L6 were also removed under these conditions . A one-step extraction of 50 S subunits at 0 degrees removed only proteins L7 and L12, while a similar one-step extraction of intact 50 S subunits at 37 degrees removed proteins L7, L12, and L10 . Two-dimensional gel electrophoresis of the protein components and measurement of the ribosome-elongation factor G-guanosine diphosphate complex formed with the various reconstituted particles showed that the binding of proteins L7 and L12 is dependent on the binding of protein L10 and in turn, that the binding of protein L10 is dependent on the binding of protein L11.

J Biol Chem, 1975 Feb 10, 250(3), 1141 - 5
Identification of a ribosomal protein necessary for thiostrepton binding to Escherichia coli ribosomes; Highland JH et al.; A ribosomal protein necessary for thiostrepton binding to Escherichia coli ribosomes has been identified using the following criteria: 1 . A loss in the thiostrepton binding ability of the ribosome was correlated with the selective removal of ribosomal protein L11 . This was achieved by a comparison of the thiostrepton binding ability of 50 S ribosomal subunits treated with 1 M NH4C1 and 50% ethanol at 37 degrees which still contained protein L11, and subunits treated successively at 0 degrees and 37 degrees in the same medium, from which protein L11 had been removed . 2 . The thiostrepton binding ability of a ribosomal core containing only seven proteins, produced by treatment of 50 S subunits with 4 M LiC1, was fully restored by the rebinding of protein L11, obtained by Sephadex G-100 fractionation of the 1 M LiC1 split protein fraction from 50 S subunits . In addition, treatment of the 1 M LiC1 split protein fraction with an IgG specific for protein L11, uniquely inhibited the restoration of activity . 3 . Thiostrepton binding to the 4 M LiC1 core, reconstituted with the 1 M LiC1 split protein fraction, was blocked by treatment with a monovalent antibody fragment (Fab) prepared against protein L11, but not by treatment with antibodies specific for the proteins of the 4 M LiC1 core . We conclude, therefore, that protein L11 is required for the ribosomal binding of thiostrepton.

J Biol Chem, 1975 Feb 10, 250(3), 1112 - 22
Degradation of abnormal proteins in Escherichia coli . Formation of protein inclusions in cells exposed to amino acid analogs; Prouty WF et al.; Cells of Escherichia coli selectively degrade proteins that have incorporated amino acid analogs . Within 1 hour after exposure of cells to canavanine, 50% of the analog-containing proteins were degraded to acid-soluble form . At the same time, no net loss of canavanine-containing protein occurred from the 100,000 X g supernatant . Instead, most of the proteins containing the analog, unlike normal ones, accumulated in particulate fractions sedimenting at 10,000 X g or 100,000 X g . They were then lost from these fractions concomitant with the degradation of the abnormal proteins . The loss of such proteins from particulate fractions accounted for all of the protein degraded to acid-soluble form . Similar observations were obtained after incorporation of other analogs or puromycin . The 10,000 X g pellets correspond to amorphous dense intracellular granules visible in electron micrographs of cells exposed to canavanine . Upon removal of the analog, these granules disappeared, simultaneously with the degradation of the analog-containing proteins . These pellets do not resemble a degradative organelle, like the lysosome; they are not osmotically sensitive, do not exclude inulin, are not enclosed by a membrane, and do not show autolytic activity . The proteins in the granules could be solubilized by sodium dodecyl sulfate but not by Triton, NaC1, dithiothreitol, RNase, DNase, or phospholipase . The proteins extracted from the pellet with sodium dodecyl sulfate tend to become particulate again upon removal of this detergent . Incorporation of canavanine caused a normally soluble polypeptide, the monomer of beta-galactosidase, to be inactive and found in the sedimentable fraction . These findings suggest that (a) the presence of amino acid analogs in proteins can make them less soluble, and (b) the inclusions are formed by the spontaneous precipitation of abnormal proteins rather than by an active granule-forming process.

J Biol Chem, 1975 Feb 10, 250(3), 1087 - 98
The nucleotide sequence in the promoter region of the fene for an Escherichia coli tyrosine transfer ribonucleic acid; Sekiua T et al.; The sequence of 29 nucleotides in the region preceding the initiation of the transcription of the Escherichia coli tyrosine tRNA gene has been determined . This is: (5') ---GGGGCGCATCATATCAAATGACGCGCCGC--- (3') (3') ---CCCCGCGTAGTATAGTTTACTGCGGCGGCG--- (5') . The general approach used for the sequence determination involved the DNA polymerase I-catalyzed elongation of suitable deoxyribopolynucleotide primers when hybridized to the 1-strand of phi80psuIII plus DNA at the appropriate site . Sequences of the newly grown oligonucleotide chains were determined by a combination of two-dimensional fingerprinting following partial exonucleolytic degradation, nearst neighbor analyses, and determination of pyrimidine tracts . Primer elongations were carried out in a controlled and stepwise manner and the newly grown oligonucleotide chains were kept short by incorporating the following features into the method: (a) the insertion of a ribonucleotide unit at or near the 3' terminus of the primers; (b) the use of a maximum of three nucleoside 5'-triphosphates in the first stage of the elongation reaction isolation of the elongated primer, and its reuse in a second step together with different sets of deoxy-nucleoside triphosphates; and (c) elongation of the primer using all of the four nucleoside triphosphates with one of the triphosphates being supplied in a limiting concentration.

J Biol Chem, 1975 Feb 10, 250(3), 1071 - 9
Purification of closed circular lambda deoxyribonucleic acid and its sedimentation properties as a function of Sodium chloride concentration and ethidium binding; Hinton DM et al.; The sedimentation of circular lambda DNA suggests that the molecular undergoes significant changes in shape and super-coiling as the NaC1 concentration increases . Closed circular lambda DNA, species I, isolated and purified from superinfected immune bacteria, sediments in sucrose gradients of low ionic strength at a rate 2.0 times faster than linear lambda DNA, species III . The addition of ethidium causes the sedimentation rate of species I DNA to decrease until enough dye is bound to remove 121 supercoils per molecule . At this point, species I co-sediments with nicked and nonsupercoiled species II . Futher additions of ethidium cause the sedimentation rate to increase until the relative rate of species I is again at least twice that of species III . This classical behavior is altered when NaC1 is present in the buffer . In 1.0 M NaC1 the changes in S are complex . Initially, species I sediments 1.55 times faster than species III . Titration with ethidium caused a decrease in S to an early minimum value, than an increase to a first maximum, followed by a decrease to the S of species II . At this point enough dye has intercalated to remove 208 superhelical turns . Further additions of dye introduce supercoils and cause S to increase again . In 0.1 to 0.4 M NaC1 the relative S of species I is 1.69 and 1.59, respectively . If titrated with ethidium, S first increases to a maximum value then decreases to the minimum rate when enough dye is bound to remove 158 and 183 supercoils, respectively . The results indicate an increase in the superhelix density from 0.026 turns per 10 base pairs in buffer alone to 0.045 in the same buffer with 1.0 M NaC1 . If this change in superhelix density results from a concomitant change in the average rotation angle between base pairs in the Watson-Crick helix, the addition of 1.0 M NaC1 alters the rotation angle by 0.68 degrees per base pair.

J Biol Chem, 1975 Feb 10, 250(3), 1061 - 70
Ethidium binding affinity of circular lambda deoxyribonucleic acid determined fluorometrically; Hinton DM et al.; Ethidium-binding isotherms for purified circular lambda DNA, isolated from a superinfected lysogen, and for linear lambda DNA, isolated from the purified phage, were constructed from fluorescence measurements of ethidium-DNA MIXTURES . The measurements were made in 0.01 M Tris-HC1-0.001 M EDTA,pH 7.1, buffer at 20 degrees and in the same buffer containing 0.1, 0.4, or 1.0 M NaC1 . When NaC1 was present, differences in the binding affinity for supercoiled and linear DNA could be quantitated . As the ethidium concentration was increased, supercoiled lambda DNA molecules bound the intercalating dye first more and then less avidly than nonsupercoiled ones . The number of potential supercoils in a circular lambda DNA molecular in the absence of dye was calculated from the amount of dye bound when it exhibited the same affinity for dye as its linear counterpart . The point of equivalent affinity shifted from 0.053 mol of dye bound per mol of nucleotide in 0.1 M NaC1 to 0.067 mol in 1.0 M NaC1 . This corresponds to the removal of 164 and 206 supercoiling turns per molecule and superhelix densities in the absence of dye equal to 0.036 and 0.045 superhelical turns per 10 base pairs . If this difference in the number of supercoils reflects a salt-dependent change in the average rotation angle between base pairs of the Watson-Crick helix the angle differs by 0.32% in the two ionic environments.

J Biol Chem, 1975 Feb 10, 250(3), 1123 - 31
In vitro synthesis of the E3 immunity protein directed by Col E3 plasmid deoxyribonucleic acid; Sidikaro J et al.; E3 colicinogenic cells are immune to colicin E3 . A protein called "E3 immunity protein" was previously isolated from E3 colicinogenic cells and shown to prevent the E3-induced in vitro inactivation of ribosomes . We now show that the structural gene for E3 immunity protein resides on the Col E3 plasmid, a plasmid which is present in E3 colicinogenic cells and carries the structural gene for colicin E3 production . For this purpose, Col E3 plasmid DNA was purified and characterized . The DNA preparation was shown to be homogeneous as judged by electron microscopy as well as agarose gel electrophoresis and has the ability to transform noncolicinogenic Escherichia coli cells into E3 colicinogenic cells with a high efficiency . The Col E3 DNA was then used as a template in a DNA-dependent in vitro protein-synthesizing system, and protein products were characterized . A radioactive protein product was detected which co-migrates with reference E3 immunity protein in urea-polyacrylamide gel electrophoresis at pH 8.7 and urea-sodium dodecyl sulfate polyacrylamide gel electrophoresis at pH 7.6 . This protein was produced only in the presence of Col E3 plasmid DNA . No such protein was produced when Col E3 plasmid DNA was omitted or replaced with chromosomal DNA . This radioactive protein was isolated and shown to be very similar to reference E3 immunity protein, as judged by the correspondence of tryptic peptides . The synthesis of immunity protein in vitro was also shown by radioimmunodiffusion . Thus, the structural gene for E3 immunity protein resides on the Col E3 plasmid . In addition, we have shown that colicin E3 protein is also synthesized in the same in vitro system using Col E3 plasmid DNA, as a template, confirming the previous notion that the structural gene for colicin E3 protein resides on the Col E3 plasmid.

C R Acad Sci Hebd Seances Acad Sci D, 1975 Feb 10, 280(6), 783 - 9
{RNA fragments, in vivo inhibitors of Shope fibroma and vaccinia virus multiplication}; Beljanski M et al.; RNA-fragments rich in purine nucleotides and resulting from degradation of ribosomal RNA from E . coli M 500 Sho-R with pancreatic RNase exhibit only in vivo an inhibitory effect on Shope fibroma and vaccinia virus multiplication.

Biochim Biophys Acta, 1975 Feb 10, 378(3), 424 - 38
DNA-binding proteins from Novikoff hepatoma cells; Johnson JD et al.; In 0.05 M NaCl, 6-8% of the total soluble proteins from Novikoff hepatoma cells bind rapidly and reversibly to columns containing either heterologous or homologous DNA adsorbed to cellulose . These proteins can be eluted by buffer containing 2.0 M NaCl . 0.5-1% of the total protein exhibits a 7-17-fold preference for rat DNA over Escherichia coli DNA . 1-1.5% of the proteins bind DNA so strongly that elution cannot be effected by 4.0 M NaCl but can be accomplished by deoxyribonuclease I treatment of the columns . DNA-binding proteins eluted by 2.0 M NaCl were labeled with 125I or 131I and characterized by sodium dodecylsulfate-polyacrylamide gel electrophoresis and isoelectric focusing . These experiments indicate that DNA-binding proteins represent a discrete subset of the total soluble protein . Many similarities were noted between the major components of the homologous and heterologous DNA-binding fractions.

J Biol Chem, 1975 Feb 10, 250(3), 835 - 42
Role of metal ions in Escherichia coli alkaline phosphatase . A study of the metal-water interaction by nuclear relaxation rate measurements on water protons; Zukin RS et al.; The binding of metal to alkaline phosphatase from Escherichia coli and the binding of water and orthophosphate to the Me-2+-enzyme binary complex have been examined by water proton relaxation rate (PRR) measurements . Titration of the three paramagnetic metals, Mn2+, Cu2+, and Co2+, into apoalkaline phosphatase and the titrations of apoenzyme into metal have been carried out . Analysis of the spin-lattice relaxation rates for these titrations and of Scatchard binding curves derived from these results, as well as EPR data, show four tight manganese sites, between two and three tight copper sites, or four cobalt sites per enzyme dimer of molecular weight 80,000 . The multiple sites for each metal are indistinguishable by these magnetic resonance techniques . Both the spin-lattice- and spin-spin-relaxation rates exhibit a negative temperature coefficient, showing that these processes are not exchange-limited . From a frequency dependence study of T-1 and from the T-1:T-2 ratio measured at 220 MHz, correlation times from the water-enzyme complexes have been estimated . For H20-Mn-2+-alkaline phosphatase, gamma c equals 1.55 times 10-9 s; for H20-Cu-2+ -alkaline phosphatase, gamma c equals 1.82 times 10-s; and for the cobalt complex, gamma c equals 1.0 times 10-12 s at 4 degrees . Assuming 1 water molecule bound per metal site, these correlation times correspond to the following water-metal distances: gamma (A) is 4.0 A for Mn-2+-H20, 3.4 A for Cu-2+-H20, and 2.8 A for Co-2+-H20 . Thus, water is shown to bind directly to the metal atoms of alkaline phosphatase . The correlation between the length of the water-metal bond and the relative activity of the various metalloenzymes support the importance of this binding in the monophosphoesterase reaction catalyzed by alkaline phosphatase . Addition of excess orthophosphate to any of the water-metalloenzyme complexes does not displace an exchangeable water molecule from the metal site . The Mn-PO-4 distance which we have reported earlier (Zukin, R.S., Hollis, D.P., and Gray, G.A . (1973) Biochem . Biophys . Res . Commun . 53, 238) to be 7.3 A is consistent with this finding and suggests a model in which Pi binds to Mn-2+-alkaline phosphatase through a water bridge.

Eur J Biochem, 1975 Feb 3, 51(1), 295 - 304
The primary structure of the major cytoplasmic valine tRNA of mouse myeloma cells; Piper PW; This paper describes the derivation of the primary structure of the major valine tRNA in the cytoplasm of mouse myeloma cells . Approximately 75% of the nucleotide sequence of this tRNA is also shared by the tRNA1-Val of yeast, this homology serving as a further indication of the extreme conservation of the structures of the tRNAs of different eukaryotic organisms . A novel feature of mouse myeloma tRNA1-Val is its loop IV sequence: -U-PSI-C-G-M1A-A-A- . This particular loop IV sequence has not previously been found in a tRNA structure . In addition, tRNA1-Val possesses some unusual nucleoside modifications . 5-Methyluridine (T) was not found to occur within loop IV of this tRNA, although this minor nucleoside is also absent from certain other mammalian tRNAs . Only one other tRNA, mammalian tRNAf-Met, has been found to possess 2-methylguanosine (m2G) in the position between the (b) and (c) stems of the cloverleaf . Numerous tRNAs have m2-2G in this location, and it would appear that the second methylation of this guanosine is characteristically absent from certain mammalian tRNA species.

Eur J Biochem, 1975 Feb 3, 51(1), 165 - 80
An investigation of the 16-S RNA binding sites of ribosomal proteins S4, S8, S15, and S20 FROM Escherichia coli; Ungewickell E et al.; The RNA binding sites of four 30-S ribosomal subunit proteins from Escherichia coli, namely S4, S8, S15, and S20 were prepared from reconstituted single protein - 16-S-RNA complexes by mild enzymic digestion of non-protected RNA regions . Oligonucleotide fingerprints of the protected RNA regions were obtained and their positions were located within the 16-S-RNA sequence . They were not completely contiguous regions of RNA; oligonucleotides had been excised from each of them . The binding sites of S4 and S20, and those of S8 and S15 showed overlapping . The specificity of the RNA binding sites was confirmed by a reconstitution method.

Eur J Biochem, 1975 Feb 3, 51(1), 155 - 64
Comparative studies on the properties of tryptophanase and tyrosine phenol-lyase immobilized directly on Sepharose or by use of Sepharose-bound pyridoxal 5'-phosphate; Fukui S et al.; Tryptophanase from Escherichia coli B/qt 7-A and tyrosine phenol-lyase (beta-tyrosinase) from Escherichia intermedia were immobilized on Sepharose 4B by several direct coupling reactions or through pyridoxal 5'-phosphate previously bound to Sepharose . The most active preparation of immobilized tryptophanase was obtained by coupling tetrameric apoenzyme to pyridoxal-P bound on Sepharose at the 6-position through a diazo linkage . This immobilization procedure involves the formation to Schiff base linkage between 4-formyl group of Sepharose-bound pyridoxal-P and the epsilon-amino group of the lysine residue at the active center of one subunit of tetrameric apo-tryptophanase, followed by the fixation of the Schiff base linkage by reduction with NaBH4 . In the case of beta-tyrosinase having two catalytic centers, however, this method was not so suitable as the case of tryptophanase . Direct coupling of the apoenzyme to CNBr-activated Sepharose or to a bromoacetyl derivative of Sepharose gave better results . In each case, the affinity for substrate or coenzyme was scarcely influenced by the immobilization . When used repeatedly in a batch system or continuously in a flow system in the absence of added pyridoxal-P, immobilized holo-tryptophanase of holo-beta-tyrosinase gradually lost its original activity; however, supplement of pyridoxal-P to the reaction system restored its initial activity . From the kinetic analyses of these phenomena, the rate constants of coenzyme dissociation from immobilized tryptophanase and beta-tyrosinase were calculated . Upon immobilization, the pH optima of both enzymes shifted 0.5 to 1.0 pH unit to the alkaline side . Both immobilized enzymes showed higher thermal stability and resistance to a denaturing agent such as guinidine-HCl than their free counterpart . Furthermore, the reactivity of sulfhydryl group of beta-tyrosinase, in connection with its coenzyme-binding property, was conveniently studied by use of the immobilized enzyme.

Eur J Biochem, 1975 Feb 3, 51(1), 117 - 27
Two-dimensional polyacrylamide-gel electrophoresis for purification of small RNAs specified by virulent coliphages T4, T5, T7 and BF23; Ikemura T et al.; RNAs synthesized in Escherichia coli infected with virulent phages T4, T5, T7 and BF23 were labelled with 32PO4 3- after phage infection . {32P}RNAs of low molecular weight were separated by two-dimensional polyacrylamide gel electrophoresis, in which electrophoresis was carried out in two dimensions at different concentrations of acrylamide . The fractionated RNAs were characterized by RNA-fingerprint patterns made after T1 ribonuclease digestion . The two-dimensional gel of 10% yields 20% acrylamide was suitable for RNA of less than 200 nucleotides, while that of 5% yields 10% was preferred for RNAs of about 150--400 nucleotides . With T4 phage, 16 RNA species were separable on a single slab gel . Among those, 11 were identified as the known RNA species, including eight T4 tRNAs, one tRNA precursor and two non-tRNA molecules . In the case of T5 and BF23, more than 20 RNA species were separated on a slab gel; 15 or more RNAs were found in the 4-S RNA region, and several in 5-S and 6-S region . The RNA-fingerprint patterns of many BF23 RNAs were very similar to those of corresponding RNAs of T5 . Pseudouridine and ribosylthymidine, minor nucleosides generally present in tRNA, were found in several BF23 4-S RNAs tested . Possibility of those BF23 4-S RNAs as tRNAs is discussed . With phage T7, three RNAs were detected, two of which were much smaller than tRNAs.

Eur J Biochem . 1975 Feb 3;51(1):UNKNOWN.
Properties of a DNA-adenylate complex formed in the reaction between mammalian DNA ligase I and DNA containing single-strand breaks; Soderhall S; The major DNA ligase from calf thymus (mammalian DNA ligase I) forms a covalent enzyme-AMP complex on incubation with ATP {Soderhall & Lindahl, J . Biol . Chem . 248, 672-675, (1973)} . The reaction of this complex with DNA has now been studied . When the ligase-adenylate complex is incubated at 0 degrees C for short time periods with DNA containing single-strand breaks, a DNA-AMP complex can be isolated from the reaction mixture by isopycnic centrifugation in CsCl . Incubation at pH 6.5 increased the amount of DNA-AMP complex that could be isolated 10-20-fold relative to that obtained at pH 7.4 . Under the same conditions, incubation of the ligase-AMP complex with DNA free from single-strand breaks did not lead to detectable DNA-AMP formation . The DNA-AMP complex was resistant to treatment with dilute acid and alkali indicating the presence of a covalent linkage . Further, this complex was sensitive to DNase but resistant to pronase and RNase . Free AMP was released on further incubation of the isolated DNA-AMP complex with thymus DNA ligase I and Mg2+, suggesting that the complex is a reaction intermediate . Degradation of the DNA-AMP complex with several reagent enzymes indicated that the AMP residues were bound at the 5' ends of the single-strand breaks in DNA by pyrophosphate bonds.

Biochem Genet, 1975 Feb, 13(1-2), 109 - 24
Overproduction of lysine by mutant strains of Escherichia coli with defective lysine transport systems; Halsall DM; Mutants selected on the basis of their resistance to S-(beta-aminoethyl) cysteine and overproduction of lysine were found to be defective in the lysine transport system . The overproduction of lysine was not due to mutation affecting either of the two regulatory enzymes aspartokinase and dihydrodipicolinic acid synthetase . Uptake of labeled lysine by the lysine-specific transport system was reduced to a negligible level, while uptake by the lysine, ornithine, arginine system was also affected . A hypothesis regarding the nature of these mutations and their effects on the regulation of lysine biosynthesis is discussed.

J Biochem (Tokyo), 1975 Feb, 77(2), 373 - 82
Mode of replication of plasmid lambda-dv-1; Matsubara K et al.; About 20 copies of plasmid lambda-dv are perpetuated per host chromosome in Escherichia coli K12 cells . The mode of DNA replication of this plasmid lambda-dv was studied, using the density-labelling technique followed by banding in DsCl . It was shown that all the copies of plasmid lambda-dv are potentially capable of replicating roughly once a cell generation . Their replication occurs by random, that is a copy of the plasmid is taken out at random for replication from a pool, to which the two replicas resulting from replication are returned . Chloramphenicol did not inhibit the initiation of a new round of replication of the plasmid molecules, indicating that selection from the replication pool does not require concomitant protein synthesis.

Proc Natl Acad Sci U S A, 1975 Feb, 72(2), 733 - 6
Preferential and cooperative binding of histone I to chromosomal mammalian DNA; Renz M; There is a strong preferential binding of histone I to lymphocyte DNA as compared to Escherichia coli DNA when large DNA fragments (2 times 10-6 daltons) are used . The binding of histone I to lymphocyte DNA and to E . coli DNA is cooperative . The distribution of preferential binding sites has been investigated on fragmented DNA . Most of the 2 times 10-6 dalton fragments were found to have at least one preferential histone I binding site, whereas most of the 2 times 10-5 dalton fragments have none.

Proc Natl Acad Sci U S A, 1975 Feb, 72(2), 683 - 7
Spectator-ion effect on the passage of ions through membranes; Kozak JJ; In this paper, we investigate the interplay between geometric and dielectric factors in influencing the image force acting on an ion passing through a membrane, for a system having the approximate dimensions of Escherichia coli . We also study the effect of one ion in a membrane on the passage of a second ion through the membrane, by calculating the radial and angular forces experienced by the second ion due to the presence of the "spectator ion." Our conclusions follow from numerical studies on expressions obtained by solving (exactly) Laplace's equation for the model assumed in this paper . The conclusions are: (i) small changes in the dielectric constant of the membrane are far more significant in determining the image force acting on an ion in a membrane than dramatic changes in the dielectric character of the regions interior and/or exterior to the cell; (ii) a spectator ion in a membrane situated near a boundary may influence in a significant way the passage of a second ion through the middle third of the membrane . We suggest that this latter result should be taken into account in discussing the mechanism of ion migration across membranes.

Proc Natl Acad Sci U S A, 1975 Feb, 72(2), 654 - 8
Nature of R-factor replication in the presence of chloramphenicol; Crosa JH et al.; Covalently closed circular deoxyribonucleic acid molecules of RSF1030, a nonconjugative R-factor, initiate and complete rounds of semiconservative replication in the absence of protein synthesis long after the bacterial chromosome has ceased its replication . RSF1030 replication under these conditions is sensitive to the inhibitor of ribonucleic acid synthesis, rifampicin . The product of this replication, a covalently closed DNA molecule, shows, in contrast to those molecules produced during the replication in a logarithmically growing culture of Escherichia coli, a transition to the open circular form upon treatment with ribonucleases of alkali . Analysis of the product resulting from alkali treatment indicates that there is a single break in one strand of the original circular duplex . This alkali-sensitive site occurs with equal probability in either of the complementary strands . These results are interpreted as a requirement for an RNA primer for the initiation of RSF1030 DNA synthesis and as showing that its removal from the covalently closed molecule is inhibited in the absence of protein synthesis.

Proc Natl Acad Sci U S A, 1975 Feb, 72(2), 423 - 7
Evidence for binding protein-independent substrate translocation by the methylgalactoside transport system of Escherichia coli K12; Robbins AR et al.; Three genes, mgl A, B, and C, are required for active transport of substrate by the methylgalactose permease of E . coli K12 . We report here that only two of these genes are required for substrate translocation, as seen by the ability or inability of isogenic mgl mutants (referred to as Tra+ and Tra minus, respectively) to grow on methyl-beta-D-galactopyranoside, supplied as sole carbon source . Individual mutants of both the Tra+ and Tra minus classes exhibited no detectable intracellular accumulation of methyl-beta-D-galactopyranoside; thus, the Tra+ phenotype cannot be explained by the mutants' levels of residual active transport . The phosphotransferase (Pts), the beta-galactoside (LacY), and the arabinose (Ara E and Ara F) transport systems are not required for substrate translocation by Tra+ cells . The Tra+ phenotype was identified with mutants defective in the mgl B, locus of the galactose-binding protein, by genetic complementation; the Tra minus phenotype was observed with both mgl A and mgl C mutants . The conclusion that the galactose-binding protein is not required for substrate translocation was supported by direct assays of the mgl mutants' binding protein activity . Mutants capable of translocation all showed reduced galactose-binding protein activity; mutants incapable of translocation exhibited binding protein activity equal to that of the mgl+ parent.

Nucleic Acids Res, 1975 Feb, 2(2), 279 - 302
Location and characteristics of ribosomal protein binding sites in the 16S RNA of Escherichia coli; Zimmermann RA et al.; Specific binding sites for five proteins of the Escherichia coli 30S ribosomal subunit have been located within the 16S RNA . The sites are structurally diverse and range in size from 40 to 500 nucleotides; their functional integrity appears to depend upon both the secondary structure and conformation of the RNA molecule . Evidence is presented which indicates that additional proteins interact with the RNA at later stages of subunit assembly.

Nucleic Acids Res, 1975 Feb, 2(2), 265 - 78
Primary sequence of the 16S ribosomal RNA of Escherichia coli; Ehresmann C et al.; Recent progress in the nucleotide sequence analysis of the 16S ribosomal RNA from E . coli is described . The sequence which has been partially or completely determined so far encompasses 1520 nucleotides, i.e . about 95% of the molecule . Possible features of the secondary structure are suggested on the basis of the nucleotide sequence and data on sequence heterogeneities, repetitions and the location of modified nucleotides are presented . In the accompanying paper, the use of the nucleotide sequence data in studies of the ribosomal protein binding sites is described.

Nucleic Acids Res, 1975 Feb, 2(2), 257 - 63
Synthesis of pppGpN type dinucleotide derivatives: the 5' end sequence of some RNAs; Simoncsits A et al.; A rapid and simple synthesis of pppGpN type /N equals C, U or A/ diribonucleotide derivatives is described by coupling guanosine 2', 3'-cyclic phosphate 5'-triphosphate with the appropriate ribonucleoside in the presence of ribonuclease T-1.

Lab Anim Sci, 1975 Feb, 25(1), 55 - 61
Colitis cystica profunda in rhesus monkeys; Scotti TM; Spontaneously occurring colitis cystica profunda, characterized by the presence of non-neoplastic glands and mucin-containing cysts in the submucosa of the large intestine, was observed post mortem in 4 of 28 rhesus monkeys (Macaca mulatta) but in none of 20 squirrel monkeys (Saimiri sciuresu) necropsied during the same period of time . An analogous lesion in the stomach was present in 2 of the monkeys with the colonic condition . The submucosal glands and cysts resulted from extension of the mucosa through the muscularis mucosae, and inflammation was considered to have played a primary role in this process . The cause of the intestinal inflammation was not determined, and there was no known exposure to toxic chemicals, including pesticides and polychlorinated biphenyls . Colitis cystica profunda affecting man and other animals has been described infrequently in the literature, but the importance of differentiating it from intestinal adenocarcinoma has been emphasized.

Am J Dis Child, 1975 Feb, 129(2), 220 - 2
Shunt fluid aspiration: an adjunct in the diagnosis of cerebrospinal fluid shunt infection; Myers MG et al.; Bacterial isolates were obtained from 42 of 59 cultures of shunt fluid aspirated from patients with infected cerebrospinal fluid shunts . The etiologic agent was recovered from 24 to 25 aspirates from patients who were not receiving antibiotics and from 18 of 34 aspirates from patients who were receiving antibiotics . Aspiration, culture, and Gram stain of shunt fluid should be considered in patients suspected of having a shunt infection.

Pediatrics, 1975 Feb, 55(2), 261 - 5
The use of indwelling radial artery catheters in neonates; Adams JM et al.; Indwelling radial artery catheterization was performed on 20 infants . In only one patient was it unsuccessful . Mean duration of indwelling catheterization was 44.1 hours . Advantages of this procedure include constant availability of arterial blood gas sampling and blood pressure monitoring . It is a relatively safe procedure offering a low incidence of clinical infection and serious vascular complications . Further experience will delineate its exact role in the care of the acutely ill neonate.

Pediatrics, 1975 Feb, 55(2), 239 - 43
Suprarenal abscess in the neonate: a case report and review of diagnosis and management; Blankenship WJ et al.; A case of unilateral suprarenal abscess is reported . This is the third such reported case, and the first reported case successfully treated with preservation of the kidney . Diagnosis and treatment were aided significantly by a thorough preoperative radiological evaluation, including angiography.

J Cell Biol, 1975 Feb, 64(2), 480 - 91
Mitosis in the cellular slime mold Polysphondylium violaceum; Roos UP; Myxamebas of Polysphondylium violaceum were grown in liquid medium and processed for electron microscopy . Mitosis is characterized by a persistent nuclear envelope, ring-shaped extranuclear spindle pole bodies (SPBs), a central spindle spatially separated from the chromosomal microtubules, well-differentiated kinetochores, and dispersion of the nucleoli . SPBs originate from the division, during prophase, of an electron-opaque body associated with the interphase nucleus . The nuclear nevelope becomes fenestrated in their vicinity, allowing the build-up of the intranuclear, central spindle and chromosomal microtubules as the SPBs migrate to opposite poles . At metaphase the chromosomes are in amphitelic orientation, each sister chromatid being directly connected to the corresponding SPB by a single microtubule . During ana- and telophase the central spindle elongates, the daughter chromosomes approach the SPBs, and the nucleus constricts in the equatorial region . The cytoplasm cleaves by furrowing in late telophase, which is in other respects characterized by a re-establishment of the interphase condition . Spindle elongation and poleward movement of chromosomes are discussed in relation to hypotheses of the mechanism of mitosis.

J Bacteriol, 1975 Feb, 121(2), 537 - 47
Sedimentation analysis of deoxyribonucleic acid from thymine-starved Escherichia coli; Nakayama H et al.; During thymine starvation, strand breaks accumulate in the chromosomal deoxyribonucleic acid (DNA) of Escherichia coli . This effect occurs to a varying extent in different strains and is particularly enhanced in strains deficient in DNA polymerase I . The inhibition of ribonucleic acid or protein synthesis suppresses the accumulation of strand breaks . In a polA strain, rifampin is more effective than chloramphenicol or puromycin in suppressing strand break accumulation . To a certain extent the pehenomenon othymineless death correlates with the appearance of strand breaks . Although the killing can not be explained by the bulk of strand breaks, it is possible that some of them represent lethal events . On the basis of our observations we proposed the following model . (i) Transcription may be accompanied by single-strand breaks in DNA . (ii) DNA polymerase I is involved in the efficient repair of these breaks . (iii) Thymine deprivation results in the accumulation of unrepaired breaks . (iv) Polymerase I-mediated repair is less affected by thymine deprivation than are the alternative pathways because it closes the breaks with short patches, requiring less thymine.

Ann Thorac Surg, 1975 Feb, 19(2), 142 - 52
The benefits of corticosteroids in endotoxic shock; Prager R et al.; PIP: 22 adult mongrel dogs were divided into 4 groups: 1) controls (fluid balanced after endotoxin injection); 2) steroid only administered (methylprednisolone sodium succinate (30 mg/kg, after endotoxin injection); 3) steroids and assisted circulation (normothermal); and 4) assisted circulation (hypothermia) . The most striking finding was the lengthened survival time of Group 2 animals . 4/6 lived for 48 hours following a mean lethal dose of E . coli endotoxin, whereas only 2/6 in Group 1 survived for this period; none of 10 in Groups and 3 and 4 . 2 Group 4 animals survived the period of perfusion but died of unknown causes, and 1 Group 3 animal developed sustained braducardia, hypotension, and cardiac arrest soon after cessation of cardiac assistance . The most consistent difference between Groups 1 and 2 was the decreased amount of fluids required by the Group 2 animals to maintain an adequate systemic arterial pressure during the observation period . Features also of interest noted were the higher cardiac outputs and the minimal evidence of pulmonary congestion or injury in the animals treated with steroids only . Results clearly demonstrate the beneficial value of pharmcological doses of corticosteroids in experimentally induced endotoxic shock .

J Virol, 1975 Feb, 15(2), 232 - 7
Canavanine-mediated depletion of polyamine pools in Escherichia coli: effect of head morphogenesis and DNA synthesis; Bolin RW et al.; We have found that L-canavanine inhibited the synthesis of polyamines in T4-infected Escherichia coli . These polyamines are known to be required for T4 DNA synthesis and may be involved in phage morphogenesis . The new data indicate that the inhibition of polyamine synthesis is not primarily responsible for the L-conavanine-mediated inhibition of DNA synthesis nor does it seem to be involved in the induction of lollipops . L-Canavanine does influence the relative amounts of putrescine and spermidine found in the phage particle, but it does not influence the amount of DNA phosphate neutralized by polyamines.

J Gen Microbiol, 1975 Feb, 86(2), 201 - 9
Sphere-rod morphogenesis of Escherichia coli; Goodell EW et al.; The morphogenetic capacity of E . coli was studied by converting the rod-shaped cells into spheres and then determining whether these spheres could revert to rods . The morphogenesis of cells was followed by immobilizing them in a viscous Methocel-containing medium . Two different types of spheres were prepared: cells which retained a mechanically intact sacculus, and osmotically sensitive sphaeroplasts lacking a sacculus . The sphaeroplasts were not able to revert to rods although they were able to synthesize a new sacculus . In contrast, spheres which had retained an intact sacculus were able to reshape themselves into rods . The were also able to form new ends at (or near) the sites of the ends on the original rods.

J Immunol, 1975 Feb, 114(2 pt 2), 856 - 62
Ly-4.2: a cell membrane alloantigen of murine B lymphocytes . II . Functional studies; McKenzie IF; The alloantigenic specificity Ly-4.2 can be detected on a proportion of lymphocytes by the antiserum (BALB/c times SWR)F1 anti-B10.D2 . In the preceding study it was shown that these lymphocytes were not thymus-derived (T) cells, as they were Thy-1 (theta)(-), and were therefore presumably B (bone marrow-derived) cells . Evidence is now presented for the reaction of the Ly-4.2 antiserum with functional B cells . Thus, the LY-4.2 and Thy-1.2 specificities were detected on antigen-binding rosette-forming cells (RFC) in mice both immune and non-immune to sheep red cells (SRC) . RFC formed to endotoxin lipopolysaccharide (LPS) were also Ly-4.2(+) . Memory cells to both SRC andLPS could be detected with anti-Ly-4.2 and anti-Thy-1.2 antisera, thereby indicating that both T and B cells are involved in memory to these antigens . Both direct and indirect antibody-forming cells (the PFC) could be inhibited, in vitro, by anti-Ly-4.2 antiserum, although it is likely that not all PFC are Ly-4.2(+) . Neither of the specificities Ly-4.2 nor Thy-1.2 were detected on the bone marrow precursor of the splenic colony forming unit (the CFU) . In an assay for B cells, the treatment of lymph node or spleen cells with anti-Ly-4.2 before transfer to irradiated recipients could inhibit the ability of these cells to make PFC to SRC, and this capacity could only be restored by bone marrow cells and not by thymus cells . These studies provide clear evidence for the presence of the Ly-4.2 specificity on antibody-forming cells and their precursor (B cells).

J Immunol, 1975 Feb, 114(2 pt 2), 742 - 6
Synergistic effects on DNA synthesis of phytohemagglutinin or concanavalin A and lipopolysaccharide in human peripheral blood lymphocytes; Schmidtke JR et al.; Mixtures of phytohemagglutinin (PHA) or Concanavalin A (Con A) and lipopolysaccharide (LPS) with human peripheral blood lymphocytes (PBL) resulted in a synergistic effect on DNA synthesis as measured by increased 3H-thymidine uptake . These data were compared to the mitogenic response of human PBL to PHA, Con A, or LPS alone . Various combinations of PHA and Con A usually suppressed DNA synthesis in human PBL . Titration experiments revealed that increasing amounts of LPS, in the presence of a constant amount of Con A, resulted in corresponding increases in the synergistic response of human PBL . Conversely, increasing amounts of Con A, in the presence of a constant amount of LPA did not lead to increases in the amount of synergism . The results are discussed in terms of the participation of additional cells in the synergistic response of human PBL.

J Bacteriol, 1975 Feb, 121(2), 735 - 6
Ultraviolet light sensitivity of Escherichia coli K-12 strains carrying ruv mutations in combination with uvrA or lon mutant alleles; Iyehara H et al.; Strains of Escherichia coli K12 have been prepared that carry the ruv mutation in combination with lon or uvrA mutant alleles . The ruv minus uvrA minus double mutant is more sensitive to ultraviolet light than the urvA minus single mutant, whereas the strain with ruv and ion mutations shows the same sensitivity to ultraviolet light as the ruv minus single mutant.

J Bacteriol, 1975 Feb, 121(2), 554 - 61
Suppression of initiation-negative strains of Escherichia coli by integration of the sex factor F; Tresguerres EF et al.; Data are presented suggesting that the most critical factor determining whether an Hfr dnaAts strain can synthesize deoxyribonucleic acid and form colonies at temperatures that are nonpermissive for corresponding F- strains is neither the site of insertion of F nor the presence of additional mutations in the F particle or the chromosome; it is whether the particle is capable of autonomous replication at the temperature used . Consequently, suppression of the DnaA phenotype in Hfr strains occurs at 40 C but not, in most of them, at 42 C without the occurrence of additional mutations . The site of insertion of F may also be important since it is shown that in one Hfr dnaA strain partial suppression does occur at 42 C . In addition, it is shown that strains exhibiting suppression by integration of F at 40 C on minimal agar plates do not do so at this temperature on nutrient agar plates.

J Bacteriol, 1975 Feb, 121(2), 524 - 30
mut-25, a mutation to mutator linked to purA in Escherichia coli; Siegel EC et al.; The mutation mut-25 that results in a mutator phenotype is closely linked to purA on the chromosome of Escherichia coli . The gene order in this region is ampA mut-25 purA . purA mut-25 double mutants retained mutator activity indicating that mut-25 is not a mutation in the purA gene . The repair mutations uvrA6, recA56, and exrA1 had no effect on mutation frequencies in mut-25 strains, and mut-25 strains were normally resistant to ultraviolet irradiation . Frequencies of host range mutations were not increased in phages T1, T2, and T7 grown on mut-25 strains . mut-25 could act trans, reverting the trpA46 mutation either on the chromosome or on an F episome . The transitions AT yields GC (adenine-thymine yields guanine-cytosine) and GC yields AT were induced by mut-25.

J Bacteriol, 1975 Feb, 121(2), 511 - 7
Enzymatic production of deoxyribonucleic acid double-strand breaks after ultraviolet irradiation of Escherichia coli K-12; Bonura T et al.; We have observed the enzymatic production of deoxyribonucleic acid (DNA) doublestrand breaks in Escherichia coli K12 after ultraviolet irradiation . Doublestrand breaks appeared in wild-type, polA1, recB21, recA, and exrA strains after incubation in minimal medium . THE UVRA6 strain showed no evidence of double-strand breakage under the same conditions . Our data suggest that uvr+ cells, which are proficient in the incision step of excision repair, accumulate double-strand breaks in their DNA as a result of the excision repair process, i.e., arising from closely matched incisions, excision gaps, or incisions and gaps on opposite strands of the DNA twin helix . Furthermore, strains deficient in excision repair subsequent to the incision step (i.e., polA, rec, exrA) showed more double-strand breaks than the wild type strain . The results raise the possibility that a significant fraction of the lethal events in ultraviolet-irradiated, repair-proficient (uvr+) cell may be enzymatically-induced DNA double-strand breaks.

J Bacteriol, 1975 Feb, 121(2), 504 - 10
Mutation affecting regulation of synthesis of acetohydroxy acid synthetase in Escherichia coli K-12; Jackson JH et al.; Altered regulation of synthesis of acetohydroxy acid synthetase (AHAS) was previously reported in a mutant of Escherichia coli strain K-12 . The mutant strain, growing in minimal medium, exhibits a partial growth limiatation and derepression of AHAS, owing to deficient synthesis of isoleucine . The genetic lesion (ilvE503) causing the isoleucine limitation was shown to cause derepression of a valine-sensitive AHAS activity . The derepression effect of the ilvE503 mutation upon synthesis of AHAS was conclusively demonstrated by introducing both the ilvE503 allele and an altered AHAS (ilv-521) into the same cell . Evidence is presented that suggests the presence of multiple genetic regions for synthesis and control of the valine-sensitive AHAS activity.

J Bacteriol, 1975 Feb, 121(2), 491 - 6
Effect of a leu-linked mutation on the valine sensitivity of acetohydroxy acid synthase activity in Escherichia coli; Kline EL et al.; A spontaneous leu-linked mutation (ilvH2015) in Escherichia coli K-12 made the strain resistant to 1 mM valine and l mM glycylvaline (Val-r) and caused the isoleucine and valine biosynthetic enzyme, acetohydroxy acid synthase, to be less sensitive to feedback inhibition by valine than the wild-type enzyme . Transfer of the ilvDAC deletion into a strain carrying ilvH2015 abolished the effect of the marker on the acetohydroxy acid synthase and rendered it as sensitive to valine as the enzyme in the isogenic control strain without the Val-r marker under both repressing and limiting conditions . In contrast, auxotrophy caused by transfer of an ilvC lesion into the Val-r strain did not interfere with the effect of ilvH2015 on valine sensitivity of acetohydroxy acid synthase . In addition, the presence of the Val-r marker produced minor but significant pleiotropic effects on several other isoleucine and valine biosynthetic enzymes but did not cause derepression of the ilv gene cluster . These studies suggest some type of interaction between a product produced by a gene close to leu and the isoleucine and valine biosynthetic enzymes.

J Bacteriol, 1975 Feb, 121(2), 401 - 5
Uptake of adenosine 5'-monophosphate by Escherichia coli; Yagil E et al.; Adenosine 5'-monophosphate is dephosphorylated before its uptake by cells of Escherichia coli . This is demonstrated by using a radioactive double-labeled culture, and with a 5'-nucleotidase-deficient, mutant strain . The adenosine formed is further phosphorolyzed to adenine as a prerequisite for its uptake and incorporation . The cellular localization of the enzymes involved in the catabolism of adenosine 5'-monophosphate is discussed.

Infect Immun, 1975 Feb, 11(2), 334 - 6
Test for enterotoxigenic Escherichia coli using Y-1 adrenal cells in miniculture; Sack DA et al.; A rapid, potentially clinically useful test for detection of enterotoxigenic Escherichia coli is described . Whole bacterial cultures of enterotoxigenic E . coli, when briefly exposed to Y1 adrenal cells in tissue miniculture, effect a rounding response in the tissue culture that can be discerned at 18 to 24 h . The tissue culture technique agreed with the rabbit ileal loop in all 58 enterotoxigenic and 52 non-enterotoxigenic E . coli strains tested.

Cancer, 1975 Feb, 35(2), 306 - 18
The central nervous system in childhood leukemia . II . Subacute leukoencephalopathy; Price RA et al.; A study was performed to evaluate the clinical and histopathologic characteristics of a distinctive degenerative lesion within the central nervous system (CNS) of children with acute lymphocytic leukemia (ALL) . Of the 231 patients in this study, 13 were found to have specific degenerative changes in telencephalic white matter . Morphologically, the principal changes consisted of diffuse reactive astrocytosis and multiple, noninflammatory necrotic foci, often containing varying amount of mineralized cellular debris . Clinical features common to all patients with this leukoencephalopathy were: 1) cranial irradiation of 2000 rads or more and 2) methotrexate administered systemically after irradiation . Comparison of selected clinical features of patients with and without leukoencephalopathy showed that methotrexate administered intravenously after a cumulative dose of CNS irradiation of 2000 rads or more can result in degeneration of CNS white matter in patients with ALL . Age at time of irradiation, bacterial infections, nutrition, and CNS leukemia were not causally related to the development of this disease . This study suggests that chemotherapeutic agents may diffuse through the blood-brain barrier following CNS irradiation of 2000 rads or more.

Am J Vet Res, 1975 Feb, 36(2), 223 - 6
Intestinal infusion of endotoxin in adrenalectomized calves; Reece WO et al.; Four adrenalectomized calves, 3 to 4 months old, were allowed to develop adrenocorticosteroid insufficiency and then were given 50 mg of Escherichia coli endotoxin by infusion via cannula into the duodenum . Several physiologic variables were studied to detect changes indicative of endotoxemia . It is concluded that adrenocorticosteroid insufficiency in calves is not an independent factor allowing an intestinal source of endotoxin to cause the development of clinical signs and physiologic changes characteristic of endotoxemia.

Int J Radiat Biol Relat Stud Phys Chem Med, 1975 Feb, 27(2), 121 - 33
Absence of ultrafast processes of repair of single-strand breaks in mammalian DNA; Palcic B et al.; Town, Smith and Kaplan (1972) reported that the yield of DNA single-strand breaks (SSB) in E . coli is largely independent of the presence of molecular oxygen during irradiation . They suggested that the oxygen enhancement ratio (o.e.r.) normally observed is due to the presence of an ultrafast repair mechanism acting (in bacterial cells) mainly on anoxically-produced breaks . To determine whether similar mechanisms exist in mammalian cells, we carried out comparable experiments on two cell-lines, one from Chinese hamster, the other from mouse . Both heat inactivation and chemical inhibition were treatment inactivated all the enzymatic processes assayed, it did not alter the o.e.r . for SSB production, which remained about 3-0 . The presence of sodium cyanide, hydroxyurea, iodoacetic acid, EDTA and quinacrine all failed to alter significantly the o.e.r . Isolated nuclei also demonstrated the full o.e.r . For these cell-lines at least, ultrafast reprir does not seem to exist . Isolated Adenovirus 2, which presumably lacks enzymic activity, demonstrated an o.e.r . of 3-6 for SSB production . From these results and others it seems unlikely that the so-called ultrafast enzymic repair is a general phenomenon accounting for the o.e.r . in a wide range of biological systems . Rather, the o.e.r . for SSB seems to result from differences in the direct physico-chemical effects of radiation under aerobic and anoxic conditions in most organisms.

J Gen Microbiol, 1975 Feb, 86(2), 333 - 42
Studies on the vitamin nutrition of the cellular slime mould Dictyostelium discoideum; Watts DJ et al.; Vitamin auxotrophs of Escherichia coli grown in the presence or absence of the corresponding vitamin were used as substrates for the growth of Dictyostelium discoideum strain Ax-2 in order to investigate some of the growth-factor requirements of this cellular slime mould . Compared with the growth yields observed on vitamin-sufficient auxotrophs and on a prototroph, the yields on lipoate-, folate-, thiamin- or biotin-depleted auxotrophs were low but could be restored by the addition of exogenous vitamin to the slime mould cultures . It is concluded that these four vitamins are essential nutrients for D . discoideum . Similar experiments with other auxotrophs were inconclusive, indicating either that the slime mould does not require pantothenate, nicotinate or vitamin B6 or that the strains of E . coli were insufficiently depleted to detect such requirements . It is also concluded that cobalamin is not an essential nutrient, since the myxamoebae grew well with E . coli which lacks this vitamin when grown in cobalamin-free media . Similar arguments suggest that ubiquinone and vitamin K are also non-essential.

J Bacteriol, 1975 Feb, 121(2), 434 - 41
Assay of deoxyribonucleic acid homology using a single-strand-specific nuclease at 75 C; Barth PT et al.; We investigated the conditions under which a crude preparation of endonuclease S1 gives maximal hydrolysis of denatured deoxyribonucleic acid (DNA) while giving minimal hydrolysis of native DNA . The hydrolysis was measured by filtering and determining the acid-insoluble reaction product using 3H-labeled substrates . We also investigated various parameters in making this measurement . Under appropriate conditions (in 1 mM ZnSO-4, 0.168 M NaCl at pH 4.8) denatured DNA is hydrolyzed within 3% of completion whereas native DNA is essentially unaffected . The reaction was applied to assay plasmid DNA homoand heteroduplexes for which the method proves to be simple, fast, and reproducible.

J Biochem (Tokyo), 1975 Feb, 77(2), 439 - 47
Mechanism of the ribosome-dependent uncoupled GTPase reaction catalyzed by polypeptide chain elongation factor G; Arai N et al.; At low NH4-+ concentrations, 50S ribosomal subunits from E . coli were fully active in the absence of 30S ribosomal subunits, in forming a complex with the polypeptide chain elongation factor G (EF-G) and guanine nucleotide (ternary complex formation), and also in supporting EF-G dependent hydrolysis of GTP (uncoupled GTPase reaction) . However, both activities were markedly inhibited on increasing the concentration of the monovalent cation, and at 160 mM NH4-+, the optimal concentration for polypeptide synthesis in a cell-free system, almost no activity was observed with 50S ribosomes alone . It was found that the inhibitory effect of NH4-+ was reversed by addition of 30S subunits . Thus, at 160 mM NH4-+, only 70S ribosomes were active in supporting the above two EF-G dependent reactions, whereas at 20 mM NH4-+, 50S ribosomes were almost as active as 70S ribosomes . Kinetic studies on inhibition by NH4-+ of the formation of 50S ribosome-EF-G-guanine nucleotide complex, indicated that the inhibition was due to reduction in the number of active 50S ribosomes which were capable of interacting with EF-G and GTP at higher concentrations of NH4-+ . The inhibitory effects of NH4-+ on ternary complex formation and the uncoupled GTPase reaction were markedly influenced by temperature, and were much greater at 0 degrees than at 30 degrees . A conformational change of 50S subunits through association with 30S subunits is suggested.

J Virol, 1975 Feb, 15(2), 259 - 67
Replication of polyoma DNA in isolated nuclei . V . Complementation of in vitro DNA replication; Otto B et al.; Nuclei from polyoma-infected 3T6 fibroblasts elongate in vitro the progeny strands of the replicative intermediates of polyoma DNA . When high concentrations of such nuclei were incubated, short DNA fragments were formed and subsequently added onto growing progeny strands . When nuclei were repeatedly washed with buffer containing detergent and then incubated at low concentrations . DNA synthesis was decreased . In particular, the joining process was reduced, resulting in an accumulation of short DNA fragments . All aspects of the synthetic capacity of the nuclei were restored by addition of cytoplasmic extract . Additions of purified enzymes (polynucleotide ligase from calf thymus or Escherichia coli together with E . coli DNA polymerase I) increased the joining function of the nuclei . The system can be used for the identification of the enzymatic steps concerned with polyoma DNA replication.

Biochem J, 1975 Feb, 146(2), 417 - 23
Metabolite transport in mutants of Escherichia coli K12 defective in electron transport and coupled phosphorylation; Rosenberg H et al.; 1 . The uptakes of Pi and serine by whole cells of mutant strains of Escherichia coli K12, grown under both aerobic and anaerobic conditions, were studied . 2 . Uptake by aerobic cells was low in a ubiquinone-less mutant but normal in two mutant strains unable to couple phosphorylation to electron transport . 3 . One of these uncoupled strains, carrying the unc-405 allele, does not form a membrane-bound Mg2+-stimulated adenosine triphosphatase aggregate, and it is concluded that the Mg2+-stimulated adenosine triphosphatase does not serve a structural role in the aerobic active transport of Pi or serine . 4 . The other uncoupled strain, in which aerobic uptake is unaffected, carries a mutation in the uncB gene, thus distinguishing this gene from the etc gene, previously shown to be concerned with the coupling of electron transport to active transport . 5 . The uptakes of Pi and serine by anaerobic cells were normal in the ubiquinone-less mutant, but defective in both the uncoupled strains . 6 . The uptake of Pi and serine by anaerobic cells of the uncB mutant could be increased by the addition of fumarate to the uptake medium . The unc-405 mutant, however, required the addition of fumarate for growth and for uptake . 7 . The uncB mutant, unlike the unc-405 mutant, is able to grow anaerobically in a minimal medium with glucose as sole source of carbon . Similarly a strain carrying a mutation in the frd gene, which is the structural gene for the enzyme fumarate reductase, is able to grow anaerobically in a glucose-minimal medium . However, a mutant strain carrying mutations in both the uncB and frd genes resembles the unc-405 mutant in not being able to grow under these conditions.

J Pathol, 1975 Feb, 115(2), 111 - 25
Comparison of the effect of various antisera and cobra venom factor on inflammatory reactions in guinea-pig skin . II . The Arthus reaction and the local Shwartzman reaction; Lewis E et al.; The ability of antisera to guinea-pig C3 to inhibit the Arthus and local Shwartzman reactions was studied . They were found to reduce the non-haemorrhagic component of the active and reversed passive Arthus reactions and to delay the appearance of the haemorrhage in the active Arthus reaction . Cobra venom factor, however, had no effect on the non-haemorrhagic components of these reactions and only delayed the appearance of the haemorrhage of the active Arthus reaction . There appeared to be a correlation between the serum complement level and the time taken for the haemorrhage to appear, and between the circulating platelet count and the extent of the non-haemorrhagic, oedematous component of the reaction . The haemorrhagic component of the local Shwartzman reaction was not affected by decomplementation with cobra venom factor . The ability of the antisera to inhibit the haemorrhage of the Shwartzman reaction was not dependent on lowering the serum complement titre . However, the haemorrhage was inhibited if the circulating platelet count was also reduced to very low numbers . Antiserum to zymosan alone had the same effect as anti-beta1C/beta1A globulin (zymosan) in blocking the reaction, although it did not alter the complement levels or the platelet counts . The possibility of an immunological cross-reactivity between zymosan and endotoxin in this action is discussed.

J Gen Microbiol, 1975 Feb, 86(2), 228 - 40
An attempt to identify the intestinal receptor for the K88 adhesin by means of a haemagglutination inhibition test using glycoproteins and fractions from sow colostrum; Gibbons RA et al.; The K88 antigen of Escherichia coli specifically adheres to the piglet intestinal cell; a solution of this antigen agglutinates guinea-pig red cells at 4 degrees C . The latter reaction was used as a model of the former, using inhibition of haemagglutination as an index of specific combination with the K88 adhesin . Inhibition was found with mucous glycoproteins and chemical modification of their heterosaccharide residues by mild acid hydrolysis, periodate oxidation or the Smith degradation procedure suggested that the terminal beta-D-galactosyl structure in a heterosaccharide sidechain of a glycoprotein might combine specifically with the K88 adhesin and inhibit haemagglutination . One serum glycoprotein (fetuin), after exposure of its subterminal beta-D-galactosyl residue, also inhibited haemagglutination, but high inhibitory activity was exhibited by some submaxillary glycoproteins in which this structure was absent or not prominent . It was concluded that in some cases inhibition of haemagglutination by glycoprotein was non-specific . No inhibition was found using glycosaminoglycans, glycogen or any simple sugar or glycoside . Sow colostrum was inhibitory but this was associated mainly with its gamma-globulin fraction . Some inhibitory activity was traced to a colostral glycopeptide fraction of low molecular weight but the smaller colostral oligosaccharides were not inhibitory; the composition of these components in sow colostrum is reported.

J Immunol, 1975 Feb, 114(2 pt 2), 734 - 7
Comparison of the ocular effects of circulating endotoxin and immune complexes: role of vasoactive amines; Howes EL Jr et al.; Both bacterial endotoxin and soluble immune complexes (BGG-anti BGG) injected i.v . in rabbits produce an alteration in ocular vascular permeability confined primarily to the vessels of the iridial portion of the ciliary processes . These effects have been measured by the ocular accumulation of 125I-albumin relative to cardiac plasma . Ten micrograms per kilogram of Escherichia coli bacterial endotoxin (055:B5) produce a consistent alteration in ocular vascular permeability over a 90-min period after injection . Fifty to 100 mmug/kg of endotoxin result in a prolonged effect that is maximum 4 hr after injection . Large quantities of immune complexes (BGG-antiBGG) prepared in 20 to 25 times antigen excess produce an anaphylaxis and approximately a 70% reduction in platelets and CH50 . The alteration in ocular vascular permeability is half as great as that produced by 10 ug/kg of endotoxin over the 90-min period after their injection . A combined treatment with antihistamine, pyrilamine maleate, and antiserotonin, methysergide maleate, results in a significant reduction in ocular 125I-albumin in normal animals, virtually prevents the ocular effect of immune complexes, and reduces the average effect of endotoxin by 30% . Increasing the quantities of injected antihistamine and antiserotonin does not have a further effect on the response to 10 ug of endotoxin.

Eur J Immunol, 1975 Feb, 5(2), 105 - 11
The immunological properties of haptens coupled to thymus-independent carrier molecules . III . The role of the immunogenicity and mitogenicity of the carrier in the induction of primary IgM anti-hapten responses; Klaus GG et al.; Hapten (DNP-lys) conjugates of two putatively nonimmunogenic polymers, hyalutonic acid and poly-gamma-D-glutamic acid, induce significant primary IgM anti-DNP responses in C3H mice . Preparations of various immunogenic (Type 3 pneumococcal polysaccharide (SIII), levan, E . coli lipopolysaccharide) and nonimmunogenic (hyaluronic acid and poly-glutamic acid) polymers were tested for their ability to act as polyclonal mitogens in vitro . In serum-containing spleen cell cultures, only lipopolysaccharide stimulated substantial cell proliferation . In serum-free medium, and using high specific activity {3H}thymidine, lipopolysaccharide, levan, SIII and to a lesser degree hyaluronic acid induced significant thymidine incorporation . However, under the latter conditions cell survival and proliferation were much less impressive . There was no apparent correlation between the capacity of various polymers to induce lymphocyte proliferation and their "potency" as carriers for the generation of a primary IgM anti-DNP response . Furthermore while low doses of lipopolysaccharide elicited "polyclonal" antibody formation in vivo, high doses of SIII, levan and hyaluronic acid did not . These results indicate that T cell-independent B cell triggering is dependent on the polymeric nature of the antigen, and that polymers need not be immunogenic or mitogenic to act as carriers for the induction of primary IgM anti-hapten antibody responses.

Biochim Biophys Acta, 1975 Jan 31, 376(1), 42 - 62
Redox properties of beta-type cytochromes in Escherichia coli and rat liver mitochondria and techniques for their analysis; Hendler RW et al.; We describe here apparatus and procedures for conducting potentiometric titrations and for analyzing the collected data in terms of the number of components present, their amounts and their midpoint potentials . Using these procedures we have determined the presence of three forms of cytochrome b1 in Escherichia coli with midpoint potentials at pH 7.1 OF about minus 50, plus 110 and plus 220 mV . We were not able to demonstrate a change in any of these potentials by the addition of phosphate, ATP, or 2, 4-dinitrophenol . We have been able to confirm the presence of two forms of cytochrome b in non-energized mitochondria and the apparent conversion of the low-potential component to a new high potential component upon energization of the mitochondria . However we cite further experimental data that question the actual conversion of one form of cytochrome b to another . An alternative interpretation based on our analysis suggests that the high voltage component may be present in a masked form in the non-energized mitochondria.

Biochemistry, 1975 Jan 28, 14(2), 235 - 41
The quaternary structure of citrate synthase from Escherichia coli K12; Tong EK et al.; A combination of equilibrium ultracentrifugation and polyacrylamide gel electrophoresis techniques has been used to establish the quaternary structure of citrate synthase from acetate-grown Escherichia coli K12 3000 . In polyacrylamide gels containing 0.1% sodium dodecyl sulfate (SDS), the pure enzyme showed one major band whose mobility was consistent with a molecular weight of 46,000 plus or minus 2000 g/mol, and a little material of 87,000 plus or minus 5000 g/mol . When first cross-linked with dimethyl suberimidate and then submitted to electrophoresis in SDS, citrate synthase showed six bands, in widely different amounts, whose apparent molecular weights were almost integral multiples of 47,000 g/mol . The dimer was the major product of the cross-linking procedure . In 6 M guanidine HCl at pH 7.0, citrate synthase behaved as a single component in high-speed sedimentation equilibrium experiments, with a weight average molecular weight of 43,400 plus or minus 300 g/mol . The molecular weight of native citrate synthase was investigated by high-speed sedimentation equilibrium ultracentrifugation under different conditions of pH and KCl concentration . In 0.02 M Tris-Cl at pH 7.0 and 7.8, the enzyme was a mixture of oligomers, with species ranging from monomer (47,000 g/mol) to greater than decamer being present . At pH 9.0, only dimer was seen (94,000 g/mol) . Large aggregates were present at pH 10.0 . The addition of small amounts of KCl, a potent activator of the enzyme, simplified the mixture of oligomers considerably at pH 7.8 . A detailed analysis of the data with 0.05 M KCl indicated that dimer and hexamer were the only species present, with marked nonideality . Increasing the KCl concentration to 0.10 M converted all the enzyme to hexamer . The amino acid composition of E . coli citrate synthase was presented . Taken together with peptide mapping experiments of others (J . A . Wright and B . D . Sanwal (1971), J . Biol . Chem . 246 1689), it indicates that the subunits have all the same or very similar amino acid sequences . The dansylation method revealed only methionine at the N-termini of the citrate synthase polypeptide chains . Citrate synthase from E . coli thus resembles the enzyme from eukaryotes in that it consists of subunits weighing just under 50,000 g/mol, although these subunits are more highly aggregated in the bacterial enzyme under most conditions . This conclusion is in disagreement with that of Wright and Sanwal (1971, see above), who reported a subunit size of 62,000 g/mol.

Biochemistry, 1975 Jan 28, 14(2), 214 - 24
Fluorescence energy transfer between ligand binding sites on aspartate transcarbamylase; Matsumoto S et al.; The method of fluorescence energy transfer is used to measure the distances between several sites on aspartate transcarbamylase . Both fluorescence steady-state and lifetime techniques are used . When the tryptophans on the catalytic subunit are the fluorescent donor groups, either pyridoxamine phosphate, covalently bound to an amino group at the active site, or 8-anilino-1-naphthalenesulfonate, noncovalently bound at the active site, is the acceptor group . The distance between tryptophan and the active site is calculated to be 2e A assuming that the fluorescence of only one tryptophan per catalytic polypeptide chain is quenched by the acceptor or 27 A assuming that both tryptophans on a catalytic chain are equally quenched . The pyridoxamine phosphate label is also used as the fluorescent donor with mercurinitrophenol bound to the sulfhydryl group of the catalytic subunit as the energy acceptor . For this pair of labels the active site is determined to be very close to the sulfhydryl group on the same catalytic chain and 26 A from the sulfhydryl groups on the other chains of the catalytic trimer . In experiments with pyridoxamine phosphate at the active site as the donor and 8-anilino-1-naphthalenesulfonate at the active site as the acceptor, a distance of 26 A between active sites of a catalytic trimer is found . No energy transfer is observed from pyridoxamine phosphate at the active site to a fluorescamine derivative of cytidine 5'-triphosphate at the regulatory site . This implies that these groups are separated by at least 42 A in the native enzyme . All of the distances are calculated using the assumption of rapid rotation of donor and acceptor dipole moments relative to the donor fluorescence lifetime . Fluorescence polarization measurements suggest this assumption does not produce a significant error in the calculated distances . The distances between the various sites are related to the subunit structure of aspartate transcarbamylase.

Biochemistry, 1975 Jan 28, 14(2), 224 - 30
Calorimetric analysis of aspartate transcarbamylase from Escherichia coli: binding of cytosine 5'-triphosphate and adenosine 5'-triphosphate; Allewell NM et al.; The binding of CTP and ATP to aspartate transcarbamylase at pH 7.8 and 8.5 at 25 degrees has been investigated by equilibrium dialysis and flow microcalorimetry . The binding isotherms for CTP at both pH 7.8 and 8.5 and ATP AT PH 8.5 can be fit by a model which assumes three tight, three moderately tight, and six weak binding sites . The binding isotherms for ATP at pH 7.8 are best fit by a model which assumes six tight and six weaker sites . Both finite differenceH binding and finite differenceS binding are negative for both nucleotides at both pH values, so that the binding is enthalpy driven . For both nucleotides, finite differenceH is the same for the first two classes of binding sites, implying that the difference in the dissociation constants of these two classes of sites is the result of entropic effects . Direct pH measurements and calorimetric measurements in two buffers with very different heats of ionization (Tris and Hepes) indicate that the binding of both nucleotides is accompanied by the binding of protons . In the pH range 6.7-8.4, the number of moles of protons bound per mole of nucleotide increases as the pH decreases.

Biochemistry, 1975 Jan 28, 14(2), 307 - 16
Priming of superhelical SV40 DNA by Escherichia coli RNA polymerase for in vitro DNA synthesis; Champoux JJ et al.; When closed circular SV40 DNA containing 58 negative superhelical turns is used as a template for RNA synthesis with Escherichia coli RNA polymerase, a fraction of the RNA product remains complexed with the DNA . The RNA in the complex is resistant to ribonuclease in high salt, and the Tm indicates that it is hydrogen bonded to the DNA . The mole ratio of RNA to DNA nucleotides in the complex ranges from 0.01 to 0.08; the RNA ranges in length from 80 to 600 nucleotides . The formation of the complex is dependent on the circular DNA being topologically underwound since no complex is formed when closed circular DNA containing zero superhelical turns is used as the template . The DNA-RNA complex can serve as a primer-template combination for in vitro DNA synthesis by E . coli DNA polymerase I . After synthesis with (alpha-32P)-labeled deoxyribonucleoside triphosphates followed by alkaline hydrolysis, the isolation of 32P-labeled ribonucleotides is evidence for a covalent linkage between the RNA and the DNA synthesized . During the in vitro DNA synthesis, the template is nicked at a low rate, and the nicked molecules support extensive DNA synthesis . This observation indicates that only limited synthesis can occur on unnicked molecules possibly owing to the topological constraints against unwinding of the helix . Possible models for in vivo priming of double-stranded DNA by E . coli RNA polymerase are discussed.

JAMA, 1975 Jan 27, 231(4), 352 - 5
Agranulocytosis caused by Chinese herbal medicines . Dangers of medications containing aminopyrine and phenylbutazone; Ries CA et al.; Four non-Chinese patients, middle-aged or older, developed agranuloctyosis while taking Chinese herbal medicines for relief of arthritis and back pain . All four developed life-threatening infections with bacterial sepsis; one died . The herbal medicines were shown to contain substantial amounts of undeclared aminopyrine and phenylbutazone, drugs that are well-known causes of agranulocytosis . These Chinese herbal medicines are widely available over the counter throughout the United States.

J Biol Chem, 1975 Jan 25, 250(2), 783 - 5
Stimulation of the uptake of soluble proteins into isolated HeLa nuclei by pancreatic deoxyribonuclease; Cox GS et al.; The uptake into isolated HeLa nuclei of radioactive cytosol proteins and purified Escherichia coli ribosomal protein L7 is stimulated up to 4-fold by pancreatic deoxyribonuclease (DNase I) . Similar effects are not observed with pancreatic ribonuclease A or phospholipase C . The results reported suggest that there is a general stimulatory effect of DNase on protein uptake by nuclei.

J Biol Chem, 1975 Jan 25, 250(2), 668 - 74
Subunit interactions in aspartate transcarbamylase . A model for the allosteric mechanism; Chan WW; The conformational changes in aspartate transcarbamylase upon binding of substrates or regulatory ligands and the effects of alterations in the subunit structure on the allosteric interactions are reviewed . The available information including recent results from studies of the c3r6 complex (c denotes the catalytic polypeptide and r, the regulatory polypeptide) is considered in terms of the existing models for the discrepancies between experimental observations and the present models could be resolved by postulating an important role for r:r interactions in the allosteric mechanism . A new model is presented in which an obligatory conformational change upon binding of substrates results in an alteration in the relative orientation of c versus r . As a consequence of symmetry conservation, the r:r domain is shifted to a position of higher potential energy . By favoring one or the other alternative r:r domains, CTP and ATP can respectively enhance and reduce the sigmoidal character of substrate saturation . The model is shown to be consistent with all of the important known properties of the enzyme . Because the heterotropic effects of CTP or ATP are postulated to operate via a mechanism separate from that for the homotropic effects of the substrates, this model accounts satisfactorily for the observation by Kerbiriou and Herve (Kerbiriou, D., and Herve, G . (1973) J . Mol . Biol . 78, 687-702) that homotropic effects can be abolished whereas heterotropic effects are retained in the altered enzyme from Escherichia coli grown in the presence of 2-thiouracil.

J Biol Chem, 1975 Jan 25, 250(2), 661 - 7
Subunit interactions in aspartate transcarbamylase . The interaction between catalytic and regulatory subunits and the effect of ligands; Chan WW; The interaction between the catalytic subunit (c3) and the regulatory subunit (r2) of aspartate transcarbamylase from Escherichia coli was studied by measuring the reversible formation of the c3r6 complex as a function of r2 concentration . Conversion to the native enzyme was prevented by using a very low concentration of c2 (40 ng per ml) in the presence of bovine serum albumin . A simple hyperbolic r2 saturation curve was obtained suggesting the presence of only one kind of c:r domain . From the association constant for the formation of c3r6, the free energy of c:r interaction can be estimated to be about -10 Cal per mole . Neither CTP nor ATP appears to affect the strength of c:r interaction in this complex . Succinate in the presence of carbamyl phosphate promotes tighter binding . At higher concentration of c3 and nonsaturating levels of r2, conversion to the native enzyme (c3r6) takes place . This renaturation process is second order with respect to the concentration of c3 and is virtually irreversible . Renaturation is inhibited by saturating levels of r2 and to some extent by both CTP and ATP . The effect of ligands on c:r interactions reported here may have significance in the allosteric mechanism of the native enzyme.

J Biol Chem, 1975 Jan 25, 250(2), 542 - 7
The structural basis for the resistance of Escherichia coli formylmethionyl transfer ribonucleic acid to cleavage by Escherichia coli peptidyl transfer ribonucleic acid hydrolase; Schulman LH et al.; Escherichia coli formylmethionly-tRNA-tMet is unique among N-acylaminoacyl-tRNAs in its resistance to cleavage by peptidyl-tRNA hydrolase . Chemical modification of tRNA-fMet with sodium bisulfite converts fMet-tRNA-fMet into a good substrate for the hydrolase . The products of the enzymatic cleavage are free tRNA-fMet and formylmethionine . Bisulfite treatment produces cytidine to uridine base changes at several sites in the tRNA structure . One of these modifications results in formation of a new hydrogen-bonded base pair at the end of the acceptor stem of tRNA-fMet . We have shown that this modification is responsible for the observed change in biological activity . Enzymatic cleavage appears to be facilitated by the presence of a 5-terminal phosphate at the end of a fully base-paired acceptor stem, because removal of the 5-phosphate group from N-acetylphenylalanyl-tRNA-Phe or bisulfite-modified fMet-tRNA-FMet reduced the rate of hydrolysis of these substrates . The unpaired base at the 5 terminus of unmodified fMet-tRNA-fMet appears to reduce susceptibility of the tRNA to hydrolytic attack both by positioning the 5-phosphate in an unfavorable orientation and by directly interfering with enzymatic binding . The unusual structure of the acceptor stem of this E . coli tRNA thus plays a critical role in maintaining the viability of the organism by preventing enzymatic cleavage of the fMet group from the bacterial initiator tRNA.

J Biol Chem, 1975 Jan 25, 250(2), 461 - 9
Deoxyribonucleic acid polymerase III of Escherichia coli . Purification and properties; Livingston DM et al.; DNA polymerase III has been purified 4,500-fold from the Escherichis coli mutant, HMS83, which lacks DNA polymerases I and II . When subjected to disc gel electrophoresis, the most purified fraction exhibits a single major protein band from which enzymatic activity may be recovered . Polyacrylamide gel electrophoresis under denaturing conditions produces two protein bands with molecular weights of 140,000 and 40,000 . The sedimentation coefficient of the enzyme is 7.0 S, and the Stokes radius is 62 A . Taken together these tow parameters indicate a native molecular weight of 180,000 . Purified DNA polymerase III catalyzes the polymerization of nucleotides into DNA when provided with both a DNA template and a complementary primer strand . The newly synthesized DNA is covalently attached to the 3' terminus of the primer strand . Because the extent of polymerization is only 10 to 100 nucleotides, the best substrates are native DNA molecules with small single-stranded regions . The most purified enzyme preparation is devoid of endonuclease activities . In addition to the two exonuclease activities described in the accompanying paper, purified polymerase III also catalyzes pyrophosphorolysis and the exchange of pyrophosphate into deoxynucleoside triphosphates . DNA polymerase III has also been isolated from wild type E . coli containing the other two known DNA polymerases . Futhermore, the enzyme purified from three different polC mutants exhibits altered polymerase III activity, confirming that polC is the structural gene for DNA polymerase III (Gefter, M., Hirota, Y., Kornberb, T., Wechsler, J., and Barnoux, C . (1971) Proc . Natl . Acad . Sci . U . S . A . 68, 3150-3153).

J Biol Chem, 1975 Jan 25, 250(2), 653 - 60
Subunit interactions in aspartate transcarbamylase . Characterization of a complex between the catalytic and the regulatory subunits; Mort JS et al.; The complex formed when excess regulatory subunits (r2) of aspartate transcarbamylase is added to a dilute solution of the catalytic subunit (c3) has been further studied . By stabilizing the complex with saturating levels or r2, it was possible to perform ultracentrifugation in sucrose density gradients . The sedimentation coefficient of the complex (7.7 plus or minus 0.2 S) is intermediate between those of the catalytic subunit (5.8 S) and of the native enzyme (11.7 S) . Consideration of the likely hydrodynamic properties of the complex suggests that this sedimentation coefficient may be consistent with the c3r6 structure previously proposed . The formation of c3r6 from c3 and r2 is readily reversible . At nonsaturating levels or r2, conversion to the native enzyme (c3r6) takes place . This conversion is inhibited by high concentrations of r2 . The c3r6 complex shows Michaelis-Menten kinetics with a low Km for aspartate and considerable substrate inhibition . The pH activity profile at high aspartate concentrations is almost identical with that of the native enzyme . All of these observations suggest that c3r6 represents the relaxed (R) state of aspartate transcarbamylase . The insensitivity of c3r6 toward CTP or ATP can also be explained by considering c3r6 as a stabilized relaxed state . These properties of c3r6 have significant implications regarding the allosteric mechanism of the native enzyme.

Science, 1975 Jan 24, 187(4173), 257 - 8
F factor promotes turnover of stable RNA in escherichia coli; Onishi Y; Male bacteria that contain and srnA- mutant allele degrade their "stable" RNA massively after RNA synthesis is blocked at 42 degrees C; a normal F- female strain shows no such RNA breakdown unless both the srnA- allele and maleness (F factor) are introduced.

J Chromatogr, 1975 Jan 22, 103(2), 355 - 63
Thermal chromatography of lysine-specific transfer ribonucleic acid from Escherichia coli B; Steinschneider A; Two Escherichia coli B tRNALys isoacceptors have been separated by thermal chromatography on hydroxyapatite demonstrating the potential of this approach in the fractionation of single-stranded polynucleotides . Elution temperatures were reproducible and depended primarily on the cation present and its concentration . Cesium phosphate was a more effective eluent than sodium phosphate . The integrity of the polynucleotide following thermal chromatography was examined by ultracentrifugation in a 60% dimethyl sulfoxide solvent, electrophoresis on polyacrylamide gels containing urea and oligonucleotide mapping . Adsorption to hydroxyapatite did not noticeably increase phosphodiester bond breakage compared with that in solution . Minimal, if any, rupture of covalent linkages was detected under the conditions of preparative separation of the isoacceptors . Several observations suggest that only one tRNALys was transcribed by the E . coli B cell.

C R Acad Sci Hebd Seances Acad Sci D, 1975 Jan 20, 280(3), 363 - 6
{RNA-fragments, necessary primers for the in vitro replication of DNA}; Beljanski M et al.; Partial degradation of ribosomal RNAs (E . coli rich in purine nucleotides) by different ribonucleases gives rise to the appearance of several families of RNA-fragments which after separation on "Sephadex G 25" were analyzed for base ratio, size and biological activity . In the presence of DNA dependent DNA polymerase, RNA-fragments act as primers for in vitro replication of DNAs from numerous sources.

Vet Rec, 1975 Jan 18, 96(3), 52 - 6
Factors influencing occurrence of colibacillosis in calves; Wray C et al.; This investigation describes some of the husbandry factors influencing the occurrence of colibacillosis in calves . Diarrhoea and mortality were usually associated with an increase in the proportion of "pathogenic" to total E coli to about 50 per cent in the faeces although there were occasions when the proportion of "pathogenic" E coli increased but no disease occurred . These increases often followed changes of diet which appeared to be more important than environmental conditions . The use of uncontaminated houses to break the cycle of infection appeared to have the greatest influence on the disease pattern.

Biochim Biophys Acta, 1975 Jan 14, 375(1), 44 - 53
Isolation and characterization of two outer membrane preparations from Escherichia coli; Mizushima S et al.; A method was developed for releasing specifically a part of outer membrane during spheroplast formation . A highly purified outer membrane (outer membrane I) was obtained from the spheroplast medium by isopycnic sucrose gradient centrifugation . The remaining outer membrane (outer membrane II) and cytoplasmic membrane was also isolated from the spheroplasts by the isopycnic centrifugation . Two outer membrane preparations were different from the cytoplasmic membrane in protein composition, enzyme localization, phospholipid composition, lipopolysaccharide content and electron micrographs . Although outer membranes I and II were almost the same in various respects, they seemed to be different from each other under electron microscope and in cardiolipin content . It is suggested that the outer membrane I and the outer membrane II, at least a part of the outer membrane II, are integrated in a different fashion in the outer-most layer of Escherichia coli cell surface.

Can J Comp Med, 1975 Jan, 39(1), 1 - 6
Clinical and clinico-pathological effects of Escherichia coli endotoxin in mature cattle; Griel LC Jr et al.; The effects of intravenous administration of Escherichia coli endotoxin were studied in eight mature lactating cows . Three cows were studied following intrammary infection with E . coli . Significant clinical findings are presented . Significant clinico-pathological findings include leukopenia, decreased blood serum calcium concentrations and increased levels of serum glutamic-oxaloacetic transaminase and serum ornithine-carbamyl transferase . Significant elevations of plasma corticosteroids were also noted.

J Biol Chem, 1975 Jan 10, 250(1), 304 - 9
Synthesis and turnover of basal level guanosine tetraphosphate in Escherichia coli; Friesen JD et al.; Cultures of escherichia coli growing exponentially in Trisacetate medium were subjected to nutritional shift-up and the pool size of guanosine 5'-3'-diphosphate-3'diphosphate (ppGpp) as well as the rates of protein synthesis and net RNA synthesis were determined . In the shift to a rich medium (glucose plus 19 amino acids plus hypoxanthine) the basal level of ppGpp falls immediately with a decay constant suggesting total inhibition of synthesis; ther is no ppGpp detectable above background for 30 to 40 min . The net rate of RNA synthesis starts to increase within 1 min of the shift-up and has reached its definite postshift value well before the pool of ppGGpp rises above background lvel . In a shift-up from Tris-acetate medium to Tris-glucose medium there is a much slower readjustment of the ppGpp pool size without the transient disappearance of the nucleotide . However, in a shift-up to Tris-acetate plus 5 amino acids, a medium which supports the same growth rate as Tris-glucose medium, a dramatic, transient drop in the ppGpp pool level was observed . Relaxed cells exhibit very similar behavior to strigent cells in the same shift-up . Our data argue strongly against an exclusive role for pGpp in regulating RNA synthesis during niutritional shift-up . The kinetic data of {3H}guanosine uptake into GTP and ppGpp pools were analyzed to determine the rate of pGpp synthesis . This rate was found to be similar during expotential growth in either Tris-acetate medium . During a shift-down from Tris-glucose to Tris-acetate medium the rate of ppGpp syntesis fell by a factor of 1.5 to 2 and the turnover rate is reduced 6- to 8-fold, suggesting that the expansion in the ppGp pool during shift-down canot be due to derepression of synthesis.

J Biol Chem, 1975 Jan 10, 250(1), 164 - 70
Properties of membrane-associated sialyltransferase of Escherichia coli; Vijay IK et al.; Membrane-associated sialyltransferase complexes in Escherichia coli K-235 can be dissociated by lipid deletion and reassembled by the addition of undecaprenyl phosphate, a unique membrane-bound lipid coenzyme . Following disruption of the cells by pressure disintegration and centrifugal fractionation, the sialyltransferase activity is assocatied with both a "particulate" and "soluble" complex . Kinetic studies as well as sugar nucleotide, metal ion, pH, ammonium sulfate, and thiol reagent requirements showed these two complexes contained functionally identical enzymatic activities . Isopycnic sucrose density gradient centrifugation studies carried out on unfractionated total membranes established that these sialytransferase activities were associated with membrane hybrids composed of different relative amounts of inner and outer membranes . Enzyme localization studies employing DPNH oxidase, a marker for the inner membrane, and relative phospholipid to protein composition determinations in the two complexes, provided added support for this conclusion . Sialyl polymer synthesis was not dependent on the incorporation of other monosaccharides and had no demonstrable metal ion requirement . Kinetic studies showed that the Km for cytidine 5-monophospho-N-acetylneuraminic acid in intact soluble and particulate enzyme preparations was 8.1 times 10-5M and 9.2 times 105M, respectively . Similarly, both enzyme complexes had nearly identical Vmax values . Following reassembly of delipidated enzyme preparations, however, there was a 10-fold increase in the Km value for the particulate enzyme and a 3-fold increase for the soluble enzyme . This increase was accompanied by an increase of approximately the same magnitude in the Vmax values . Since the lipid coenzyme was limiting in intact enzyme preparations, the increase in Vmax reflected an increase in the concentration of the active lipid in reconstituted complexes . Sialyl polymer synthesis in intact membrane preparations was stimulated by the exogenous addition of lipid . Insertion of the carrier lipid was dependent on temperature . At 37 degrees, a 120% increase in sialytransferase activity was observed while only a 35% increase was observed at 30 percent . At 20 degrees, no stimulation occurred . Fluidity of the lipid phase is apparently required for proper function of this membraneassociated enzyme complex . Thus, at 20 degrees, a temperature below the membrane lipid transition temperature, the lipids are relatively immobile.

Science, 1975 Jan 10, 187(4171), 27 - 35
Genetic regulation: the Lac control region; Dickson RC et al.; The nucleotide sequence of the lac promoter-operator region has been determined . The 122 base pairs comprising this region include the recognition sites for RNA polymerase, the positive regulatory protein, CAP, and the negative regulatory protein, the repressor . Identification of mutant variants of the sequence combined with the in vitro biochemical studies of others has allowed us to tentatively identify the recognition site for each of these proteins, and to suggest how CAP might act at a distance to affect the interaction of RNA polymerase with the promoter.

J Biol Chem, 1975 Jan 10, 250(1), 322 - 30
Effect of N-bromosuccinimide modification on dihydrofolate reductase from a methotrexate-resistant strain of Escherichia coli . Activity, spectrophotometric, fluorescence and circular dichroism studies; Williams MN; When dihydrofolate reductase from a methotrexate-resistant strain of Escherichia coli B, MB 1428, is treated with approximately a 5 mol ratio of N-bromosuccinimide (NBS) to enzyme at pH 7.2 and assayed at the same pH, there is a 40% loss of activity due to the modification of 1 histidine residue and possibly 1 methionine residue before oxidation of tryptophan occurs . The initial modification is accompanied by a shift of the pH for maximal enzymatic activity from pH 7.2 to pH 5.5 Upon further treatment with N-bromosuccinimide, the activity is gradually reduced from 60 to 0% as tryptophan residues become oxidized . An NBS to enzyme mole ratio of approximately 20 results in 90% inactivation of the enzyme . When the enzyme is titrated with NBS in 6 M guanidine HCl, 5 mol of tryptophan react per mol of enzyme, a result in agreement with the total tryptophan content as determined by magnetic circular dichroism . The 40% NBS-inactivated sample posses full binding capacity for methotrexate and reduced triphosphopyridine nucleotide, and the Km values for dihydrofolate and TPNH are the same as for the native enzyme . After 90% inactivation, only half of the enzyme molecules bind methotrexate, and the dissociation constant for methotrexate is 40 nM as compared to 4 nM for native enzyme in solutions of 0.1 M ionic strength, pH 7.2 Also, TPNH is not bound as tightly to the modified enzyme-methotrexate complex as to the unmodified enzyme-methotrexate complex . Circular dichroism studies indicate the 90% NBS-inactivated enzyme has the same alpha helix content as the native enzyme but less beta structure, while the 40% inactivated enzyme is essentially the same as the native enzyme . Protection experiments were complicated by the fact that NBS reacts with the substrates and cofactors of the enzyme . Although protection of specific residues was not determined, it was clear that TPNH was partially protected from NBS reaction when bound to the enzyme, and the enzyme, and the enzyme was not inactivated by NBS until the TPNH had reacted.

J Biol Chem, 1975 Jan 10, 250(1), 149 - 55
A deoxyribonucleic acid ligase from nuclei of rat liver . Purification and properties; Zimmerman SB et al.; A DNA ligase has been extensively purified from nuclei of rat livers . The ligase seals single strand nicks in DNA with any of the four usual bases on either the 3 or 5 sides . It requires ATP and a divalent cation (Mg-2plus or Mn-2plus) for activity . At low Mg-2plus concentrations the activity is greatly stimulated by a variety of monovalent cations . Relatively small excesses of either monovalent or divalent cation above the amounts which give maximal activity lead to inhibition of activity . Poly(G) and poly(I) inhibit ligase activity; several other polyribonucleotides are not inhibitory . Low concentrations of inorganic pyrophosphate are inhibitory . The molecular weight of the ligase is estimated from gel filtration to be about 10 times 10-4.

J Biol Chem, 1975 Jan 10, 250(1), 141 - 8
Nicotinamide adenine dinucleotide phosphate-malic enzyme of rat liver . Purification, properties, and immunochemical studies; Li JJ et al.; Rat liver malic enzyme (EC 1.1.1.40) was purified from livers of rats fasted and refed a high sucrose diet containing 1% desiccated thyroid powder . The purification was accomplished by a six-step procedure . The specific activity of the purified enzyme was increased 181-fold above that of the initial high speed supernatant of liver extracts . Slight additional purification of malic enzyme was achieved with preparative disc electrophoresis . The specific activities of the purified rat liver malic enzyme from the least two steps were between 28.0 and 30.5 units per mg of protein . Homogeneity of the purified enzyme was determined by disc and starch gel electrophoresis as well as sedimentation velocity and sedimentation equilibrium studies . The molecular weight and S20, w values of rat liver malic enzyme are 268,000 and 10.2, respectively . Amino acid analysis based on milligram of protein hydrolyzed yielded higher amounts of leucine and glutamic acid but lower quantities of alanine and voline per subunit than the corresponding Escherichia coli enzyme...

J Biol Chem, 1975 Jan 10, 250(1), 127 - 31
Threonine deaminase from Escherichia coli . II . Maturation and physical properties of the enzyme from a mutant altered in its regulation of gene expression; Calhoun DH et al.; The biosynthetic L-threonine deaminase (L-threonine hydrolase deaminating, EC 4.2.1.16) has been purified from Escherichia coli K12 regulatory mutant CU18 . This mutant has properties that follow the predictions of the autogregulatory model previously proposed for the control of synthesis of the isoleucine-valine biosynthetic enzymes . The autoregulatory model specifies that L-threonine deaminase participates in the control of the expression of the ilv ADE gene cluster as well as the ilv B gene and ilv C gene, which constitute three separate units of regulation . The single mutation in strain CU18 results in altered regulation of ilv gene expression and in the production of an altered L-threonine deaminase . The immature form of the enzyme purified from mutant CU18 exhibits an altered response to L-valine, a maturation-inducing ligand . The native form of the mutant is altered in its apparent Km for L-threonine and in its response to the effects of L-valine and L-isoleucine upon catalytic activity . The mutant and wild type L-threonine deaminases differ in the apoenzyme formed as a consequence of alkaline dialysis . Dialysis of the mutant enzyme yields an apoenzyme mixture, apparently of dimers and monomers, while the wild type enzyme yields only dimers . The CU18 L-threonine deaminase, is however, indistinguishable from the wild type enzyme in molecular weight and subunit composition.

Biochim Biophys Acta, 1975 Jan 6, 378(1), 80 - 91
Escherichia coli mutants deficient in RNA accumulation at high temperature; Chaney SG et al.; A selection technique is described which has permitted isolation of 10 mutants that continue to form protein, but are deficient in the accumulation of rRNA at 42 degrees C . DNA-RNA hybridization experiments have demonstrated that most of these mutants are specifically defective in their ability to synthesize rRNA at the restrictive temperature . The bulk of the pulse-labeled RNA formed in such mutants at 42 degrees C appears to be mRNA, with normal instability as measured in the presence of rifampicin . The remaining mutants appear to synthesize normal levels of rRNA, but that rRNA does not accumulate in a stable form . Since all of these mutants continue to form protein, and most do not accumulate significant levels of ppGpp at 42 degrees C, it appears likely that the shut-off of rRNA synthesis at 42 degrees C does not act through a lesion in the rel locus . Thus, these mutants may reveal another element(s) required to promote ribosomal RNA formation.

Biochim Biophys Acta, 1975 Jan 6, 378(1), 64 - 72
Aminoacylation of tRNA-Leu species from Escherichia coli and from the cytoplasm, chloroplasts and mitochondria of Phaseolus vulgaris by homologous and heterologous enzymes; Guillemaut P et al.; Leucyl-tRNA synthetase from Phaseolus vulgaris chloroplasts could be separated from its cytoplasmic counterpart upon chromatography on hydroxyapatite, but the cytoplasmic and mitochondrial leucyl-tRNA synthetases could not be distinguished . The tRNALeu species from the various plant cell compartments and from Escherichia coli were aminoacylated using either homologous or heterologous enzymes; the levels of aminoacylation and the profiles of the leucyl-tRNAs upon reverse-phase chromatography were studied . Cytoplasmic tRNALeu species could be aminoacylated by the cytoplasmic or by the mitochondrial enzymes and in both cases yielded two peaks upon reverse-phase chromatography (RPC-5) . But they could not be charged by the chloroplast-specific or by the E . coli enzynes . Mitochondrial tRNALeu species could be charged by the mitochondrial or by the cytoplasmic enzymes and in both cases yielded four peaks upon reverse phase (RPC-5) chromatography . But they could not be aminoacylated using the chloroplast-specific or the E . coli leucyl-tRNA synthetases . Chloroplastic tRNALeu species can be divided into two classes: the first class contains four isoacceptor species which can be charged by the cytoplasmic or mitochondrial enzymes, but not by the chloroplast-specific or the E . coli enzymes; the second class contains three chloroplast-specific tRNALeu species which can be charged by the chloroplast-specific or the E . coli enzymes but not by the cytoplasmic or the mitochondrial enzymes . There are five isoacceptor tRNALeu species in E . coli; all are charged by the E . coli or the chloroplast-specific enzymes, while only one is aminoacylated by the plant cytoplasmic or mitochondrial enzymes.

Eur J Biochem, 1975 Jan 2, 50(2), 419 - 24
Determination of the kinetic constants of glucose-6-phosphate 1-epimerase by non-linear optimization; Chance EM et al.; 1 . The overall kinetic constants of the reversible anomerisation of d-glucopyranose 6-phosphate from alpha to beta non-enzymatically as well as catalysed by glucose-6-phosphate 1-epimerase are determined by application of a novel computerized non-linear optimization technique . 2 . The non-enzymic rate constants for the anomerisation of d-glucopyranose 6-phosphate from alpha to beta and reverse are 0.0658 and 0.0389s-minus 1, respectively . The Michaelis constants of the enzymic reaction are (see journal for formulas) with the turnover numbers of 1950s-minus 1 and 446s-minus 1 for the conversion of d-glucopyranose 6-phosphate from alpha to beta and reverse, respectively.

Eur J Biochem, 1975 Jan 2, 50(2), 343 - 9
Properties of ATP and UTP analogues with P-S-C-5' bonds; Stutz A et al.; Analogues of ATP and UTP bearing C-5'-S-P ester bonds were found not to be substrates but weak competitive inhibitors of Escherichia coli RNA polymerase . The K-i values of the analogues obtained in the transcription of poly{d(A-T)} or poly(dT) under various conditions are in the order of millimolar . Evidence was derived from proton magnetic resonance spectra that nucleotides with C-5'-S-P bonds do not exist in gauche-gauche conformation normally adopted by natural occurring nucleotides . This leads us to assume that the gauche-gauche conformation is an essental prerequisite for substrates of RNA polymerase . Ado-5'-S-PPP substituted for ATP as substrate of hexokinase from yeast rather effectively thus indicating that a distinct stereochemical orientation of the alpha-phosphate ester bond is not a stringent requirement for substrates of this enzyme.

Eur J Biochem, 1975 Jan 2, 50(2), 425 - 30
The threonine-sensitive homoserine dehydrogenase and aspartokinase activities of Escherichia coli K12 . Specific inactivation of the homoserine dehydrogenase activity by the affinity label, 2-amino-4-oxo-5-chloropentanoic acid; Hirth CG et al.; 2-Amino-4-oxo-5-chloropentanoic acid inactivates specifically the homoserine dehydrogenase activity of the bifunctional enzyme, aspartokinase I--homoserine dehydrogenase I . The aspartokinase activity remains essentially untouched and retains its threonine sensitivity . The inactivation of the dehydrogenase requires the covalent binding of one equivalent of the analogue per subunit . Alkylation does not affect the tetrameric state of the protein . The alkylating agent, a substrate analogue, meets the qualitative and quantitative requirements of an affinity label.

Eur J Biochem, 1975 Jan 2, 50(2), 431 - 5
The threonine-sensitive homoserine dehydrogenase and aspartokinase activities of Escherichia coli K12 . Distribution and accessibility to antibodies of some epitopes of the bifunctional enzyme; Costrejean JM et al.; In the presence of l-threonine, the allosteric effector, most of the antigenic determinants situated in the aspartokinase region of the wild-type enzyme become unavailable to the antibodies raised against a fragment of the enzyme containing this region and devoid of homoserine dehydrogenase activity . The cross-reactivities of the antibodies raised against this fragment (extracted from a nonsense mutant) and a fragment endowed with homoserine dehydrogenase activity but devoid of aspartokinase activity (obtained by limited proteolysis) with the corresponding antigens were studied . The conclusion is drawn that the two fragments, which share an overlapping sequence of molecular weight about 17,000, share at least two antigenic determinants.

Intervirology, 1975-76, 6(2), 98 - 107
phi gamma: A coliphage coliphage related to, but distinct from the phi 80 virion; Calberg-Bacq CM et al.; The coliphage phi gamma, though capable of genetic recombination with phi 80, is morphologically distinct from the phi 80 virion . It has a prolate head (58.4 X 46.7 nm) bearing a tail (143 nm) which is strikingly flexible . On the basis of their buoyant density in CsC1, both infectious and transducing phi gamma particles form a single population . This density value is slightly higher than that of the phi 80 virion . The phi gamma chromosome is a double-stranded linear DNA molecule of 13.4 mum in length (corresponding mol . wt.: 27.6 X 10(6)) . From its melting temperature and its buoyant density in CsC1, phi gamma DNA appears to have a base composition very close to that of Escherichia coli DNA.

Acta Med Acad Sci Hung, 1975, 32(2), 123 - 7
Pernicious anaemia: its pathogenesis in the light of the beneficial effect of duodenal intrinsic factor; Horanyi M et al.; Oral ingestion of powdered duodenal mucosa with small doses of vitamin B12 proved efficient in four cases of untreated pernicious anaemia . The erythropoietic as well as the other responses were similar as those elicited by any reliable anti-pernicious drug . It has thus been confirmed that the growth factor demonstrated earlier in human duodenal juice by means of E . coli mutant corresponds in fact to the intrinsic factor . It is suggested that besides the deficiency of gastric intrinsic factor the duodenal intrinsic factor and the immune mechanisms neutralizing it are also involved in the pathogenesis . It has been shown that the aqueous extracts of hog stomach and duodenum differ in addition to their growth factor and nitrogen contents, also in aminoacid composition.

Acta Microbiol Pol A, 1975, 8(3), 169 - 77
Isolation and some properties of colicin V preparations; Kucharzewska T et al.; E . coli strain CLI(V) produces colicin V which can exist in two chemically different forms . A heat-stable, liposaccharide-protein complex is present as a main component of the cell wash . An intracellular colicin is a heat-labile and seems to be a simple protein . Preliminary experiments have shown that colicin V inhibits simultaneously synthesis of protein, RNA and DNA . Its mode of action is similar to colicins: E1, B, K and A.

Adv Exp Med Biol, 1975, 53, 123 - 36
Alterations in chromatin functions during aging in vitro; Ryan JM; Age-associated alterations in the chromatin functions of human diploid cells have been observed . These alterations include: (1) a decline in the rate of histone acetylation; (2) a reduction in the rate of RNA synthesis as measured by (a) the rate of 3H-uridine incorporated into the RNA of young and old cells; (b) comparison of the template activity of isolated chromatin from young and old cells using E . coli RNA polymerase and (c) measurement of chromatin template activity using the endogenous RNA polymerase of young and old cells . It is suggested that the nondividing state of old cells may be the result of the inability to synthesize specific RNA molecules (and perhaps specific proteins) necessary for the cell to continue through the cell cycle.

J Supramol Struct, 1975, 3(5-6), 426 - 47
Studies on the primary structure of 14 proteins from the large subunit of Escherichia coli ribosomes with an improved protein sequenator and with mass spectrometry; Wittmann-Liebold B et al.; Fourteen proteins from the large subunit of Escherichia coli ribosomes were analyzed in an improved sequenator . In addition to our previously described modifications of a Beckman sequenator, new valves which work free of a dead volume were constructed . By this and the previous improvements (e.g., a new vacuum system with a recorder, cool traps, automatic conversion) much better results were obtained than before . It was even possible to use (in addition to the standard methods, e.g., thin-layer chromatography and amino acid analysis) mass spectrometry without preceding gas chromatography for identification of the released PTH amino acids . Our experience with the various methods, especially mass spectrometry, is described and the techniques are compared . The results obtained by the described methods on the amino acid sequences of the 14 ribosomal proteins are summarized.

J Virol, 1975 Jan, 17(1), 94 - 105
T7 protein synthesis in F-factor-containing cells: evidence for an episomally induced impairment of translation and relation to an alteration in membrane permeability; Blumberg DD et al.; T7 infection of F-factor-containing PIFA+, B+ cells is abortive . In spite of the presence of mRNA for all three classes of T7 proteins, only the earliest of the T7 proteins are synthesized . A crucial question is whether the failure of T7 to develop in PIFA+, B+ cells is the result of an inability to translate the late classes of T7 mRNA or, as has been recently suggested (Britton, and Haselkorn, 1975; Condit, 1975), whether it is the result of a more generalized alteration in membrane permeability . We have examined the effects of the wild-type PIFA+, B+ spisome and two sipsomal mutations (pifA- and pifB-) on in vitro translation and membrane permeability . In vivo the episomal mutations allow partial or complete T7 development to occur . We demonstrate that cell-free protein-synthesizing systems from T7-infected PIFA+, B+ cells show a three- to fivefold decrease in the rate of translation of both natural and synthetic mRNA . In addition, ribosomes from T7-infected PIFA+, B+ cells are defective in their ability to bind Fmet tRNAf in response to natural mRNA . By contrast, cell-free extracts from T7-infected pifA-(PIFA-, B+) celld retain the ability to bind Fmet defective T7-infected PIFA+, B+ rigosomes can be restored to full activity by a trypsin-sensitive fraction from uninfected PIFA+, B+ or T7-infected PIFA-, B+ cells . Despite the differences in translational capacity of these extracts, both T7-infected PIFA+, B+ and PIFA-, B+ cells display the same permeability lesions as measured by the loss of ATP from the cells into the supernatant . Mutation of the episome of pifB- prevents the loss of ATP from the cells after T7 infection.

Immunol Commun, 1975, 4(5), 407 - 17
Blastogenesis of human lymphocytes by endotoxin; Hsu SH; Lipopolysaccharides of S . typhimurium, S . enteritidis and E . coli at wide range of concentrations were used to induce blastogenesis in human and mouse (nude) lymphocytes . Human lymphocytes from peripheral blood showed positive response to at least one source of LPS (stimulation index of 2-9) . The optimum concentration resulting in maximum stimulation varied with different individual, sources and concentrations of LPS used . Lymphocytes from cord blood failed to respond to LPS, but had positive response to PHA . All three LPS produced about equally strong mitogenic effects on mouse spleen cells.

Biochimie, 1975, 57(6-7), 711 - 48
The determination of the primary structure of the 16S ribosomal RNA of Escherichia coli . III . Further studies; Ehresmann C et al.; In this paper, we describe in detail the recent progress in the nucleotide sequence analysis of the 16S ribosomal RNA from E . coli . The sequence which has been partially or completely determined so far encompasses 1520 nucleotides, i.e . about 95 percent of the molecule . Possible features of the secondary structure are suggested on the basis of the nucleotide sequence and data on sequence heterogeneities, repetitions and the location of modified nucleotides are presented.






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