J Bacteriol, 1975 Aug, 123(2), 481 - 91 Ribonucleic acid destruction and synthesis during intraperiplasmic growth of Bdellovibrio bacteriovorus; Hespell RB et al.; During growth of Bdellovibrio bacteriovorus on (2-14C)uracil-labeled Escherichia coli approximately 50% of the radioactivity is incorporated by the bdellovibrio and most of the remainder is released as free nucleic acid bases . Kinetic studies showed that 50 and 30S ribosomal particles and 23 and 16S ribosomal ribonucleic acid (RNA) of E . coli are almost completely degraded by the first 90 min in a 210- to 240-min bdellovibrio developmental cycle . Synthesis of bdellovibrio ribosomal RNA was first detected after 90 min . The specific activity and the ratio of radioactivity in the bases of the synthesized bdellovibrio RNA was essentially the same as those of the substrate E . coli . The total radioactivity of the bdellovibrio deoxyribonucleic acid (DNA) exceeded that in the DNA of the substrate E . coli cell, and the ratio of radioactivity of cytosine to thymine residues differed . Intraperiplasmic growth of B . bacteriovorus in the presence of added nucleoside monophosphates (singly or in combination) significantly decreased the uptake of radioactivity from (2-14C)uracil-labeled E . coli; nucleosides or nucleic acid bases did not . It is concluded that the RNA of the substrate cell, in the form of nucleoside monophosphates, is the major or exclusive precursor of the bdellovirbrio RNA and also serves as a precursor for some of the bdellovibrio DNA. J Bacteriol, 1975 Aug, 123(2), 443 - 8 Random replication of the stringent plasmid R1 in Escherichia coli K-12; Gustafsson P et al.; The R-factor R1 is present in a low number of copies per genome (near unity, so-called stringent control of replication) . The replication of R1 was studied in a density-shift experiment . One generation after the shift about 20% of the R1 copies had not replicated, whereas about 20% had replicated at least twice . The results are in quantitative accordance with a random replication of R1 in which the replicating molecules are taken from a cytoplasmic plasmid pool and transferred back to the pool after replication . This is analogous to the results obtained by Bazaral and Helinski (1970) and Rownd (1969) for plasmids that are present in 10 to 20 copies per genome (so-called relaxed control of replication) . Hence, there seem to be no difference between stringent and relaxed plasmids with respect to selection of plasmid molecules for replication . However, we cannot tell whether all R1 copies in a cell replicate during a fraction of or throughout the cell cycle . The random selction of plasmid copies for replication has to be considered when models for control of replication are constructed. Infect Immun, 1975 Aug, 12(2), 240 - 4 Heat-stable Escherichia coli enterotoxin production in vivo; Whipp SC et al.; Hysterectomy-derived, colostrum-deprived piglets were infected with enterotoxigenic Escherichia coli on day 4 of life . Samples of feces and intestinal contents were collected and tested in infant mice for enterotoxic activity . Positive enterotoxic responses were observed in mice given filtrates of feces and intestinal contents from piglets infected withe enterotoxigenic E . coli known to produce heat-stable enterotoxin but not heat-liabile enterotoxin in vitro . It is concluded that heat-stable enterotoxigenic E . coli induce diarrhea by production of heat-stable enterotoxin in vivo. Gastroenterology, 1975 Aug, 69(2), 303 - 9 Complications of endoscopic retrograde cholangiopancreatography . Analysis of 300 consecutive cases; Zimmon DS et al.; A retrospective analysis of complications arising from 300 consecutive attempts at endoscopic retrograde cholangiopancreatography (ERCP) in 278 patients was undetaken to determine the rate and severity of complications . An over-all complication rate of 5% (15 cases) was documented . Complications were categorized in terms of those arising from endoscopy itself or from the administration of pharmacological agents (7 cases), and those observed after the injection of radioopaque contrast into the biliary tree or pancreas (8 cases) . Complications which might be considered coincidental to a patient's underlying illness were not excluded . Complications were significantly more frequent after injection of diseased duct systems . Brief, self-limited pancreatitis after retrograde pancreatography occurred in 5 of 90 patients with pancreatic disease . No cases of pancreatitis were observed after retrograde pancreatography in 102 patients without pancreatic disease X2 = 5.82, P less than 0.025) . Sepsis occurred after retrograde cholangiography in 3 of 56 patients with extrahepatic biliary obstruction . In the absence of extrahepatic obstruction, cholangiography was performed without complication in 85 cases (X2 = 3.62, P less than 0.1), although 25 of these had intense cholestasis due to hepatic parenchymal disease . This analysis provides the basis for modifications of ERCP technique and management that may reduce the future incidence of complications . This study suggests that the incidence and severity of complications that arise from ERCP compare favorably with procedures of equivalent diagnostic yield. Clin Chem, 1975 Aug, 21(9), 1245 - 52 An analytical system for rapid separation of tissue nucleotides at low pressures on conventional anion exchangers; Khym JX; An analytical anion-exchange procedure has been developed for the rapid separation of acid-soluble nucleotides (the so-called "free" or tissue nucleotides) . It permits assay at low pressures (275-415 kPa; 40-60 psi) in less than 1 h on 10-cm columns of Aminex resins (conventional styrene-type anion exchangers) with alkaline citrate solutions as the eluent . Separation variables have been investigated, to determine optimum conditions for the routine analysis of samples containing tissue nucleotides . Also described here is a simple solvent-extraction procedure for removing HCIO4 or CCI3CO2H quantitatively from cell extracts that contain acid-soluble nucleotides: they are removed from aqueous acid solutions with a water-insoluble amine dissolved in a water-immiscible solvent. J Exp Med, 1975 Aug 1, 142(2), 473 - 82 The lipoprotein of the outer membrane of Escherichia coli: a B-lymphocyte mitogen; Melchers F et al.; The lipoprotein of the outer membrane of Escherichia coli is a B-cell mitogen in mice . Polyclonal activation of B lymphocytes was measured by an increase in thymidine uptake, by the development of plaque-forming cells against densely coupled trinitrophenylated sheep red cells, and by selectively increased rates of synthesis and secretion of leucine-labeled IgM . Murein-free and muropeptides-containing lipoprotein are effective in B-cell activation, while free murein is inactive . Removal of ester-linked fatty acids from the amino-terminal end of the lipoprotein by alkaline hydrolysis abolishes the mitogenicity of the lipoprotein . B lymphocytes from high responder (C3H/Tif and BALB/c nu/nu) or from low responder (C3H/HeJ) mice to the mitogen lipopolysaccharide (LPS) both respond well to the lipoprotein . Anti-immunoglobulin antibodies inhibit the mitogenic stimulation of B cells by lipoprotein . A complex of structures including the Ig-receptor molecules, the LPS receptor, and the lipoprotein receptor appear involved in the regulation of mitogenic stimulation of B cells to proliferation and differentiation to IgM-secreting cells. Chem Biol Interact, 1975 Aug, 11(2), 91 - 9 The interaction of natural tetra-azacyclopentazulene dyes with DNA and their effects on the DNA and RNA polymerase reactions; Quadrifoglio F et al.; The interaction of two natural tetra-azacyclopentazulene dyes with native calf thymus DNA was studied by means of microcalorimetric, viscosimetric, and spectroscopic measurements . The results are consistent with the hypothesis of an intercalative-binding . However, comparison of calorimetric studies shows that the changes in enthalpy associated with the interaction of these compounds with DNA are, in absolute value, significantly lower than those found with known intercalating agnets (daunomycin, ethidium bromide) . The influence of these dyes on the template capacity of DNA in the in vitro synthesis of nucleic acids was also determined . Under the conditions used, these compounds selectively inhibited DNA synthesis . No appreciable inhibitory effect upon E . coli RNA polymerase was observed . Both compounds had greater inhibitory effect on rat liver high molecular weight DNA polymerase than E . coli DNA polymerase I . Zoanthoxanthin was a more effective inhibitor than 3-norzoanthoxanthin. Proc Natl Acad Sci U S A, 1975 Aug, 72(8), 2950 - 4 Interaction of elongation factor Tu with 2'(3')-O-aminoacyloligonucleotides derived from the 3' terminus of aminoacyl-tRNA; Ringer D et al.; The interaction between Escherichia coli elongation factor-Tu-GTP complex and chemically synthesized 2'(3')-O-aminoacyldinucleoside phosphates with the nucleotide sequence of the 3' terminus of aminoacyl-tRNA (AA-tRNA) has been studied . It was found that C-A-Phe, C-A-Pro, and C-A-Asp interact with EF-Tu-GTP, causing the release of GTP bound to the enzyme . The specificity of this interaction closely resembles that of AA-tRNA since C-A and C-A(Ac-Phe) as well as the corresponding tRNAs are inactive . The 3'-O-aminoacyl derivative C-2'-dA-Phe does not interact with EF-Tu-GTP, whereas the 2'-O-aminoacyl derivative C-3'-dA-Phe is almost as active as the 2'(3')-O-aminoacyl derivative, C-A-Phe . C-A-Phe also interacts with the EF-Tu-GDP complex in a manner similar to its interaction with EF-Tu-GTP . It is concluded that interaction of 2'(3')-O-aminoacyloligonucleotides possessing the sequence of the 3' terminus of AA-tRNA is analogous to the interaction of that terminus with EF-Tu and it is suggested that EF-Tu is specific for 2'-O-AA-tRNA. Biotechnol Bioeng, 1975 Aug, 17(8), 1157 - 81 Kinetic behavior of microencapsulated beta-galactosidase; Wadiak DT et al.; Escherichia coli beta-D-galactosidase (E.C . 3.2.1.23) was immobilized in cellulose nitrate membrane microcapsules and the reaction kinetics with o-nitrophenyl-beta-D-galactopyranoside (ONPG), lactose, and whole milk were studied using both continuous stirred tank and packed bed reactor configurations . The results of the experiments gave effectiveness factors of 0.3 for ONPG, 0.6 to 0.7 for lactose in solution, and close to unity for lactose in milk . Using a coupled mass transfer and kinetic model, it was possible to estimate the permeability of the microcapsule membrane from the reactor data . Membrane permeabilities on the order of 5 X 10(-3) and 3 X 10(-4) cm/sec were estimated for ONPG and lactose, respectively . It was determined that the membrane was the limiting mass transfer resistance for the overall reaction . The analysis showed that within the microcapsule, the reaction is reaction rate limited for lactose and slightly diffusion limited for ONPG. Zentralbl Bakteriol {Orig B}, 1975 Aug, 160(6), 601 - 27 {Test methods for surgical hand disinfection (author's transl)}; Reber H et al.; As exemplified by a test preparation, methods for assessing the effect of hand disinfection on the resident flora are put up for discussion . The test of a method for hand disinfection must make allowance for conditions prevailing in practice . Accordingly, the following steps should be taken into consideration: A . Single use 1 . Immediate disinfecting effect 2 . Duration of the disinfecting effect B . Repeated use 3 . Course of the initial colony count 4 . Immediate disinfecting effect 5 . Behaviour of colony count after interruption of the disinfecting method applied . For the purpose of comparing the total count, as with all other disinfecting methods, the colony count must be determined by fractional collection methods; to this end, Traub's procedure may be modified, possibly using the plastic bag method described by Gaschen . A careful statistical evaluation with appropriate transformation of the results is indispensable. Eur J Biochem, 1975 Aug 1, 56(1), 183 - 92 Synthesis of mitochondrial proteins in an Escherichia coli cell-free system directed by yeast mitochondrial DNA; Scragg AH et al.; The optimum conditions for transcription in vitro of yeast mtDNA into biologically relevant RNA by Escherichia coli RNA polymerase holoenzyme and yeast mitochondrial RNA polymerase was found to critically depend on salt concentration . RNA was transcribed (at 0.25 M KCl concentration) from high-molecular-weight mtDNA which was then translated in an E . coli (S-30) cell-free protein synthesising system . Efficient translation of mitochondrial RNA was achieved using conditions which had also been determined to be optimal in other systems . Identification of the polypeptides produced in the translation system was made using antiserum raised against mitochondrial membranes . Electrophoresis of the completely dissociated antigen-antibody complexes using dodecylsulphate-polyacrylamide gels revealed that the system in vitro produced polypeptides of similar molecular weight to those synthesised in vivo by cycloheximide-inhibited whole cells. J Med Chem, 1975 Aug, 18(8), 849 - 51 Aromatic esters of 5-O-desosaminylerythronolide A oxime; LeMahieu RA et al.; Several substituted aromatic esters of the C-3 hydroxyl of 5-O-desosaminylerythronolide A oxime were prepared . Ribosomal binding studies showed that meta substituents on the aromatic ring gave the most active analogs . The esters described were all inactive in vivo at the maximum level tested. J Med Chem, 1975 Aug, 18(8), 784 - 7 Synthesis and biological activity of 5-fluoro-4'-thiouridine and some related nucleosides; Bobek M et al.; The synthesis of a series of 4'-thio-5-halogenopyrimidine nucleosides, including the 5-fluoro, chloro, bromo and iodo derivatives, has been carried out by condensation of the 2,4-bis-O-trimethylsilyl derivatives of the corresponding pyrimidine bases with the protected 4-thio-D-ribofuranosyl chloride . Among these, the alpha and beta anomers of 4'-thio-5-fluorouridine inhibited the growth of leukemia L1210 cells at concentrations of 4 x 10(-7) and 2 x 10(-7) M, respectively, and that of S . faecium at 4 x 10(-9) and 6 x 10(-10) M, respectively . These compounds retained marked activity against strains of S . faecium resistant to 10(-3) M 5-fluorouracil or 5-fluorouridine . As determined in S . faecium cultures, 4'-thio-5-fluorouridine decreased the total protein content of the cells more markedly than it did their RNA or DNA content . X-Ray crystallography showed that substitution of sulfur for the oxygen in the carbohydrate ring markedly changes the conformation of that moiety. Aust J Exp Biol Med Sci, 1975 Aug, 53(4), 305 - 13 Antibody response in sows following intramammary or intramuscular immunization with a killed Escherichia coli vaccine; Watson DL; Pregnant sows were immunized either intramuscularly or intramammarily with a killed E . Coli vaccine . Samples of blood and mammary secretion were assayed for antibody to E . Coli cells and E . coli enterotoxin . The association of each class of immunoglobulin with antibody to the bacterial cells was also estimated . It was found that immunization by either method increased direct agglutination titres and antiglobulin enhancement titres in serum and mammary secretion . There were no major differences in these parameters between the two groups of sows . In serum and mammary secretion from both groups of sows IgG was quantitatively the most important class of immunoglobulin associated with antibody to E . coli cells . Immmunization had the effect of increasing the percentage of IgG and IgM associated with antibody in blood serum from about 5% to about 20% . In contrast, the percentage of IgA in serum with antibody activity against E . coli was around 20% prior to immunization and did not alter significantly after immunization . Samples of blood and mammary secretion from sows immunized intramammarily were more effective in neutralizing E . coli enterotoxin than corresponding samples from sows immunized intramuscularly; the biological significance of this finding is at present uncertain. Eur J Biochem, 1975 Aug 1, 56(1), 289 - 94 Ribonucleotide reductase from Escherichia coli: demonstration of a highly active form of the enzyme; Eriksson S; Ribonucleotide reductase from Escherichia coli consists of two nonidentical subunits, proteins B1 and B2 . The activity of the enzyme in crude extracts prepared from mechanically disrupted bacteria is very low . Enzyme activity is stimulated 5 to 10-fold by addition of an excess of either subunit . Concentrated extracts from cells lysed gently on Cellophane discs (Schaller et al.) contained 10 to 20-fold higher activity than extracts from mechanically disrupted cells . This activity was not further stimulated by either B1 or B2 . The system is suitable for complementation tests for the analysis of temperature-sensitive mutants affecting the ribonucleotide reductase system . Concentrated high-speed supernatants from E . coli treated with lysozyme (Wickner et al.) also contained a high ribonucleotide reductase activity, which was stimulated slightly or not at all by addition of B1 and B2 . This active form of the enzyme was unstable and could not be purified . The results suggest that the intracellular form of the enzyme consists of a tight complex of proteins B1 and B2, possibly stabilized by other intracellular structures. Appl Microbiol, 1975 Aug, 30(2), 267 - 70 Chemostat culture of Escherichia coli K-12 limited by the activity of alkaline phosphatase; King SL et al.; The growth-limiting reaction of a chemostat culture of Escherichia coli K-12 was the hydrolysis of beta-glycerophosphate by alkaline phosphatase . The culture was buffered at pH 5.2 where alkaline phosphatase was unable to supply phosphate to the cell at a rate sufficient to sustain the maximum rate of growth . Alkaline phosphatase activity in this system is discussed in terms of the so-called Flip-Flop mechanism. J Immunol, 1975 Aug, 115(2), 425 - 30 Activation by some T-independent antigens and B cell mitogens of the alternative pathway of the complement system; Bitter-Suermann D et al.; A number of T-independent antigens and B cell mitogens were examined for their ability to activate C3 via the alternative pathway of the complement system . Loss of hemolytically active C3, generation of anaphylatoxin activity, and immunoelectrophoretic conversion of C3 and factor B, were checked in normal and C4-deficient guinea pig serum, and, in some cases, in normal human serum . As judged by their activity in these assays, 10 lipopolysaccharides of different origin and constitution, pneumococcus type III polysaccharide, levan, dinitrophenylated aminoethyl-dextran, dinitrophenylated (D-glutamic acid, D-lysin) copolymer, polymerized flagellin, and pokeweed mitogen were all capable of initiating the alternative pathway, but differed with respect to their potency, their relative activity in the presence or absence of C4, and their ability to inhibit C3-turnover at high concentrations . Polyvinylpyrrolidone of intermediate molecular weight (4 x 10(4) daltons) was only active if the most sensitive assay was used (anaphylatoxin generation) . Other species of polyvinylpyrrolidone, depolymerized pneumococcal polysaccharide, aminoethyl-dextran, {D-glutamic acid, D-lysin} copolymer, phytohemagglutinin and concanavalin A failed to activate C3 . C3-consumption by concanavalin A was due to nonspecific binding. J Bacteriol, 1975 Aug, 123(2), 391 - 9 Regulation of the lysine biosynthetic pathway in Escherichia coli K-12: isolation of a cis-dominant constitutive mutant for AK III synthesis; Cassan M et al.; A method for isolating regulatory mutants for the synthesis of lysine biosynthetic enzymes in Escherichia coli is described . One of them is identified as a cis-dominant constitutive mutant for the synthesis of the lysine-sensitive asportokinase AK III (lysC gene). Proc Natl Acad Sci U S A, 1975 Aug, 72(8), 2895 - 9 Location of histones on simian virus 40 DNA; Polisky B et al.; The physical location of histone molecules in a simian virus 40 DNA-histone complex isolated from purified virions was examined using site-specific restriction endonucleases . The complex contains four host histone species but lacks histone F1 . Histones prevent complete cleavage of SV40 DNA by two restriction enzymes, HindIII and EcoRI . From the pattern of DNA fragments resulting from cleavage of the histone-DNA complex by the HindIII endonuclease, which makes six breaks on purified SV 40 DNA, we have concluded that histones are randomly arranged on SV40 DNA relative to restriction enzyme cleavage sites . The EcoRI endonuclease, which makes one break in SV40 NDA, was used to determine the degree of physical coverage of the SV 40 DNA molecule by histones . We observed that 80% of the EcoRI sites in the complex are accessible to the enzyme while 20% are "closed." This degree of coverage is consistent with the mass ratio of DNA:histone in the complex as revealed by the buoyant density of the formaldehyde-fixed complex . We conclude that the histones in the complex are located randomly on the SV 40 genome and cover approximatley 20% of the DNA . These results suggest that the histone species F2b, F2al, F2a2, and F3 are bound without regard to nucleotide sequence of SV 40 DNA. J Cell Physiol, 1975 Aug, 86(1), 91 - 104 Studies on the regulation of the three enzymes of the Leloir pathway in cultured mammalian cells . I . Effect of substitution of galactose for glucose as the sole hexose in the medium in human diploid cell strains and in a rat hepatoma line; Stern ES et al.; In human diploid cell strains, the substitution of galactose for glucose as the sole hexose in the medium had no measurable effect on the specific activity of the cell protein for any of the three enzymes of the Leloir pathway . These enzymes are galactokinase, alpha-D-galactose-1-phosphate: UDP glucose uridyl transferase and UDP galactose 4-epimerase . A cell strain from a patient with galactosemia had no detectable activity for the transferase . The substitution of galactose for glucose in the medium of these cells (which has been shown to cause the cells to accumulate galactose-1-phosphate) also failed to affect cellular activity for the three enzymes . Similarly, the three activities failed to respond to the substitution of galactose for glucose in cultures of a rat hepatoma line . Cells of this line have been shown by others to perform a number of the tissue-specific functions of liver . The failure of galactose to stimulate increasd cellular activity for the three enzymes represents a striking difference between the behavior of these enzymes in human diploid cell strains and their behavior in E . coli. J Cell Physiol, 1975 Aug, 86(1), 105 - 10 Studies on the regulation of the three enzymes of the Leloir pathway in cultured mammalian cells . II . A search for quantitative interrelationships between the three enzyme activities; Stern ES et al.; Studies on a normal human diploid cell strain revealed that the specific activity of the cell protein, for each of the three enzymes of the Leloir pathway, changed significantly as the cells grew . The kinetics of change in specific activity varied according to the enzyme being studied, and the kinetics for each enzyme varied from experiment to experiment . Within each experiment, there was no consistent correlation between specific activity for any one enzyme and specific activity for the other two . The ratios between the specific activities did not tend to remain constant as the absolute levels of specific activity changed . Hence, the activities did not behave coordinately . The kinetics of change in these ratios varied from experiment to experiment . The failure of galactose to stimulate increased cellular activity for the three enzymes (shown in the preceding paper), and the absence of a coordinate relationship between the activities, represent a striking difference between the behavior of these enzymes in human diploid cell strains and their behavior in E . coli. Cancer Res, 1975 Aug, 35(8), 1907 - 14 Complete and apparently specif local tumor regression using syngeneic or xenogeneic "tumor-immune" RNA extracts; Schlager SI et al.; Syngeneic and xenogeneic RNA-rich extracts of lymphoid tissues were used in an immunotherapeutic regimen to treat strain 2 guinea pigs that were given intradermal injections of a uniformly lethal dose (1 x 10(6)) of line 10 diethylnitrosamine-induced transplantable hepatoma cells . When 1 X 10(7) syngeneic nonsensitive peritoneal exudate cells, 2.5 mg RNA from line 10-immune strain 2 guinea pigs or line 10-immune Rhesus monkeys, and 1.0 mg of a line-10 tumor-specific antigen preparation were injected s.c . under the tumor cells injected 5 days previously, complete local tumor regression in all treated animals was observed . If either nonsensitive peritoneal exudate cells, RNA, or line 10 tumor-specific antigen was omitted, or if Escherichia coli RNA or RNA from animals sensitized to a different tumor (line 1) was used, little or no tumor regression was observed, suggesting that the action of the RNA may have resulted in an antitumor response specific for the noplasm being treated . The long-term tumor-free survival of all treated animals indicates that the action of the RNA is systemic, since metastases are known to occur frequently by the time our therapeutic regimen was given . Also, in testing the biological activity of the "tumor-immune" RNA in the in vitro cell-migration-inhibition assay, both the syngeneic and xenogeneic RNA extracts could transfer tumor-specific immunological sensitivity, as demonstrated by the elaboration of migration-inhibitory factor by the RNA-treated nonsensitive peritonial exudate cells in the presence of the line 10 tumor-specific antigen. Proc Natl Acad Sci U S A, 1975 Aug, 72(8), 3039 - 43 Arrangement of sequences in the inverted terminal repetition of adenovirus 18 DNA; Garon CF et al.; In contrast to the single-stranded circular molecules produced with denatured DNA from other adenoviruses, there was associated with nearly all circular molecules of adenovirus type 18 a visible, duplex projection . These projections had a mean contour length of 0.31 +/- 0.12 mum, equivalent to approximately 3% of genome length . Individual projections ranged in size from 0.1 to 2 mum . Alkaline sucrose gradient purification of single-stranded molecules did not affect formation of these projections, and treatment of a preparation of circular molecules with Neurospora crassa single-strand specific nucleases yielded 0.34 +/- 0.09 mum duplex fragments . Single-stranded circles did not form if a limited number of nucleotides were removed from the 3' ends of native molecules by Escherichia coli exonuclease III digestion prior to denaturation and annealing . In addition, preformed single-stranded circles could be converted to linear molecules by similar treatment . Based on the formation of specific heteroduplex structures when preparations of native DNA were denatured and reannealed and the absence of branches on linear, single-stranded molecules, we conclude that projections are generated by unusually long, inverted terminal repetitions . The repetitious sequences occur in place of rather than in addition to regular sequences . These data provide direct, visual evidence for the arrangement of bases in the inverted terminal repetition of adenovirus DNA. Proc Natl Acad Sci U S A, 1975 Aug, 72(8), 2945 - 9 Protein kinase and its regulatory effect on reverse transcriptase activity of Rous sarcoma virus; Lee SG et al.; We have studied the effect of protein phosphokinase (EC 2.7.1.37; ATP:protein phosphotransferase) and phosphoprotein phosphatase (EC 3.1.3.16; phosphoprotein phosphohydrolase) on reverse transcriptase (RNA-dependent DNA nucleotidyltransferase) activity of Rous sarcoma virus . Protein kinase from Rous sarcoma virus-transformed chick embryo fibroblasts was purified by DEAE-cellulose chromatography, Sephadex gel filtration, and isoelectric focusing . Purified reverse transcriptase from Rouse sarcoma virus was preincubated with protein kinase and ATP under conditions allowing incorporation of phosphate into substrate protein . After the preincubation, reverse transcriptase activity was assayed in the presence of poly(rA).oligo(dT) as template . A 2- to 5-fold increase of reverse transcriptase activity was found after the preincubation of reverse transcriptase with protein kinase and ATP . Incubation of reverse transcriptase with heat-treated, inactive protein kinase and ATP had no effect on transcriptase activity . When the transcriptase preparation was incubated with protein kinase and {gamma-32P}ATP and subsequently purified by chromatography on phosphocellulose and Sephadex gel filtration, significant amounts of 32P-labeled proteins were found in the fractions exhibiting reverse transcriptase activity, suggesting 32P incorporation into transcriptase or transcriptase-associated proteins . A 20-60% decrease of reverse transcriptase activity was observed after incubation of reverse transcriptase with phosphatase . The results suggest that phosphorylative modification of reverse transcriptase may be critical in the regulation of reverse transcriptase-catalyzed DNA synthesis. J Natl Cancer Inst, 1975 Aug, 55(2), 433 - 42 Inhibition of purified DNA polymerase of RNA tumor viruses by fluoranthene derivatives and analogues of tilorone hydrochloride; Green M et al.; At concentrations of 7 times 10(-6) to 7 times 10(-5) M, derivatives consisting of the polycylic ring structures fluoranthene, fluorenone, fluorene, anthraquinone, xanthenone, and dibenzofuran with appropriate amine side chains inhibited by over 90% the purified RNA-directed DNA polymerase of avian myeloblastosis virus acting on poly(deoxyadenylate-deoxythymidylate) {poly(dA-dT)} . Of these, only the fluoranthene derivatives were strong inhibitors of the viral DNA polymerase directed by polyadenylate-oligodeoxythymidylate {poly(A)-(dT)12-18} . Low levels of fluoranthene derivatives (1 times 10(-5) M) also strongly inhibited polymerase with polyinosinate-oligodeoxycytidylate {poly(I)-(dC)12-18}, activated calf thymus DNA, and viral 70S RNA as templates, but not with polycytidylate-oligodeoxyguanylate as template . A comparison of the activity of 11 fluoranthene derivatives with different side chains showed that the structure of the amine side chain influenced both the extent of antipolymerase activity with a given template and the relative inhibition with different synthetic DNA and RNA templates . The naturally occurring polyamines, spermine, spermidine, and putrescine, did not inhibit the activity of the viral DNA polymerase . Studies on the mechanism of action indicated that the synthetic derivatives inhibited polymerase activity by binding to the template and not to the enzyme: 1) inhibition by fluoranthene derivatives was overcome by the addition of excess template including poly(dA-dT), poly(A)-(dT)12-18, poly(I)-(dC)12-18, viral 70S RNA, and activated calf thymus DNA; 2) the degree of inhibition by fluoranthene derivatives was unaffected by the addition of the creased viral DNA polymerase; 3) with the same template, Escherichia coli DNA-directed RNA polymerase and the viral RNA-directed DNA polymerase were inhibited to about the same extent; and 4) the derivatives formed a complex with DNA, poly(I), and poly(A) that was stable to exclusion chromatography on Sephadex G-100 . Several derivatives also had biologic activity, since they blocked the ability of the murine sarcoma virus to transform cells. Zentralbl Bakteriol {Orig A}, 1975 Aug, 232(4), 454 - 61 {The cathode bound group antigen of dysentery-provoking escherichieae (author's transl)}; Stenzel W; Antigens from disrupted cells of dysentery-provoking and of non-enteropathogenic Escherichieae were submitted to immunoelectrophoresis on cellulose acetate stripes at pH 8.0 . Among 6 immune sera produced for this purpose by immunizing rabbits against desintegrated dysentery bacteria, only one contained a precipitine reacting with an antigen similar to the "generic antigen" of BELAYA . This - at pH 8.0 - cathode-bound group antigen (KGA) could not only be found in virulent but also in 5 attenuated cultures and in 5 from 6 avirulent strains of several dysentery types . Only the - apathogenic - type culture 1111/55 of dysentery-provoking E . coli O 136 showed no KGA-reaction . Some sources of methodical errors responsible for false outcomes of immunopherogrammes have been discussed. Biochemistry, 1975 Jul 29, 14(15), 3350 - 6 Demonstration of two reaction pathways for the aminoacylation of tRNA . Application of the pulsed quenched flow technique; Fersht AR et al.; A rapid mixing and quenching device is described which operates efficiently in the range of 150 msec to several minutes as well as the usual time scale of 5-150 msec of the conventional apparatus . This has been used to measure the initial rate of acylation of tRNATyr by the tyrosyl-tRNA synthetase of Escherichia coli during the first turnover of the enzyme, and also the rate constants of the partial reactions of amino acid activation and transfer to the tRNA . It is shown that at saturating conenctration of tRNA the reaction proceeds by a ternary complex mechanism . The rate-determining step is either the aminoacyltion process or a step preceding it . At low concentrations of tRNA the reaction proceeds by the stepwise process of formation of tyrosyl adenylate followed by acylation of the tRNA . The rate constants for these partial reactions are faster than that for the ternary complex reaction . But the prior binding of tRNA greatly decreases the rate of tyrosyl adenylate formation . Both pathways are probably important at physiological concentrations . 88% of the tyrosine from the tyrosyl adenylate complex is transferred to tRNA . The presence of added tyrosine and ATP reduces this to 78% . However, the addition of aliquots of ATP to a mixture of enzyme, tyrosine, and a saturating concentration of tRNA (i.e., ternary complex conditions) leads to at least 0.97 mol of tRNA being acylated/mol of ATP hydrolyzed . Trapping experiments show that the 12% of adenylated that is not transferred to tRNA is hydrolyzed on the enzyme rather than expelled into solution. Biochim Biophys Acta, 1975 Jul 27, 397(1), 220 - 30 Purification of biosynthetic threonine deaminase from Escherichia coli; Koerner K et al.; Biosynthetic threonine deaminase (L-threonine hydro-lyase (deaminating), EC 4.2.1.16) was purified to apparent homogeneity from cell extracts of Escherichia coli by chromatographic procedures using valine-Sepharose, isoleucine-N-hexamethyleneamine-Sepharose, and hydroxyapatite with an overall yield of 40% . Analytical ultracentrifugation shows a molecular weight of 214 000 . In sodium dodecyl sulfate gel electrophoresis, the enzyme migrates as a single band corresponding to a molecular weight of about 50 000 . These data confirm that the enzyme is a tetramer . The sedimentation coefficient, s-020,w, determined by differential sedimentation experiments is 9.2 S . The enzyme shows absorption maxima at 415 and 280 nm . Determination of pyridoxal phosphate by three indenpendent methods shows the presence of two molecules of pyridoxal phosphate per enzyme molecule, the different methods being in excellent agreement equilibrium dialysis experiments establish the presence of two isoleucine binding sites . The Scatchard plot suggests non-cooperativity of these sites . The association constant for isoleucine is 1.2 - 10(5)M-1. J Biol Chem, 1975 Jul 25, 250(14), 5647 - 54 The 30 S ribosomal precursor RNA from Escherichia coli . A primary transcript containing 23 S, 16 S, and 5 S sequences; Ginsburg D et al.; The 30 S ribosomal precursor RNA has been prepared from Escherichia coli AB301/105 (RNase III-) labeled with 32-PO4 in the presence of chloramphenicol . Direct nucleotide sequence studies yield the following information . 1 . The major 5'-terminal sequence in our precursor preparations is pppA-C-U-G- . 2 . Treatment of the precursor RNA with purified ribonuclease III in vitro releases species sedimenting near 23 S and 17 S, neither of which retain the pppA- end, plus a collection of small fragments with chain lengths of less than 400 nucleotides . 3 . The RNase III product sedimenting near 17 S (16 SIII) appears identical with the 17 S RNA typically isolated from pulse-labeled or chloramphenicol-treated cells or from several mutants deficient in ribosome assembly: fingerprint analysis reveals the presence of the same additional RNase T1 oligonucleotides and the 5' terminus (pU-G-) previously described for 17 S RNA . A 3'-terminal T1 oligonucleotide (which was not previously identifiable in the case of the 17 S precursor) has been isolated from 16 S III and its sequence determined: C-U-C-A-C-A-C-A . 4 . 5 S rRNA sequences are contained in an RNase III-released fragment of approximately 300 nucleotides . This molecule lacks the 5' terminus of the mature 5 S RNA . The implications of these findings with respect to the control of ribosomal RNA synthesis, the pathways of rRNA processing in vivo, and the specificity of RNase III cleavage of natural substrates are discussed. J Biol Chem, 1975 Jul 25, 250(14), 5614 - 21 Inhibition of thymidylate synthetase and dihydrofolate reductase by naturally occurring oligoglutamate derivatives of folic acid; Friedkin M et al.; Naturally occurring oligoglutamate derivatives of folic acid in extracts of Escherichia coli have been isolated on the basis of their inhibitory actions toward thymidylate synthetase and dihydrofolate reductase . The inhibitor of thymidylate synthetase has been identified as N-5-formyl-H4pteroyloligoglutamate (approximately 5 amino acid residues) . It is 150-fold more inhibitory than the monoglutamate . Synthetic N-5-formyl derivatives containing 3 to 6 glutamyl residues were prepared and found to be 67- to 200-fold more inhibitory than the monoglutamate . N-5-Formimino-H4pteroyltriglutamate is one-twentieth as inhibitory as the corresponding N-5-formyl derivative . The inhibitor of mouse leukemia dihydrofolate reductase has been identified as N-10-formylpteropentaglutamate . It is approximately 7 times as inhibitory as N-10-formylpteroylmonoglutamate . It is 4,400 times as inhibitory toward mouse leukemia dihydrofolate reductase compared with the enzyme from E . coli . Lysine analogs of N-5-formyl-H4folate containing alpha0glutamyl groups in peptide linkage to the epsilon-amino group of lysine were relatively poor inhibitors of thymidylate synthetase . The inhibitory action of folic acid oligoglutamates on E . coli thymidylate synthetase was subject to reversal with 0.4 M NaCl, an effect that was more marked with various pteroyloligoglutamates than with H4homopteroylmonoglutamate and N-5, N-8-deaza-N-10-methylpteroylmonoglutamate. J Biol Chem, 1975 Jul 25, 250(14), 5609 - 13 Escherichia coli succinic thiolinase . Stoichiometry of phosphorylation and coenzyme A binding; Bowman CM et al.; Equilibrium and covalent binding studies of succinic thiokinase from Escherichia coli indicates that there can be a stoichiometric relationship between coenzyme A binding and the phosphoyrlation capacity of the enzyme . A comparison of homogeneous enzyme preparations has revealed that enzyme of high specific activity exhibits greater binding capacity and that this property is proportional to enzyme activity . Phosphorylation capacity was related to specific activity in highly active enzyme preparations, but leveled off at 1 mol of phosphorus/mol of thiokinase . These studies show that the "dimer of dimers" structure of succinic thiokinase contains the expected two active sites and that this enzyme does not demonstrate "half-the-sites" reactivity . A coenzyme A binding site of lower affinity can be detected in some enzyme samples of lower specific activity . Binding of coenzyme A to the higher affinity sites may involve positive cooperativity . ATP, unlike ADP, does not bind to phosphorylated enzyme. J Biol Chem, 1975 Jul 25, 250(14), 5556 - 62 Purification and characterization of homogeneous initiation factor M2A from rabbit reticulocytes; Merrick WC et al.; Rabbit reticulocyte initiation factor M2A has been prepared in homogeneous form . The final preparation was purified 2,300-fold and ran as a single band on polyacrylamide gel electrophoresis in three different buffer systems: alkaline, sodium dodecyl sulfate, and acidic 6.5 M urea . IF-M2A also ran as a single band in polyacrylamide gel isoelectric focusing experiments with an apparent pI of 6.45 . The molecular weight of IF-M2A was approximately 125,000 based on determinations by low speed equilibrium centrifugation (118,000), sodium dodecyl sulfate gel electrophoresis (130,000) and s20,w combined with Stokes radius (124,000) . The amino acid composition of IF-M2A revealed three unusual features: a) the basic amino acids represented 19.4 mol %; b) glutamicacid (plus glutamine) constituted 18.8 mol%; c) tryptophan and cysteine residues were only 0.4 and 0.7 mol%, respectively . Homogeneous IF-M2A was tested in several initiation assays using either natural or artificial mRNAs . In each assay tested, homogeneous IF-M2A fully substituted for cruder preparations and at concentrations commensurate with its increased purity . IF-M2A was also examined for ribosome-dependent GTP hydrolysis, an assay requiring ribosomes but no other initiation factors . Analysis of the data yielded a Km for GTP of 10 muM and a Vmax for hydrolysis of 1.20pmol/mug IF-M2A/min . In addition, IF-M2A mediated GTP hydrolysis required both 40 S and 60 S subunits for maximal activity . The possibility that IF-M2A is a factor required for the joining of 40 S and 60 S subunits is discussed. J Biol Chem, 1975 Jul 25, 250(14), 5352 - 8 Resolution of D-serine dehydratase by cysteine . An analytical treatment; Schonbeck ND et al.; A general method is presented for analysis of the resolution of pyridoxal-P-requiring enzymes by carbonyl reagents . The method is useful for accurately determining the very small equilibrium constants (KP) which characterize the dissociation of cofactor from many pyridoxal-P-requiring enzymes . The analysis also establishes the minimum number and relative stabilities of distinct enzymic species involved in the resolution process . Analysis of the resolution of D-serine dehydratase by L-and D-cysteine resulted in the establishment of an enzyme bound thiazolidine derivative as an intermediate in the pathway for resolution . The over-all equilibrium constant (KR) for the reaction, D-serine dehydratase + cystein in equilibrium KR thiazolidine derivative +D-serine apodehydratase was determined . At pH 7.80, T/2 0.33, 25 degrees, KR equal to 1.08 times 10-minus 3 . A value of 7.0 nM for the equilibrium constant for the dissociation of D-serine dehydratase to apoenzyme and free pyridoxal-P was determined from the ratio KR/KT, where KT is the equilibrium constant for the formation of a thiazolidine derivative from free pyridoxal-P and cysteine . An estimate of 14 nM for KP was also obtained from partial resolution of D-serine dehydratase by high dilution . The difficulties associated with this direct determination of KP from the dependence on the enzyme concentration of the activity of very dilute solutions of enzyme are discussed. J Biol Chem, 1975 Jul 25, 250(14), 5501 - 7 Reaction heat variation with pH in formation of the trypsin-soybean inhibitor complex; Barnhill MT Jr et al.; The heat of reaction between beta-trypsin and Kunitz soybean inhibitor (STI) hasbeen measured at 5 degrees and 25 degrees from pH 4 to 8.5 . Corresponding measuremenportion of tRNA-Gly2-GGA/G molecules isolated from E . coli cells . The missense suppressor mutation, glyTsuA36(HA), results in a C yields U base substitution at the 3' end of the anticodon of tRNA-Gly2-GGA/G(nucleotide position 38) . Asecondary effect of this base substitution is the modification of the A residue directly adjacent to the 3' end of the anticodon of tRNA-Gly2-suA36(HA), suggesting that the enzymes responsible for this modification recognize the anticodon sequencesof prospective tRNA substrates . The creation of a missense-suppressing tRNA, tRNA-Gly2-suA36(HA), by an alteration of the anticodon sequence of tRNA-Gly2-GGA/G is analogous to mechanisms whereby other suppressor tRNAs have arisen . The high degree of nucleotide sequence homology between the amino acid acceptor stems and anticodon regions may be recognized by the glycyl-tRNA synthetase; the involvement of theanticodon region in the synthetase recognition process is supported by the greatly decreased rate of aminoacylation of tRNA-Gly2-suA36(HA). J Biol Chem, 1975 Jul 25, 250(14), 5289 - 94 Multiple forms of beta-ketoacyl-acyl carrier protein synthetase in Escherichia coli; D'Agnolo G et al.; Two forms of beta-ketoacyl-acyl carrier protein (ACP) synthetase (designated I and II) have been identified in extracts of Escherichia coli . Synthetase I corresponds to the condensing enzyme that was studied earlier (GREENSPAN, M.D., ALBERTS, A.W., and VAGELOS, P.R . (1969) J . Biol . Chem . 244, 6477-6485); synthetase II represents a new form of the enzyme . Synthetase II was isolated as a homogeneous protein . It differs from synthetase I in having a higher molecular weight (76,999 versus 66,000), a lower pH optimum (5.5 to 6.1 versus 7.2), and a greater resistance to denaturation by heat . Synthetase II is similar to synthetase I in that both are inactivated by iodoacetamide, and prior incubation of the enzymes with fatty acyl thioesters prevents the inhibitory effect of iodoacetamide . Both also react with a fatty acyl thioester to form an acyl-enzyme intermediate, and the latter reacts with malonyl-ACP to form a beta-ketoacyl thioester . Specificity studies indicated that synthetase II, like synthetase I, has similar affinities with saturated and cis unsaturated fatty acyl thioesters of ACP that are intermediates in the synthesis of saturated and unsaturated fatty acids, respectively . The two synthetases differ only with respect to reactivity with palmitoleyl thioesters: synthetase II has a lower Km and higher Vmax than synthetase I with palmitoleyl-ACP . This finding suggests that synthetase II functions specifically in the elongation of palmitoleyl-ACP to form cis-vaccenyl-ACP . An investigation of synthetases I and II in two classes of unsaturated fatty acid auxotrophs revealed that synthetase I is absent in one class, fabB . Addition of wild type synthetase I to fabB fatty acid synthetase, which synthesizes only saturated fatty acids, permitted this fatty acid synthetase to synthesize unsaturated fatty acids . These experiments indicate that synthetase I plays a critical role in the synthesis of unsaturated fatty acids. J Biol Chem, 1975 Jul 25, 250(14), 5283 - 8 Beta-Ketoacyl-acyl carrier protein synthetase . Characterization of the acyl-enzyme intermediate; D'Agnolo G et al.; Beta-Ketoacyl-acyl carrier protein (ACP) synthetase catalyzes the condensation reaction of fatty acid synthesis in Escherichia coli . The homogeneous enzyme reacts with hexanoyl-CoA to form hexanoyl-enzyme which was isolated and characterized . Hexanoyl-enzyme contains 2 mol of hexanoate/mol of enzyme (molecular weight 66,000); it is liable at alkaline pH, and it reacts with neutral hydroxylamine to form hexanoyl hydroxamic acid . Hexanoate was cleaved from the enzyme when hexanoyl-enzyme was subjected to performic acid oxidation . These properties indicate that hexanoyl-enzyme is a thioester . Studies of the circular dichroism spectra of fully acylated and nonacylated forms of the enzyme indicated that the secondary structure of the enzyme is relatively unperturbed by the presence of the hexanoyl groups . An alpha helical content of 65% was estimated for the enzyme from the circular dichroism spectrum . Hexanoyl-enzyme is active in both partial reactions that comprise the beta-ketoacyl-ACP synthetase reaction; it reacts with ACP to form hexanoyl-ACP and with malonyl-ACP to form beta-ketooctanoyl-ACP . Although the hexanoate of hexanoyl-enzyme is transferred very rapidly to ACP, the physiological acceptor in this reaction, it is also transferred very slowly to CoA, dithiothreitol, and 2-mercaptoethanol, indicating that the enzyme can react nonspecifically with a number of unrelated mercaptans. J Biol Chem, 1975 Jul 25, 250(14), 5542 - 55 The nucleotide sequence of a precursor to the glycine- and threonine-specific transfer ribonucleic acids of Escherichia coli; Chang S et al.; The primary nucleotide sequence of an Escherichia coli tRNA precursor molecule has been determined . This precursor RNA, specified by the transducing phage lambdah80dglyTsuA36 thrT tyrT, accumulates in a mutant strain temperature-sensitive for RNase P activity . The 170-nucleotide precursor RNA is processed by E . coli extracts to form mature tRNA Gly 2 suA36 and tRNA Thr ACU/C . The sequence of the precursor is pG-U-U-C-C-A-G-G-A-U-G-C-G-G-G-C-A-U-C-G-U-A-U-A-A-U-G-G-C-U-A-U-U-A-C-C-U-C-A-G-C-C-U-N-C-U-A-A-G-C-U-G-A-U-G-A-U-G-C-G-G-G-T-psi-C-G-A-U-U-C-C-C-G-C-U-G-C-C-C-G-C-U-C-C-A-A-G-A-U-G-U-G-C-U-G-A-U-A-U-A-G-C-U-C-A-G-D-D-G-G-D-A-G-A-G-C-G-C-A-C-C-C-U-U-G-G-U-mt6A-A-G-G-G-U-G-A-G-m7G-U-C-G-G-C-A-G-T-psi-C-G-A-A-U-C-U-G-C-C-U-A-U-C-A-G-C-A-C-C-A-C-U-UOH(tRNA sequences are italicized) . It contains the entire primary nucleotide sequences of tRNA Gly2 suA36 and tRNA Thr ACU/C, including the common 3'-terminal sequence, CCA . Nineteen additional nucleotides are present, with 10 at the 5' end, 3 at the 3' end, and the remaining 6 in the inter-tRNA spacer region . RNase P cleaves the precursor specifically at the 5' ends of the mature tRNA sequences. J Biol Chem, 1975 Jul 25, 250(14), 5426 - 37 Studies and sequences of Escherichia coli 4.5 S RNA; Griffin BE; 4.5 S RNA, a biologically stable species with electrophoretic properties intermediate between 5 S and transfer RNAs, has been isolated from Escherichia coli and characterized . No function has yet been found for this molecule . Its primary structure and behavior suggests an unusually stable and possibly unique secondary structure . Even from single species of E . coli, there is some sequence heterogeneity within the molecule . The sequence of a major species from MRE 600 is: (see article) . Methods for getting sequence overlaps on this highly structured RNA are described, and a possible functional role for 4.5 S RNA is discussed. Nature, 1975 Jul 24, 256(5515), 288 - 92 Existence and possible roles of transcriptional barriers in T7 DNA early region as shown by electron microscopy; Darlix JL et al.; Transcription of T7 DNA in vitro by Escherichia coli RNA polymerase in the absence or presence of termination factor rho has been studied extensively using electron microscopy . It is demonstrated that sequences located between the early genes behave as transient transcriptional barriers in the absence of rho and are recognised both as rho-dependent terminators and as RNaseIII cleavage sites. Biochim Biophys Acta, 1975 Jul 23, 395(4), 478 - 89 30 S pre-ribosomal RNA of Escherichia coli:primary and secondary processing; Nikolaev N et al.; The metabolism of newly-formed labeled 30 S pre-ribosomal RNA was studied in the Escherichia coli mutant strain AB301-105 . Detailed kinetic analysis in rifampicin-treated cells showed that precursors of 23 S and 16 S rRNA are formed from the 30 S RNA species, even in phosphate-limited cultures . To establish the order fo segments along 30 S pre-rRNA, it was allowed to accumulate in replicate chloramphenicol-treated cultures, and pulse-labeled after the addition of rifampicin in segments that progressively contained more 3'-distal label with time . The purified RNAs from the various cultures were then cleaved to a limited extent by RNAase III; the specific activity of the fragments yielded an order from the 5'-end of 17.5S-X-25S, where X refers to some of the sequences released as additional small fragments . More extensive treatment the 25-S and 17.5-S pre-rRNA chains with RNAase III yielded additional fragments and produced RNA species with the mobility of the 23-S and 16-S RNA precursors made in normal cells treated with chloramphenicol . A processing scheme is suggested that distinguishes between early, site-specific cleavage of recognition sites in the RNA itself ('primary processing'), and later, "secondary processing' reactions . The secondary processing reactions are blocked chloramphenicol-treated wild-type cells. Biochim Biophys Acta, 1975 Jul 23, 395(4), 446 - 54 The ATP-dependent DNAase from Escherichia coli rorA: a nuclease with changed enzymatic properties; Van Dorp B et al.; The ATP-dependent DNAases from Escherichia coli wild-type and rorA were isolated and purified and their enzymatic properties were compared . The enzymes were found to differ in the amount of ATP that is consumed during DNA degradation . This difference can be influenced by the reaction conditions and the nature of the substrate. S Afr Med J, 1975 Jul 19, 49(31), 1259 - 62 The effect of vehicle composition on the release of chloramphenicol from creams and eye ointments; McCarthy TJ; The effects that formulation of topical vehicle can have on the bio-availability and effectiveness of active ingrediants are discussed with reference to steroids, salicylic acid and benzocaine . Results obtained for chloramphenicol-containing preparations are presented, and both dissolution curves and cup-plate assays demonstrate that chloramphenicol has far superior release (and hence activity) from creams than from ophthalmic ointments . The latter, however, produce adequate zones of inhibition, and are long-acting. Biochemistry, 1975 Jul 15, 14(14), 3144 - 51 Synthesis properties of the naturally occurring N-{(9-beta-D-ribofuranosylpurin-6-yl)-N-methylcarbamoyl}-L-threonine (mt-6A) and other related synthetic analogs; Dutta SP et al.; The naturally occurring modified nucleoside, N-{(9-beta-D-ribofuranosylpurin-6-yl)-N-methylcarbamoyl}-L-threonine (mt6A), and the corresponding glycine analog mg6A were synthesized from N6-methyl-2',3',5'-tri-O-acetyladenosine and the appropriately blocked isocyanates derived from threonine and glycine . The natural mt6A isolated from Escherichia coli tRNA (F . Kimura-Harada et al . (1972), Biochemistry 11, 3910), from wheat embryo tRNA (R . Cunningham and M . W . Gray (1974), Biochemistry 13, 543), and from rat liver tRNA (Rogg et al . (1975), Eur . J . Biochem . 53, 115) was found to be identical with the synthetic mt6A in paper and thin-layer chromatography and electrophoresis . Several analogs of the parent 6-ureidopurine ribonucleoside, N-{(9-beta-D-ribofuranosylpurin-6-yl)carbamoyl}-L-thronine (t6A), were also prepared . Starting from 2',3',5'-tri-O-acetylguanosine and 2',3',5'-tri-O-acetylcytidine and the above isocyanates, the t6A analogs, N-{(9-beta-D-ribofuranosyl-6-oxo-1H-purin-2-yl)carbamoyl}-L-threonine (t2G) and N-{(1-beta-D-ribofuranosyl-2-oxypyrimidin-4-yl)carbamoyl}-L-threonine (t4C), were prepared . Also synthesized were the corresponding glycine analogs, g2G and g4C, from guanosine and cytidine, respectively . The 2'-deoxyribosyl analog, N-{(9-beta-D-2'-deoxyribofuranosylpurin-6-yl)carbamoyl}-L-threonine (2'-deoxy-t6A), and the arabinosyl derivative, N-{(9-beta-D-arabinofuranosylpurin-6-yl)carbamoyl}-L-threonine (t6AraA), were synthesized from the appropriate urethane and the requisite amino acid . The ureido group in mt6A could not be hydrolyzed by the enzymes urease, peptidase, and protease . Various chemical and biological properties of the naturally occurring mt6A and the related analogs are discussed. Eur J Biochem, 1975 Jul 15, 55(3), 573 - 82 Quaternary structure of polynucleotide phosphorylase from Escherichia coli: evidence of a complex between two types of polypeptide chains; Portier C; A new form of polynucleotide phosphorylase containing alpha and beta subunits was isolated (form B) by purification without preparative electrophoresis; this form was compared to the enzyme obtained by preparative electrophoresis purification (form A) . The Stokes radius of these two forms are very different: 6.4 nm for form A and 8.7 - 9.0 nm for form B; on the other hand the sedimentation coefficients are close: 8.9 S and 9.9 S respectively . Such a result suggests that form B is very asymmetric . The apparent molecular weights, calculated from the Stokes radii and from the sedimentation coefficients, are approximately 365000 for form B and 252000 for form A . The latter is homogeneous on polyacrylamide gel, whereas the former yields two components, one of which behaves similarly to form A . Finally, whereas form A is composed of only one type of subunit, alpha, form B contains two types of chains: alpha (Mr 86000 +/- 5000) and beta (Mr 48000 +/- 2000) in stoichiometric proportions . From these results we believe that one should assume for form B the existence of a complex between form A and beta chains; the role of the latter still remains to be specified. Eur J Biochem, 1975 Jul 15, 55(3), 561 - 71 {Mechanism for the condensation reaction of fatty-acid biosynthesis (author's transl)}; Arnstadt KI et al.; The spontaneous hydrogen-deuterium exchange of the methylene group of malonyl-thioesters was investigated by nuclear-magnetic-resonance (NMR) spectroscopy using the model compound S-malonyl-N-acetylcysteamine . The half life of the methylene proteins is 12 to 16 min in 0.1 M K-phosphate buffer at pH 6.5 to 7.0 at 25 degrees C, the conditions of maximal activity of fatty acid synthetase from yeast . Proton catalysis was used for the quick preparation of deuterium- and tritium-labeled malonylthioesters . Compared with malonyl-CoA, dideutero-malonyl-CoA had no primary isotope effect on the reaction velocity of the yeast enzyme catalysed fatty acid synthesis, in which the rate limiting step is the condensation reaction . Although deuterium oxide had a solvent isotope effect, there was no difference in reaction velocities between malonyl CoA and dideuteromalonyl CoA in deuterium oxide . The condensation reaction was investiaged separately from the overall fatty acid synthesis using beta-ketoacyl-acyl-carrier-protein (ACP) synthetase (condensing enzyme) of Escherichia coli . The condensation reaction with deuteromalonyl-ACP had no kinetic isotope effect, in agreement with the observations on the overall reaction . However, in this case no solvent isotope effect was observed with 2H2O . When the condensation reaction was carried out in the presence of tritiated water, there was no incorporation of label into the reaction product acetoacetyl-thioester, excluding proton exchange with the solvent . The results exclude a mechanism for the condensation reaction involving a malonyl carbanion and its acylation as intermediates in the sense of an organic-chemical malonic ester synthesis, and they indicate that the condensation reaction follows a concerted mechanism: The formation of the new carbon-carbon bond is coupled with the cleavage of the carboxyl bond of the malonyl group. Eur J Biochem, 1975 Jul 15, 55(3), 517 - 29 Equivalent and non-equivalent binding sites for +RNA on aminoacyl-tRNA synthetases; Krauss G et al.; Complexes between tRNAPhe (yeast), tRNASer (yeast) and tRNATyr (Escherichia coli) and their cognate aminoacyl-tRNA synthetases have been studied by sedimentation velocity runs in an analytical ultracentrifuge . The amount of complex formation was determined by the absorption and the sedimentation coefficients of the fast-moving boundary in the presence of excess tRNA or excess synthetase respectively . The same method has been applied to unspecific combinations of tRNAs and synthetases . Inactive material of tRNA or synthetase does not influence the results . 1 . Two moles of tRNAPhe can be bound to one mole of phenylalanyl-tRNA synthetase with a binding constant greater than 10(6) M-1 . The binding constants for both tRNAs are very similar; the binding sites are independent of each other . Omission of Mg2+ does not prevent binding . 2 . Two moles of tRNASer can be bound to one mole of Seryl-tRNA synthetase; the binding of the first and second tRNA is non-equivalent, K1 greater than 10(6) M-1, K2 is determined to be 1.3 X 10(5) M-1 at pH 7.2 . Omission of Mg2+ prevents complex formation . 3 . Tyrosyl-tRNA synthetase behaves very similarly to seryl-tRNA synthetase . The binding constant for the weakly bound tRNA is 2.3 X 10(5) M-1 at pH 7.2, and 2.5 X 10(6) M-1 at pH 6.0 . No complexes are observed in the absence of Mg2+ . 4 . Unspecific binding was only obtained with phenylalanyl-tRNA synthetase . It binds tRNASer (yeast), tRNAAla (yeast) and tRNATyr (E . coli) with a binding constant about 100 times lower compared to its cognate tRNA . The binding data are discussed with respect to the tertiary structure of the tRNAs, the subunit structure of the synthetases and the possible physical basis for the non-equivalence of binding sites. Am J Obstet Gynecol, 1975 Jul 15, 122(6), 788 - 9 Neonatal perforation of the colon; Georgy FM; PIP: A case study of a 25-year-old white women, gravida 3, para 2 with an estimated 32-week gestation delivered a 3 pound cyanotic infant . The placenta was grossly infected with E . coli . The newborn had abdominal distention, very poor ability for ingestion of food, persistent greenish vomitus and X-ray revealed free air in the peritoneal cavity . A diagnosis of perforated bowel was made . A colectomy and sigmoidostomy were performed on the infant but he died . Pathology of the colon showed necrotizing bacterial colitis and autopsy revealed diffuse inflammation in multiple organs of the digestive tract and pancrease . 70 cases of perforated bowel in the neonate have been reported with 11 survivors . It was suggested that the cause of perforated colon in the neonate was an infected intrauterine environment . Early diagnosis was instrumental in the survival of these infants . The classic clinical picture should alert the obstetrician and the pediatrician to this condition . Biochemistry, 1975 Jul 15, 14(14), 3195 - 203 Cooperative interactions in hybrids of aspartate transcarbamylase containing succinylated regulatory polypeptide chains; Nagel GM et al.; Succinylated derivatives of the regulatory subunit of aspartate transcarbamylase of Escherichia coli were prepared by treating the intact enzyme with succinic anhydride followed by dissociation of the modified protein into catalytic and regulatory subunits which were separated by ion exchange chromatography . The succinylated regulatory subunits were used in hybridization experiments with native subunits to study the organization of the six regulatory polypeptide chains in the intact enzyme . Rapid mixing of succinylated and native regulatory subunits with native catalytic subunits yielded a four-membered hybrid set of reconstituted enzyme-like molecules; hence, the assembly process involves three regulatory combining units and the six regulatory polypeptide chains in the intact enzyme must be arranged as three dimeric subunits . When the modified and native regulatory subunits were incubated together for only brief periods (less than 1 min) followed by the addition of catalytic subunits, the resulting hybrid set was complex with no resolution of discrete species . Apparently the isolated regulatory dimers dissociate readily and reversibly into single polypeptide chains due to relatively weak intra-subunit bonding domains . In contrast, after reconstitution of enzyme-like molecules, the incorporated succinylated regulatory subunits did not exchange with free subunits . Enzyme-like molecules containing three extensively succinylated regulatory subunits show reduced binding of the inhibitor, CTP, and lack both the homotropic and heterotropic effects characteristic of native aspartate transcarbamylase . Preparations containing only slightly succinylated regulatory subunits showed only little inhibition by CTP and considerable cooperativity . The decrease in homotropic effects in these reconstituted molecules correlated with the reduction in the succinate-promoted change in the sedimentation coefficient . Reconstituted enzyme-like molecules containing regulatory subunits which had been extensively succinylated in the presence of CTP retained their binding capacity even though they were only slightly inhibited by CTP and exhibited reduced cooperativity . Hybrid molecules containing both native and succinylated regulatory subunits also possessed reduced allosteric behavior. Biochim Biophys Acta, 1975 Jul 14, 399(1), 10 - 22 Isolation and initial characterization of glutathione-deficient mutants of Escherichia coli K 12; Apontoweil P et al.; The thiol-oxidizing agent "diamide" (CH3)2NCON equal to NCON(CH3)2 was used to isolate mutants of Escherichia coli K 12 deficient in the biosynthesis of glutathione . A colony-colour technique has been developed for identification of colonies of these mutants . Four glutathione-deficient mutants were isolated . They show normal growth rates in minimal medium without GSH supplementation, indicating that glutathione is not involved in essential metabolic process . In one mutant, glutathione synthetase was entirely inactive . Three mutants were deficient in gamma-glutamylcysteine synthetase; in two of them, this resulted in a complete lack of GSH . These mutants were found to be more susceptible than their parent strains to a wide rang of chemical agents, but did not show a greater sensitivity to X-rays . It must be concluded that the protective role of glutathione is only significant when a chemical challenge is present. Biochim Biophys Acta, 1975 Jul 14, 399(1), 1 - 9 Glutathione biosynthesis in Escherichia coli K 12 . Properties of the enzymes and regulation; Apontoweil P et al.; The synthesis of glutathione in Escherichia coli K 12 was studied in crude, cell-free extracts . The pH optima and the apparent Km values for the substrates have been determined for both synthesizing enzymes, gamma-glutamylcysteine synthetase and glutathione synthetase . gamma-Glutamylcysteine synthetase was found to be approximately twice as active as glutathione synthetase . In a growing culture, the cellular level of GSH showed a considerable increase up to 6.6 mumol per ml cell pellet in the stationary growth phase . GSSG was not detectable . The levels of the enzymes remained constant, indicating that glutathione biosynthesis depends at least in the beginning on the availability of the component amino acids . The pathway is controlled by feedback inhibition and not by repression. Proc Natl Acad Sci U S A, 1975 Jul, 72(7), 2582 - 6 Proton nuclear magnetic resonance of spin-labeled Escherichia coli tRNAf1MET; Daniel WE Jr et al.; Thiouridine at position 8 (s4U8) of tRNAf1Met was spin-labeled with the nitroxide free radical, N-(1-oxyl-2,2,5,5-Tetramethyl-3-pyrrolidinyl) bromacetamide, for proton nuclear magnetic resonance spectroscopic studies . The well-resolved methyl peak of ribothymidine is unperturbed, but the peak tentatively assigned to the C-5 methylene group of dihydrouridine is considerably broadened in spin-labeled tRNAf1Met . Of the approximately 27 slowly exchanging protons observed in the region between 11 and 15 ppm downfield from 4,4-dimethyl-4-silapentane-1-sulfonic acid, the equivalent of about five protons apparently disappeared in spin-labeled tRNAf1Met . The well-resolved single proton at 14.8 ppm was missing not only in the paramagnetic species, but also in the diamagnetic reduced form of spin-labeled tRNAf1Met, and was unequivocally identified as a hydrogen bond involving s4U8 by comparison of several forms of tRNAf1Met specifically modified at s4U . Evidence that the perturbation of a second single proton resonance at 14.6 ppm (shift and broadening) is coupled to the loss of a tertiary hydrogen bond involving residue 8, arises from the same modified forms . The resolved resonances in the methyl and N-H regions, particularly the resonance at 14.6 ppm as well as the four N-bonded proton resonances at higher field which are broadened solely due to their proximity to the unpaired electron of the spin label, provide specific indicators of the geometry of tRNAf1Met structure in solution . Their observability by nuclear magnetic resonance spectroscopy opens up the possibility of monitoring distance changes among the base residues of spin-labeled tRNAf1Met upon its interaction with aminoacyl-tRNA synthetase and other enzymes. Mol Gen Genet, 1975 Jul 10, 138(4), 351 - 8 Enhanced uptake of donor DNA by Ca2+ treated Escherichia coli cells; Sabelnikov AG et al.; The first steps in E . coli transformation have been studied . It was found that the cells attain the ability of enhanced uptake of donor exogenous DNA upon Ca2+ treatment . At saturating DNA concentrations the cells were capable to take up irreversibly about 6-10(8) dalton of donor DNA per cell (if to assume that all the cells are competent) . About 75% of this DNA was found to be attached to the cytoplasmic membrane . Employing Poisson approximation for distribution of the donor-marker DNA molecules on recipient cells it was found that the efficiency of a marker recombination and expression is about 2-10(-5).The first steps in E . coli transformation have been studied . It was found that the cells attain the ability of enhanced uptake of donor 0exogenous Mol Gen Genet, 1975 Jul 10, 138(4), 323 - 31 Conjugational behaviour of N plasmids in Escherichia coli K12; Dennison S et al.; R factors of the N incompatibility group apparently transfer at low frequencies (often 10(-6)-10(-5) per donor cell) in 30 min liquid matings . However, the level of transfer is greatly increased when donor-recipient mixtures are held on solid medium selective for the recipient only, prior to transconjugant selection . Increase in transconjugant recovery is still donor-dependent at plating and therefore arises from mating on the plate . The process limiting N-mediated conjugation in liquid is probably mating pair formation, suggesting that the increase in yield of transconjugants may result from provision of a solid substrate for mating pair formation rather than from the delay in selection per se . The kinetics of N plasmid transfer on solid medium resemble those of traniments suggest that RP4, the prototype P incompatibility group plasmid, shows a stimulation of transfer on solid medium similar to the N plasmids studied . We suggest that N, and probably P, plasmids are (like F and the col V's) naturally derepressed for fertility, but this is masked by the imcompetence in liquid matings of donors carrying them. J Biol Chem, 1975 Jul 10, 250(13), 5175 - 82 Effect of estrogen on gene expression in the chick oviduct . V . Changes in the number of RNA polymerase binding and initiation sites in chromatin; Schwartz RJ et al.; Estrogen administration to chicks results in an increase in the chromatin template activity of oviduct target tissue as assayed under standard in vitro assay conditions . However, the results obtained by the simple measurement of template activity may be a complicated function of the number of available RNA polymerase initiation sites, the rate of RNA chain elongation, and the rate of reinitiation . In the present study, we have measured separately the change in both the number of chain initiations as well as the rate of RNA chain propagation under conditions in which reinitiation was eliminated . Chromatin prepared from either estrogen-treated or control oviducts both supported an RNA chain elongation rate of six nucleotides per s and a chain size of approximately 700 nucleotides . Thus, both the elongation rate and size of the average product remained relatively constant following estrogen stimulation . In contrast, within 8 hours after a single injection of estrogen to unstimulated chicks, the concentration of RNA polymerase needed to saturate chromatin binding sites was increased to 150% in comparison to control values, and by 24 hours the level of polymerase bound to chromatin was twice that of the untreated control chick chromatin . With daily injections of estrogen, polymerase binding continued to rise . Coincident with the over-all increase in chromatin-bound polymerase was an increase in rifampicin-insensitive initiation sites and newly synthesized RNA chains . Unstimulated chick oviduct chromatin initiated 10,000 RNA chains/pg of DNA, while 24 hours of steroid treatment increased the number of initiated chains to 34,000 chains . These data demonstrate that the estrogen-induced increase in chromatin transcriptive activity was due to an increased number of polymerase binding and initiation sites on the chromatin template without a detectable change in the rate of RNA chain elongation. J Biol Chem, 1975 Jul 10, 250(13), 5165 - 74 Effects of estrogen on gene expression in the chick oviduct . IV . Initiation of RNA synthesis on DNA and chromatin; Tsai M-J et al.; The interaction of Escherichia coli RNA polymerase (ribonucleoside triphosphate:RNA nucleotidyltransferase, EC 2.7.7.6.) with chick oviduct DNA and chromatin has been studied in order to quantify the RNA polymerase binding sites and rifampicin-resistant initiation sites . The number of high affinity binding sites at saturation was calculated from the number of molecules of enzyme bound to DNA or to chromatin . The number of rifampicin-resistant initiation sites was calculated from the total quantity and the number average chain length of RNA synthesized under the conditions in which only RNA polymerase already in a stable preinitiation complex could avoid inhibition by the drug . Using an assessment of the temperature requirement for the information of this preinitiation complex, we were able to differentiate initiations at single-stranded or nicked DNA regions from initiations at double-stranded DNA regions . This method enabled us to rule out the possibility that RNA chain initiation on chromatin was due to initiation at nicked or end regions of DNA . In addition, we also demonstrated that not all RNA polymerase molecules bound to DNA or chromatin are located at a site at which RNA chain initiation can immediately begin . This methodological approach should allow investigators to monitor the individual reactions and factors involved in in vitro transcription of eukaryotic DNA and chromatin. J Biol Chem, 1975 Jul 10, 250(13), 4911 - 7 The involvement of guanosine 5-diphosphate-3-diphosphate in the regulation of phospholipid biosynthesis in Escherichia coli . Lack of ppGpp inhibition of acyltransfer from acyl-ACP to sn-glycerol 3-phosphate; Lueking DR et al.; The response of the Escherichia coli sn-glycerol-3-phosphate acyltransferase to guanosine 5-diphosphate-3-diphosphate (ppGpp) has been determined in vitro employing palmityl coenzyme A (CoA) and palmityl acyl carrier protein as acyl substrates . Levels of ppGpp which cause significant inhibition of enzyme activity with palmityl-CoA as substrate have no effect on enzyme activity when palmityl acyl carrier protein is employed as acyl donor . The inhibition of enzyme activity observed with palmityl-CoA as acyl substrate was dependent upon the relative concentrations of MgCl2 and ppGpp (MgCl2 to ppGpp ratio) employed . With palmityl-CoA as acyl donor, PPGpp inhibited the production of lysophosphatidic acid but not phosphatidic acid . With palmityl acyl carrier protein as acyl substrate, ppGpp had no influence upon the distribution of the reaction products. J Biol Chem, 1975 Jul 10, 250(13), 5109 - 13 Transmission of stability (telestability) in deoxyribonucleic acid . Physical and enzymatic studies on the duplex block polymer d(C15A15) - d(T15G15); Burd JF et al.; The properties of the duplex block polymer d(C15A15) - d(T15G15) were examined by thermal denaturation and nuclease susceptibility studies in the absence and presence of drugs (actinomycin and netropsin) which bind specifically to only one end of the block polymer . The nucleotide composition of one region of this synthetic double-helical DNA affected the properties of a contiguous but remote region . Furthermore, the binding of actinomycin influenced the properties of both the binding and nonbinding regions . These findings suggest a mechanism for gene regulation at a distance. Biochim Biophys Acta, 1975 Jul 8, 396(1), 17 - 23 Energy-linked and energy-independent transhydrogenase activities in Escherichia coli vesicles; Houghton RL et al.; Active transport vesicles of Escherichia coli were shown to possess low levels of energy-independent and energy-dependent nicotinamide nucleotide transhydrogenase activities . Breakage of such vesicles in a French pressure cell resulted in a fraction which had an 8-10-fold increased respiration- and ATP-driven transhydrogenase activities . Stimulation of the ATPase activity in vesicles with Triton X-100 was also paralledled by a 2-fold increase in the energy-independent transhydrogenase . Disruption of the vesicles similarly resulted in increases in the energy-independent transhydrogenase, NADH and succinate oxidase activities but a decrease in succinate supported proline uptake . In the light of these findings, the "sidedness' of the vesicle membranes is discussed. Biochim Biophys Acta, 1975 Jul 7, 395(3), 306 - 13 Physiological transition of a coliphage lambda DNA replication; Takahaski S; The "rolling-circle" replicative intermediate (sigma-type molecules) which is normally produced in the late stage of coliphage lambda DNA replication can be found during the first round of lambda DNA replication if cells infected with lambda replication mutant Ots28 are incubated at the nonpermissive temperature until the late stage of the latent period of lambda infection . After shifting to the permissive temperature, the vast majority of replicating forms are sigma-type rolling circle even during the first round of DNA replication . Concatemeric lambda DNA molecules, produced from these sigma-type intermediates, are efficiently packaged into progeny phage, indicating that in the first round of lambda DNA replication, double-branched theta-type molecules are not required for production of viable progeny phage. Biochim Biophys Acta, 1975 Jul 7, 395(3), 239 - 45 Analysis of the breaking sites in the physical degradation of DNA; Bertazzoni U; The 3'-termini of procaryotic and eucaryotic DNA fragments obtained by shearing, sonication and irradiation have been analyzed . Analysis of the 3'-ends of DNA fragments was done by 32-P-labeling with terminal transferase, hydrolysis of products by spleen DNAase and spleen exonuclease and separation of labeled 3'-terminal nucleotides on polyethyleneimine plates . In the case of sonication the deviation from DNA base composition of 3'-terminal nucleotides is very close to random for Escherichia coli and Haemophilius influenzae DNA, whereas for calf thymus, mouse and yeast mitochondrial DNAs a significant increase in dA and decrease in dC was observed . Shearing at high salt released random 3'-termini in E . coli DNA and 3'-termini with 4 to 6% deviation from base composition in calf thymus DNA . In the case of gamma-irradiation no real differences have been found between E . coli and calf thymus DNA and 3'-terminal nucleotide composition is very close to random . Mechanical breakage of eucaryotic DNA seems to release 3'-termini having a composition which differs slightly but significantly from the average base composition of the DNA. Biochim Biophys Acta, 1975 Jul 7, 395(3), 221 - 8 Determination of UTP and ATP pool sizes in human tonsillar lymphocytes by using Escherichia coli RNA polymerase; Sasvari-Szekely M et al.; The present paper describes a rapid, specific and sensitive method for quantitating ribonucleoside triphosphates (ATP and UTP) in cell extracts . The principle of the method is based on the synthesis of a ribonucleotide polymer in the presence of UTP, ATP and poly(dA-dT) as template . A method for calculation is also described, making the determination of UTP and ATP pool sizes in the cells possible under the same experimental conditions . The calculation takes into account the isotope dilution effect caused by the intracellular ATP . Our experiments show that the neutralized perchloric acid soluble fraction of human tonsillar lymphocytes contains no inhibitors for the RNA polymerase test . According to our results, this cell extract contains 80 pmol of UTP and 340 pmol of ATP per mug RNA. Biochim Biophys Acta, 1975 Jul 7, 395(3), 381 - 7 Specific conversion of s4 U to U in Escherichia coli tRNA by iodate oxidation; Wong KL et al.; The minor nucleoside 4-thiouridine in Escherichia coli tRNA is transformed selectively to uridine by iodate oxidation at acidic pH . The four major nucleotides were found to be inert under these conditions . The iodate oxidation appears to be more specific than the previous conversion methods reported, and has the advantage that it does not affect the chargeability of most tRNA. Mol Biol (Mosk), 1975 Jul-Aug, 9(4), 552 - 5 {Secondary structure of DNA from T4 and T6 phages}; Mokul'skaia TD et al.; The secondary structure of NaDNA from E . coli T4 and T6 phages has been studied by the X-ray diffraction method . Molecules of these DNAs as well as T2 phage DNA molecules contain hydroxymethylcytosine glucosylated at different position instead of cytosine . At high relative humidity these DNAs are shown to exist in B-conformaion . As humidity decreases the transformation into T=conformation takes place in the T4 phage DNA whereas in the T6 phage DNA changes of secondary structure similar to B-T transformation occur which do not result however in the appearance of all the characteristics of the T-conformation. Biochem J, 1975 Jul, 150(1), 9 - 12 Regulatory state of ribosomal genes and physiological changes in the concentration of free ribonucleic acid polymerase in Escherichia coli; Bremer H et al.; The concept of promoter efficiency is introduced as frequency of RNA chain initiation at a given promoter normalized to the intracellular concentration of free (but functional) RNA polymerase . Previous observations from this laboratory on the synthesis of ribosomes and beta-galactosidase are used to show that during a nutritional shift-up from succinate minimal to glucose-amino acids medium (3-fold increase in steady-state growth rate) the concentration of free (active) RNA polymerase decreases to one-quarter of the pre-shift value and the promoter efficiencies of the genes for ribosomal RNA and ribosomal proteins increase 9- and 6-fold respectively . This extent of control of ribosomal genes is much greater than expected on the basis of the increase in the rate of ribosome synthesis (3-fold). Biochem J, 1975 Jul, 150(1), 13 - 20 Synthesis time of beta-galactosidase in Escherichia coli B/r as a function of growth rate; Dalbow DG et al.; By analysing the kinetics of beta-galactosidase accumulation after induction, the synthesis time of beta-galactosidase in Escherichia coli B/r was found to be 75s in rapidly growing cells (1.36 and 2.1 doublings/h), and 90s in slowly growing cells (0.63 doubling/h) . These values correspond to peptide-chain-elongation rates of 16 and 13 amino acids/s respectively, in agreement with previous findings, indicating that the peptide-chain growth rate is constant (presumably maximal) in fast-growing bacteria, but decreased in slowly growing bacteria {Forchhammer & Lindahl (1971) J . Mol . Biol . 55, 563-568}. J Biochem (Tokyo), 1975 Jul, 78(1), 243 - 6 Formation of a binary complex between elongation factor G and guanine nucleotides; Arai N et al.; The interaction of the polypeptide chain elongation factor G (EF-G) from E . coli with guanine nucleotides was investigated using the hydrophobic dye, 1-anilino-8-naphthalensulfonic acid . It was found that the fluorescence intensity of the hydrophobic dye elicited in the presence of EF-G was diminished markedly by addition of GTP, and to a lesser extent, by addition of GDP . Direct evidence for the formation of the binary complexes, EF-G-GTP and EF-G-GDP, was provided by gel filtrations of EF-G on Sephadex G-25 columns equilibrated with buffers containing radioactive GTP and GDP, respectively. J Biochem (Tokyo), 1975 Jul, 78(1), 235 - 7 Enzyme-linked immunoassay . I . Novel method for synthesis of the insulin-beta-D-galactosidase conjugate and its applicability for insulin assay; Kato K et al.; Pork insulin was subjected to mercaptosuccinylation and then coupled to beta-D-galactosidase {EC 3.2.1.23} from Escherichia coli using N,N'-o-phenylenedimaleimide . The competitive binding of the conjugate and insulin to anti-insulin antibody was tested . Results showed that formation of an insulin-beta-D-galactosidase conjugate could be used for immunoassay of insulin. J Biochem (Tokyo), 1975 Jul, 78(1), 123 - 9 Effects of growth conditions on thymidine nucleotide pools in Escherichia coli; Hosono R et al.; The cellular levels of thymidine nucleotide derived from {3H}thymine or {3H}thymidine were followed under various environmental conditions with a thymine-requiring mutant of Escherichia coli . It was shown that the pool sizes varied greatly with the growth conditions; that is, with growth temperature, inhibition of DNA synthesis of replacement of thymine with thymidine . In the strain used here, the level of compound X, presumably dTDP-sugar, was very much higher than those of other thymidine nucleotides . It is suggested that the conversion of thymine to thymidine is rate-limiting, while the conversions of thymidine to dTMP, and of dTMP to dTDP are more rapid than other steps in the salvage pathway of thymidine nucleotide. Eur J Biochem, 1975 Jul 1, 55(2), 431 - 7 Altered alpha subunits in phenylalanyl-tRNA synthetases from p-fluorophenylalanine-resistant strains of Escherichis coli; Hennecke H et al.; Three different phenylalanyl-tRNA synthetases have been purified to near homogeneity, one from a wild-type strain of Escherichia coli and the others from two independently isolated p-fluorophenyalanine-resistant strains . The mutant enzymes were not able to use p-fluorophenylalanine as a substrate for activation and attachment to tRNA . They proved to be indistinguishable from the wild-type enzyme by several electrophoretic and immunological criteria . The alpha and beta subunits of all three enzymes have been prepared by a method described in this paper . The isolated subunits per se did not reveal any significant enzyme activity, but combined they were able to form active phenylalanyl tRNA synthetase after a defined reconstitution process . Mixed reconstitution experiments between wild-type and mutant subunits indicate that the mutant alpha subunit is responsible for p-fluorophenylalanine resistance and therefore seems to carry the phenylalanine-binding site or to participate in its formation. Eur J Biochem, 1975 Jul 1, 55(2), 343 - 54 The energy-coupling controlled efflux of 2-keto-3-deoxy-D-gluconate in Escherichia coli K 12; Lagarde AE et al.; Experiments were devised to test the plausibility and the predictions of a efflux rate equation which was previously derived {10}9 1 . 2-Keto-3-deoxy-D-gluconate transport system conforms with universal laws relating zero-trans influx, influx at steady-state, steady-state levels of accumulation to external and internal substrate concentrations . 2 . Full-time-course uptake kinetics fit the linearized graphical representation that can be inferred from the integrated rate equation . 3 . Influx does not depend upon internal substrate concentration nor upon energy-coupling . 4 . Zero-trans outflux (leak inot empty medium) is a first-order process (rate constant: 0.02 min-1) and not mediated by the carrier . Absence of cis-competition with D-glucuronate is in agreement with a simple diffusion mechanism . 5 . Outflux increases when external substrate concentration is raised (counterflow) . Outflux at steady-state equilibrates influx, and is a first-order process (rate constant: 0.15 min-1) . 6 . Uncoupling with azide leads to accelerate zero-trans outflux by a factor of 2-3 . No further acceleration is obtained when other classical uncouplers are used . The process remains first-order, independent of the amount of carrier, and is accelerated by the presence of internal D-glucuronate as a result from trans-inhibition of the recapture . 7 . Exchange outflux is all the more accelerated by azide as the carrier is less saturated . The process is clearly carrier-mediated and the outflux rate obeys a Michaelis law with respect to internal concentration . V is equal to V for influx . 8 . Homo and hetero-overshoot experiments are in agreement with the participation of the carrier for mediating influx as well as outflux . 9 . The kinetics of D-glucuronate outflux in a strain lacking the specific hexuronate permease but carrying the 2-keto-3-deoxy-D-glucuronate permease are similar to those obtained with 2-keto-3-deoxy-D-gluconate . We draw the conclusion that energy-coupling promotes the adjustment of outflux without interfering with influx rate . It apparently acts by reducing, in a continuous range, the affinity of the carrier facing inwards . The discussion is focused on the comparison with previously published models and on possible molecular mechanisms. Eur J Biochem, 1975 Jul 1, 55(2), 323 - 32 Two ribose-5-phosphate isomerases from Escherichia coli K12: partial characterisation of the enzymes and consideration of their possible physiological roles; Essenberg MK et al.; Two physically and genetically distinct forms of ribosephosphate isomerase have been identified in Escherichia coli K12 . The constitutive ribosephosphate isomerase A has a Km for ribose 5-phosphate (4.4 +/- 0.5 mM) six times greater than that of the inducible ribosephosphate isomerase B (0.83 +/- 0.13 mM) . Treatment of the enzymes with 1.25 mM iodoacetate resulted in 100% loss of activity for ribosephosphate isomerase B, whereas ribosephosphate isomerase A was unaffected . Various cellular metabolites were tested and found to be without significant effect on either enzyme . The two enzymes could be separated by filtration on Sephadex G75 superfine and their apparent molecular weights were 45000 for ribosephosphate isomerase A and 32000-34000 for ribosephosphate isomerase B . Under certain conditions the two enzymes showed different patterns of heat inactivation but the results with ribosephosphate isomerase A varied in an unusual way with the protein concentration . Ribosephosphate isomerase B was formed inducibly in a mutant lacking ribosephosphate isomerase A but there was no evidence for the production of ribosephosphate isomerase B in wild-type cells . The formation of ribosephosphate isomerase B was not a consequence of the ribosephosphate isomerase B mutation, since strains could be constructed which formed both enzymes constitutively in the anticipated amounts . The ribosephosphate isomerase formed by a secondary mutant obtained from a ribosephosphate-isomerase-A-negative strain was identified as ribosephosphate isomerase B on the basis of its Km, elution profile from Sephadex G75, inhibition of iodoacetate, and heat inactivation . The ribosephosphate isomerases of another Escherichia coli K12 strain, X289, were investigated, since their properties were reported to be different from many of these described here for ribosephosphate isomerases A and B . In our hands strain X289 contained two ribosephosphate isomerases apparently identical to ribosephosphate isomerases A and B . The evidence to date suggests that ribosephosphate isomerase A catalyses the formation of ribose 5-phosphate from ribulose 5-phosphate and also participates in the reverse reaction during ribose and adenosine catabolism . The normal physiological role of the inducible ribosephosphate isomerase B is still uncertain. Transplantation, 1975 Jul, 20(1), 68 - 74 Inhibition of tumor growth in syngeneic chimeric mice mediated by a depletion of suppressor T cells; Rotter V et al.; Syngeneic chimeric (lethally irradiated and reconstituted with syngeneic bone marrow cells) mice manifested an increased resistance to the development of Lewis lung carcinoma . In addition, these mice had a higher response to polyvinylpyrrolidone and a reduced reactivity to T mitogens . The present findings suggest that syngeneic chimeric mice lack suppressor T cells shown to regulate the development of Lewis lung tumor and the response to polyvinylpyrrolidone . Other components of the T cell population, such as helper cells responding to sheep red blood cells or cells involved in allograft rejection, assayed in these syngeneic chimeras were found unaffected . The fact that chimeric mice are deficient in a certain suppressor T cell population whereas other T activities are normal suggests the existence of different cell lines within the T cell population. Proc Natl Acad Sci U S A, 1975 Jul, 72(7), 2743 - 7 Cluster of genes in Escherichia coli for ribosomal proteins, ribosomal RNA, and RNA polymerase subunits; Lindahl L et al.; The transducing phage lambdarifd18 isolated by Kirschbaum and Konrad {(1973 J . Bacteriol . 116, 517-526} was found to carry structural genes for several 50S ribosomal proteins and 16S and 23S rRNA . It has previously been demonstrated {Kirschbaum & Scaife (1974) Mol . Gen . Genet . 132, 193-201} that this phage carries genes for the DNA-dependent RNA polymerase (nucleosidetriphosphate:RNA nucleotidyltransferase; EC 2.7.7.6) subunits beta and beta' . Thus, the region of the E . coli chromosome carried by lambdarifd18 contains a cluster of genes essential for transcription and translation. Pol J Pharmacol Pharm, 1975 Jul-Aug, 27(4), 439 - 43 Release of a prostaglandin E-like substance into mixed venous blood during endotoxin hypotension in cats; Korbut R et al.; An iv injection of E . coli endotoxin (10--15 mg/kg) produced biphasic hypotensive reaction in cats . The second phase of hypotension developed 5--10 min after the administration of endotoxin and was paralleled by an appearance of a substance contracting the chick rectum and rat colon which were bathed in mixed venous blood . On the basis of differential sensitivity of the assay organs to prostaglandins it was concluded that a prostaglandin E-like substance (PGEs) was released . Its peak concentration in blood was 1--3-5 ng/ml in terms of PGE2-like activity . Indomethacin (10 mg/kg) interrupted the liberation of PGEs into blood and at the same time a slow but steady increase in arterial blood pressure occured . The amelioration of endotoxin hypotension by indomethacin was abolished by an infusion of PGE2 at a rate of 20--100 ng/kg/min . It is suggested that the second phase of endotoxin hypotension in cats is mediated by a release of PGEs. Yale J Biol Med, 1975 Jul, 48(3), 211 - 6 An ultrastructural study of the renal medulla in experimental acute pyelonephritis; Dublin M et al.; Experimental acute pyelonephritis was produced in rats by a combination of intravenous administration of Escherichia coli, strain IMRU-54, and temporary unilateral mechanical ureteral obstruction . Structural alterations of the renal medulla were studied by light and electron microscopy . Major cellular alterations occurred in the vasa recta . Tubular and interstitial cells demonstrated minimal alterations after the brief period of acute inflammation . Polymorphonuclear leukocytes within tubular lumina contained structures resembling E . coli in nonprotoplasts-like form . Numerous protoplast-like organisms, to the exclusion of any other structural forms, were detected within the interstitium of the inner medulla . Nonprotoplast-like structures resembling E . coli were rarely observed in interstitium of the inner medulla . Following relief of ureteral obstruction, clearance of acute inflammation was rapid . In conclusion, hemoatogenous acute pyelonephritis induced by E . coli, IMRU-54, is able to inflict cytological and ultrastructural damage to structural elements of the inner and outer medulla of rats . Vasa recta incurred prominent alterations in endothelia and basement membranes, whereas tubular epithelia and interstitial cells had relatively good structural preservation . The data suggest that intravenously administered E . coli is capable to revert to a protoplast-like structure in the inner medulla. Mol Biol Rep, 1975 Jul, 2(2), 95 - 100 Complementary binding of oligonucleotides with 16S RNA and ribosomal ribonucleoproteins; Kopylov AM et al.; The accessibility of single-stranded sequences in 16S RNA in free state and in ribonucleoprotein particles (RNP) to complementary binding with isoplith fractions of oligonucleotides was studied . RNP had different protein composition and corresponded to intermediate stages of E . coli 30S subunit assembly in vitro . Gel-filtration was used to detect the most strong binding . It was found that S4 essentially inhibited the hexamer binding to RNA . 'Core' proteins bound to 16S RNA strongly increased the shielding of single-stranded regions while 'split' proteins insignificantly changed the hexamer binding . Nevertheless evidence is presented that 'split' proteins might also interact directly with 16S RNA in the 30S subunit. Clin Nephrol, 1975 Jul, 4(1), 8 - 12 The qualitative nitroblue tetrazolium (NBT) test as a means to differentiate between infection and rejection in renal transplant patients; Sher R et al.; In order to determine whether immunosupression depresses the response of the NBT test to bacterial infections and to note the effect of allograft rejection on this test, a prospective study was carried out on 30 renal transplant recipients . 12 of 30 renal transplant patients developed bacterial infections and in these patients NBT readings were elevated . 12 of the remianing 18 patients who developed rejection episodes showed normal NBT results . All patients were on high doses of steroids and other immunsuppressive agents . We conclude that the NBT test may be of value in diagnosing bacterial infection in the immunsuppressed allograft recipient, and may also be an useful adjunct in the differentiation between allograft infection and bacterial infection. Biochemistry, 1975 Jul, 14(13), 2907 - 14 Photoaffinity labeling of the ribosomal A site with S-(p-azidophenacyl)valyl-tRNA; Schwartz I et al.; S-(p-Azidophenacyl)valyl-tRNA, an analog of valyl-tRNA which has a photoaffinity label attached to its 4-thiouridine residue, was bound to the ribosomal A site at 10 mM Mg2+ . Binding was stimulated 25-fold by the presence of elongation factor EFTu . Photoactivation of the p-azidophenacyl group by irradiation resulted in covalent linking of 6% of the noncovalently bound tRNA to the ribosomes . Covalent linking was dependent on the simultaneous presence of ribosomes, poly(U2,G),EFTu.GTP, required irradiation, and did not occur when S-(phenacyl)valyl-tRNA, a nonphotolyzable analog, replaced S-(p-azidophenacyl)valyl-tRNA . The attached tRNA was distributed approximately equally between both the 30S and 50S subunits . At the 30S subunit, 30% of the tRNA was bound to protein while 70% was linked to 16S RNA . At the 50S subunit, however, negligible binding to the 23S RNA was observed . More than 90% of the tRNA was attached to low molecular weight material according to sodium dodecyl sulfate-sucrose gradient analysis, and more than 87% of this fraction consisted of tRNA-protein complexes as assayed by phenol solubility and electrophoretic mobility before and after protease treatment . These results, in conjunction with our previous report (I . Schwartz and J Ofengand (1974), Proc . Natl . Acad . Sci . U.S.A . 71, 3951) which showed that covalent linking of this same tRNA derivative at the ribosomal P site resulted in attachment solely to the 16S RNA, demonstrate that 16S, but not 23S or 5S rRNA, is an important component of the tRNA binding site in the region of the 4-thiouridine residue and furthermore show that ribosomal A and P sites are topologically distinct. Am J Surg, 1975 Jul, 130(1), 63 - 7 Biology of infections of split thickness skin grafts; Bacchetta CA et al.; The purpose of this study was to identify determinants of split thickness skin graft infection . The bacterial count of the experimental wounds was proportional to the incidence of infection in split thickness skin grafts . When the wound was heavily contaminated with 107 organisms, infection developed under most grafts . Graft take frequently occurred in wounds subjected to a lower level of inoculum . The importance of bacterial counts as a determinant of potential skin graft infection was also suggested by a clinical study . We now routinely use quantitative bacterial counts to identify the granulating wounds that are ready for grafting . The type of organism played no significant role in the development of infection. Nucleic Acids Res, 1975 Jul, 2(7), 1005 - 22 The binding of polyamines and of ethidium bromide to tRNA; Sakai TT et al.; The binding of spermidine and ethidium bromide to mixed tRNA and phenylalanine tRNA has been studied under equilibrium conditions . The numbers and classes of binding sites obtained have been compared to those found in complexes isolated by gel filtration a low ionic strength . The latter complexes contain 10-11 moles of either spermidine or ethidium per mole of tRNA; either cation is completely displaceable by the other . In ethidium complexes, the first 2-3 moles are bound in fluorescent binding sites; the remaining 7-8 molecules bind in non-fluorescent form . At least one of the binding sites for spermidine appears similar to a binding site for fluorescent ethidium . Similar results are found with E . coli formylmethionine tRNA . Spermine, in excess of 18-20 moles per mole tRNA, causes precipitation of the complex . Putrescine does not form isolable complexes with yeast tRNA and displaces ethidium less readily from preformed ethidium-tRNA complexes . Under equilibrium conditions, in the absence of Mg++, there are 16-17 moles of spermidine bound per mole of tRNA as determined by equilibrium dialysis . Of these, 2-3 bind with a Ksence of 9 mM Mg++, the total number of binding sites is decreased slightly and there appears to be only one class of sites with a Ka = 600 M(-1) . Quantitatively similar results are obtained for the binding of spermidine to yeast phenylalanine tRNA . When the interaction between ethidium bromide and mixed tRNA is studied by equilibrium dialysis or spectrophotometric titration, two classes of binding sites are obtained: 2-3 molecules bind with an average Ka = 6.6 x 10(5) M(-1) and 14-15 molecules bind with an average Ka = 4.1 x 10(4) M(-1) . Spermidine, spermine, and Mg++ compete effectively for both classes of ethidium sites and have the effect of reducing the apparent binding constants for ethidium . When the binding of ethidium is studied by fluorometry, there are 3-4 highly fluorescent sites per tRNA . These sites are also affected by spermidine, spermine and Mg++ . Putrescine has little effect on any of the classes of binding sites . These data are consistent with those found under non-equilibrium conditions . They suggest that polyamines bind to fairly specific regions of tRNA and may be involved in the maintenance of certain structural features of tRNA. Mutat Res, 1975 Jul, 29(1), 21 - 31 Phileomycin-induced lethality and DNA degradation in Escherichia coli K12; Nakayama H; The cell lethality and DNA fragmentation caused by phleomycin (PM) were studied in E . coli K12 strains with special reference to the effects of repair or recombination deficiences and metabolic inhibitors . (1) Unlike excision-defective derivatives of E . coli B, uvrA, uvrB, and uvrC mutants of strain K12 showed no peculiarities compared with wild type in regard to cell survival . Likewise, mutant alleles at uvrD and polA loci had no effect . In contrast, rec mutants were more sensitive to PM-killing than were rec+ strains . (2) PM-induced strand breakage in DNA was observed in all strains tested including the above-mentioned mutants . There was no significant distinction between the uvr mutants and the wild type strain, indicating that the uvr-endonuclease was not responsible for the strand breaks . Involvement of endonuclease I was also ruled out . (3) At least some of the PM-induced breaks were repairable . (4) PM-induced lethality and strand breakage were totally dependent on energy supply . Inhibition of protein synthesis resulted in a partial and parallel suppression of the two effects . Our results suggest that the lethality is due to DNA strand breakage and the repair of such damage is postulated to be controlled by rec genes. Mutat Res, 1975 Jul, 29(1), 1 - 20 Study of genetic effects of high energy radiations with different ionizing capacities on extracellular phages; Bresler SE et al.; The inactivating and mutagenic action of high-energy radiations with different ionizing capacities (gamma-rays, protons, alpha-particles and accelerated ions of 12C and 20Ne) was studied by using coliphages lambda11 and SD as subjects . In particular the role of irradiation conditions (broth suspension, pure buffer, dry samples) and of the host functions recA, exrA and polA was investigated . The dose-response curve of induced mutagenesis was studied by measuring the yield of vir mutants in lambda11 and plaque mutants in SD . The following results were obtained . (1) The inactivation kinetics of phages under the action of gamma-rays and protons was first order to a survival of 10(-7) . Heavy ions also showed exponential inactivation kinetics to a survival of 10(-4) . At higher doses of 20Ne ion bombardment some deviation from one-hit kinetics was observed . For dry samples of phages the dimensions of targets for all types of radiation were approximately proportional to the molecular weights of phage DNA's . For densely ionizing radiation (heavy ions) the inactivating action was 3-5 times weaker than for gamma-rays and protons . (2) Mutagenesis was observed for all types of radiation, but heavy ions were 1-5-2 times less efficient than gamma-rays . For both phages studied the dose-response curve of mutagenesis was non-linear . The dependence on the dose was near to parabolic for lambda11 . For SD a plateau or maximum of mutagenesis was observed for the relative number of mutants at a survival of about 10(-4) . (3) Host-cell functions recA and exrA were practically indifferent for survival of gamma-irradiated phage lambda11, but indispensable for mutagenesis . Mutation recAI3 abolished induced vir mutations totally and exrA- reduced them significantly . The absence of the function polA had a considerable influence on phage survival, but no effect on vir mutation yield (if compared at the same survival level) . (4) In conditions of indirect action of gamma-rays no vir mutations were induced . This is regarded as evidence that the single-strand breaks formed under indirect action conditions cannot serve as pre-mutational damage in DNA. J Pharm Sci, 1975 Jul, 64(7), 1235 - 7 Genetic effects of providone-iodine; Wlodkowski TJ et al.; Povidone-iodine is capable of specifically altering the DNA of living cells . This alteration may result in the induction of mutations of the base-substitution type . Because of the known relationship between mutagenic potential and the ability to induce cancer in animals, the present findings raise serious questions concerning the safety of this topical disinfectant. J Gen Microbiol, 1975 Jul, 89(1), 57 - 66 Genetical studies of serum resistance in Escherichia coli; Taylor PW; One induced and one spontaneous serum-resistant mutant were derived from smooth serum-sensitive Escherichia coli strains . There was little difference in the yield or the O-side-chain-sugar to core-sugar ratio of lipopolysaccharides from the mutants compared with the parental strains . The mutations to serum resistance were not accompanied by increases in either the amount or haemagglutination-inhibiting activity of acidic polysaccharides extracted from the strains . Two colonially rough forms, designated 17fa and 17gb, were isolated from an aged culture of serum-resistant mutant 17 . Escherichia coli 17fa appeared to be a som mutant and was rapidly killed by serum . Escherichia coli 17gb retained serological O-specificity, and 17gb lipopolysaccharide contained a full complement of O-side-chains; the strain was killed by serum but only after a delay of 1 h . Escherichia coli K-negative O8 donor Hfr59, which was serum-resistant, was crossed with rough serum-sensitive E . coli strain F470 and his+ recombinants were selected . The majority of his-+ recombinants inherited a full complement of lipopolysaccharide O-side-chains but were killed by serum after a delay of 1 to 2 h . These results suggest that lipopolysaccharide O-side-chains are responsible for a delay in the killing by normal human serum of smooth E . coli strains but that other factors determine full serum resistance . No evidence was found of a role for K-antigens in serum resistance. J Exp Med, 1975 Jul 1, 142(1), 253 - 8 Genetical control of B-cell responses . IV . Inheritance of the unresponsiveness to lipopolysaccharides; Coutinho A et al.; The inheritance of B-cell responsiveness to lipopolysaccharide (LPS) was studied in 55 crosses between mice of the low-responder strain C3H/HeJ and the high-responder strains B10.5M and C3H/Tif . F1 hybrid mice between the low-and the high-responder strains, showed in every case responses which were intermediate between the responses obtained with each parent . The responsiveness among F2 hybrid and backcross mice to either high- or low-responder parents, segregated into intermediate, high, or low categories, respectively . The present results are compatible with the hypothesis that responsiveness to LPS is determined by one single, codominantly expressed, autosomal gene . The capacity to develop a specific thymus-independent response to a hapten-LPS conjugate, also under genetical control, was found to segregate together with the capacity to develop polyclonal responses to LPS. J Immunol, 1975 Jul, 115(1), 135 - 8 Studies on leukocyte migration inhibiton: the role of E rosette-forming cells in specific antigen-induced inhibition of migration; Fimmel PJ; The role of E rosette-forming cells in leukocyte migration inhibition was investigated with human buffy coat cells fractionated by combined E rosette formation and Ficoll-Hypaque (FH) density gradient centrifugation . The migration of unfractionated dextran-sedimented leukocytes and FH-separated mononuclear cells (MN) were equally inhibited in the presence of Escherichia coli somatic antigen (E . coli), purified protein derivative of tuberculin (PPD) and influenza virus antigen (Flu) . Polymorphonuclear leukocytes (PMN) separated from whole leukocytes by one FH density gradient fractionation contained greater than 1.0% E rosette-forming cells and were inhibited by E . coli and PPD . PMN submitted to three transits of FH (DF III) contained less than or equal to 0.02% E rosette forming cells and were unaffected by each of the three antigens . The migration of mixtures of 10% MN and 90DF III cells were inhibited in the presence of specific antigen, while MN depleted of E rosette-forming cells (less than or equal to 0.5%) pooled with equal numbers of DF III cells did not respond to any antigen by migration inhibition . These results show that PMN purified by three transits of FH are unable to respond to specific antigen by inhibition of migration from microcapillary tubes, and specific antigen-induced inhibition of migration of peripheral blood leukocytes does not occur in the absence of D rosette-forming cells in normal subjects. J Cell Sci, 1975 Jul, 18(2), 315 - 26 Fine structure of an organelle associated with the nucleus and cytoplasmic microtubules in the cellular slime mould Polysphondylium violaceum; Roos UP; Polysphondylium violaceum was grown in association with Escherichia coli . Vegetative amoebae and pseudoplasmodia were fixed under different conditions and processed for electron microscopy . An electron-opaque body (nucleus-associated body, NAB) lies in the cytoplasm near the tapered end of interphase nuclei . The NAB consists of a disk-shaped, multilayered core, approximately 200 nm in diameter and 150 nm thick, embedded in a granular matrix from which electron-opaque nodules protrude . The nodules are termination points of microtubules radiating from the NAB into the cytoplasm or running along the nucleus . On the average there are 16 nodules per NAB . One or two microtubules terminate in each nodule . Spindle pole bodies, arising by duplication of the NAB at the beginning of mitosis, are unstructured foci for spindle microtubules in mitotic cells . It is suggested that cytoplasmic microtubules do not determine cell shape, but they probably cause the tapering deformation of the nucleus . They may, furthermore, represent a storage form of subunits for utilization during the formation of the mitotic spindle . The nodules of the NAB are potential nucleation sites of cytoplasmic microtubules during interphase . Spindle pole bodies presumably ac