Biokhimiia, 1994 Nov, 59(11), 1621 - 37 {Nucleotide sequence of the Escherichia coli B38 flagellin gene}; Zheleznaia LA et al.; The fliC gene of the E . coli B38 flagellin has been cloned and its nucleotide sequence determined using the terminator method . According to the sequencing data, the flagellin contains 565 amino acid residues which exceeds by 65 residues the number of amino acid residues in the earlier decoded E . coli K12 flagellin . Strong homology was observed in the two flagellins among the 160 initial and 89 tail-ended residues, whereas the central, variable parts showed no homology . Similar to the K12 flagellin, the B38 flagellin has no serines, cysteines or tryptophans . The variable part of the fliC E . coli B38 gene contains a Chi-site which initiates the genetic recombination in E . coli and related species. Bioconjug Chem, 1994 Nov-Dec, 5(6), 660 - 5 Preparation and characterization of a bifunctional fusion enzyme composed of UDP-galactose 4-epimerase and galactose-1-P uridylyltransferase; Tamada Y et al.; A fusion enzyme consisting of UDP-galactose 4-epimerase and galactose-1-P uridylyltransferase with an intervening Ala3 linker was constructed by in-frame fusion of E . coli gene galT to the 3'-terminus of the E . coli gene galE that had been extended with the coding sequence for three alanine residues, all contained within a high-expression plasmid . The fusion enzyme was expressed in E . coli and purified 24-fold to about 98% homogeneity by chromatography on hydroxylapatite and Q-Sepharose . On the basis of the comparison of the elution profile for enzyme activities upon gel permeation chromatography (Sephacryl S-400) with the molecular weight of 80,000 determined by sodium dodecyl sulfate polyacrylamide gel electrophoresis, the fusion enzyme appears to exist in monomeric, dimeric, and tetrameric forms, all of which exhibit both enzymatic activities . The Km values of the fusion enzyme for substrates were similar to those for the corresponding native enzymes, except for UDP-glucose, but the kcat values were smaller than those for the native enzymes . The fusion enzyme shows kinetic advantages in that the initial velocity to produce glucose-1-P from UDP-galactose and galactose-1-P is about 20% faster than that for a mixture of equal activities of the separate enzymes. Antimicrob Agents Chemother, 1994 Nov, 38(11), 2623 - 7 Comparison of inhibition of Escherichia coli topoisomerase IV by quinolones with DNA gyrase inhibition; Hoshino K et al.; In order to examine the inhibitory activities of quinolones against topoisomerase IV, both subunits of this enzyme, ParC and ParE, were purified from Escherichia coli . The specific activity of topoisomerase IV decatenation was found to be more than five times greater than that of topoisomerase IV relaxation . Thus, the decatenation activity of topoisomerase IV seems the most relevant activity for use in studies of drug inhibition of this enzyme . Although topoisomerase IV was less sensitive to quinolones than DNA gyrase, the 50% inhibitory concentrations for decatenation were significantly lower than those for type I topoisomerases . Moreover, there was a positive correlation between the inhibitory activity against topoisomerase IV decatenation and that for DNA gyrase supercoiling . These results imply that topoisomerase IV could be a target for the quinolones in intact bacteria and that quinolones could inhibit not only supercoiling of DNA gyrase but also decatenation of topoisomerase IV when high concentrations of drug exist in bacterial cells. Mol Biol Cell, 1994 Nov, 5(11), 1253 - 63 Nuclear mRNA accumulation causes nucleolar fragmentation in yeast mtr2 mutant; Kadowaki T et al.; We have identified a set of genes that affect mRNA transport (mtr) from the nucleus to the cytoplasm of Saccharomyces cerevisiae . One of these genes, MTR2, has been cloned and shown to encode a novel 21-kDa nuclear protein that is essential for vegetative growth . MTR2 shows limited homology to a protein implicated in plasmid DNA transfer in Escherichia coli . PolyA+RNA accumulates within the nucleus of mtr2-1 in two to three foci at 37 degrees C . mRNA, tRNA, and rRNA synthesis continue as do pre-mRNA splicing, tRNA processing, and rRNA export at 37 degrees C . Under these conditions the polyA tail length increases, and protein synthesis is progressively inhibited . Nucleolar antigens also redistribute to two to three nuclear foci at 37 degrees C, and this redistribution depends on ongoing transcription by RNA polymerase II . Surprisingly, these foci coincide with the sites of polyA+RNA accumulation . Comparable colocalization and dependance on RNA polymerase II transcription is seen for the mtr1-1 mutant . The disorganization of the nucleolus thus depends on mRNA accumulation in these mutants . We discuss the possible functions of MTR2 and the yeast nucleolus in mRNA export. Anal Biochem, 1994 Nov 1, 222(2), 479 - 82 Preparation of high-molecular-weight DNA: application to mycobacterial cells; Imai T et al.; A method for isolating high-molecular-weight DNA from bacteria is described . A special feature of the method is the treatment of whole bacterial cells with an organic solvent (chloroform-methanol (2:1, v/v) or ethanol-ether (1:1, v/v)) prior to DNA extraction from the cells . The DNA preparations obtained from organic solvent-pretreated bacterial cells such as Mycobacterium smegmatis, M . phlei, and Escherichia coli contained highly polymerized DNA, as revealed by pulse-field gel electrophoresis . The size and yield of the DNA obtained from E . coli pretreated with the organic solvent were quite similar to that of the DNA obtained from protoplasts . The results strongly suggest that the organic solvent pretreatment is effective for extracting very large DNA from bacterial cells and especially from bacteria whose protoplasts cannot be easily formed. Anal Biochem, 1994 Nov 1, 222(2), 456 - 60 Differential suppression of background mammalian lysosomal beta-galactosidase increases the detection sensitivity of LacZ-marked leukemic cells; Hendrikx PJ et al.; A method is described for the detection of Escherichia coli beta-galactosidase-expressing leukemic cells in ex vivo bone marrow samples . 4-Methylumbelliferyl-beta-D-galactopyranoside is used as a substrate in a kinetic assay . D-Galactose is used to suppress endogenous lysosomal beta-galactosidase activity, yielding a sixfold increase in sensitivity . With this assay, the detection limit is one leukemic cell per 10(4) normal bone marrow cells. Thromb Res, 1994 Nov 1, 76(3), 253 - 67 Fluorescence studies on plasminogen activator inhibitor 1: reactive centre cysteine mutants remain active after fluorophore attachment; Strandberg L et al.; To investigate structural-functional aspects of plasminogen activator inhibitor 1 (PAI-1) we have taken advantage of the lack of cysteines in the PAI-1 molecule and replaced Ser344 (P3) and Asn329 (P18) with cysteine residues, thereby creating unique attachment sites for extrinsic fluorescent probes . After expression in E . coli and purification to homogeneity, both of the mutant proteins were found to have similar biochemical characteristics as wild type PAI-1 (wtPAI-1) . Following labelling with 4-chloro-7-nitrobenzofurazan (NBD) and 2-(4'-iodoacetamido-anilino)naphtalene-6-sulfonic acid (IAANS) the mutant inhibitors showed similar inhibitory activities and heat stability as wtPAI-1 . The purified complex between uPA and NBD-labelled P3cys mutant was found to be extremely stable, suggesting that no slow cleavage or reversible reaction occurs in complexes that have been properly formed . The rate of labelling of both mutants was decreased when the mutants were in the latent form indicating that these cysteine residues may be less accessible in the latent configuration . The PAI-1 mutants labelled with both NBD and IAANS could convert from the active to the latent form, but P3cys labelled with the larger IAANS chromophore showed a two fold decrease in the rate of conversion to latency, suggesting that a large chromophore in the P3 position may interfere with the active to latent conversion . The fluorescence spectra of the two NBD labelled mutants were similar, but the intensity was three times higher for the P3cys mutant than for P18cys . No significant spectral changes could be seen when the P3cys mutant was transferred to latency . In contrast, the P18cys mutant showed a major change in the excitation spectra characteristic of migration of the NBD chromophore from a thiol to an amine . Complex formation with uPA had no effect on the fluorescence spectrum of P18cys-NBD while the spectrum of P3cys-NBD revealed changes consistent with a restriction of the mobility of NBD probe in the uPA-PAI-1 complex. Hum Exp Toxicol, 1994 Nov, 13(11), 764 - 75 Evaluation of the genetic toxicity of the peroxisome proliferator and carcinogen methyl clofenapate, including assays using Muta Mouse and Big Blue transgenic mice; Lefevre PA et al.; The rodent liver carcinogen and hepatic peroxisome proliferator methylclofenapate (MCP) has been evaluated for genetic toxicity in a range of in vitro and rodent genotoxicity assays . It gave a negative response in each of the following assays: mutagenicity to S . typhimurium and E . coli (+/- S9 mix, plate and pre-incubation assays), clastogenicity to cultured human lymphocytes and CHO cells (+/- S9 mix), a mouse bone marrow micronucleus assay (24h and 48h sampling), a rat liver assay for UDS in vivo (12h sampling), assays for lac I (Big Blue) and lac Z (Muta Mouse) mutations in the liver of transgenic mice, and an assay of the ability of MCP to modify the mutagenicity to the liver of dimethylnitrosamine in both transgenic mutation assays . The micronucleus and UDS assays were conducted using a single administration of MCP at its maximum tolerated dose, while the transgenic assays were conducted using nine daily administrations of MCP at its cancer bioassay dose level . These nine daily administrations were shown to double the weight of the liver of non-transgenic, Big Blue and Muta Mice, as well as leading to a dramatic proliferation of peroxisomes (electron microscopy) in the livers of each strain . These changed parameters had returned to control levels when the mutation analyses were conducted (10 days after the final dose of MCP) . Despite the liver enlargement observed following MCP administration, no evidence of mitotic activity was observed in treated livers, although an increased number of cells were undergoing replicative DNA synthesis during the final 3 days of the 9 days of administration (BUdR assessment of S-phase) . Liver biochemistry parameters (ALT, AST, AP, CK, GGT and albumin) were unaffected by the chronic (9 day) administration of MCP indicating an absence of hepatic toxicity . These combined observations favour a non-genotoxic mechanism of action for the hepatic carcinogenicity of MCP . The clastogenicity in vitro of the perixisome proliferator Wyeth 14,643 has been confirmed in CHO cells, but it is noted that this chemical is more soluble than is MCP . In particular, at the highest dose level at which MCP could be tested, Wy 14,643 was also non-clastogenic. Hum Exp Toxicol, 1994 Nov, 13(11), 759 - 63 Selective inhibition of gastrointestinal beta-glucuronidase by poly(vinylbenzyl D-glucaro(1,4)lactonate) . Part 2 . Poly(vinylbenzyl D-glucaro(1,4) lactonate) in vitro inhibition studies; Sacco C et al.; In vitro inhibition studies with beta-glucuronidase from purified E . coli and mouse intestinal contents indicated that the polymer, poly(vinylbenzyl D-glucaro(1,4)lactonate, is an effective beta-glucuronidase inhibitor . Purified E . coli beta-glucuronidase was inhibited by 99.6% with 177 mM D-glucaro(1,4)lactone using the polymer-inhibitor . Similarly, 95% inhibition of beta-glucuronidase activity of mouse intestinal contents was obtained with 177 mM and 50% inhibition was obtained with 31.5 mM D-glucaro(1,4)lactone based on the modified polymer . The structural requirements of an effective beta-glucuronidase inhibitor based on the structure of the polymer-inhibitor are also discussed. Virus Res, 1994 Nov, 34(2), 178 - 86 High level expression of the envelope glycoprotein (gp53) of bovine viral diarrhoea virus (Singer) and its potential use as diagnostic reagent; Yu M et al.; A 1.74-kb cDNA fragment containing the gp53 coding region has been cloned from bovine viral diarrhoea virus (BVDV) strain Singer by reverse transcription polymerase chain reaction (RT-PCR) . Sequence analysis indicated that gp53 of BVDV strains Singer, NADL and SD-1 shared extensive sequence homology at both the RNA (85-94%) and protein (82-91%) levels . Nineteen cysteine residues and five potential N-linked glycosylation sites were identified within the sequenced region, all of which were conserved . These observations suggest that although the homology at the nucleotide sequence level may vary, there was strong structural conservation among bovine viral diarrhoea virus envelope proteins . Full-length gp53 was expressed in Escherichia coli as a fusion protein with glutathione-S-transferase (GST) . The N-terminal half of gp53 was also synthesised in E . coli as a 28-KDa recombinant protein using the T7 RNA polymerase-directed expression system . Both recombinant proteins were expressed at high levels (approximately 30-50 mg/l) . The recombinant proteins were recognised in ELISA and Western blot analyses by polyclonal serum raised against a mixture of BVDV and classical swine fever virus (CSFV) . Rabbit antiserum raised against the 28-kDa recombinant protein reacted with different BVDV strains in ELISA and immunofluorescent antibody test, but not with CSFV in the same tests . These results demonstrated that the bacterial recombinant proteins have similar immunological properties to that of the native viral protein and, in conjunction with its homologous antisera, can be useful as diagnostic reagents. Vet Immunol Immunopathol, 1994 Nov, 43(4), 401 - 11 Caprine arthritis encephalitis virus infection changes caprine blood monocyte responsiveness to lipopolysaccharide stimulation in vitro; Werling D et al.; The effects of caprine arthritis encephalitis virus (CAEV) infection on cytokine activity of caprine monocytes stimulated with Escherichia coli lipopolysaccharide (LPS) were examined . Compared with supernatants from LPS-stimulated monocytes of CAEV-negative goats, supernatants from CAEV-positive goats stimulated less proliferation of murine thymocytes in the MTT (3-(4,5-dimethylthiazol-2-yl)2,5-diphenyl tetrazolium bromide) assay, showed about 50% less IL-1 activity on the IL-1-dependent cell line LBRM-33 1 A-5, and showed about 200% more tumor necrosis factor (TNF) activity on the TNF-sensitive murine fibroblast cell line L-929 . These results indicate that CAEV infection changes caprine monocyte cytokine responsivity. Pediatr Pathol, 1994 Nov-Dec, 14(6), 1017 - 28 Role of asphyxia and feeding in a neonatal rat model of necrotizing enterocolitis; Caplan MS et al.; Necrotizing enterocolitis (NEC) is a common gastrointestinal disorder affecting premature infants . To investigate critically the importance of the purported risk factors of NEC (formula feeding, asphyxia, bacteria, and prematurity), we developed a neonatal rat model that closely mimics the human disease . Full-term and premature newborn rats were stressed with formula feeding, asphyxia, and/or exogenous bacterial colonization and subsequently evaluated grossly and histologically for the development of intestinal injury . We found that most animals treated with asphyxia, formula feeding, and bacteria developed NEC (77%) and died (86%) by 96 h . All maternally fed animals treated with asphyxia and bacterial colonization survived and had normal intestinal histology . Furthermore, asphyxia was a critical instigating factor, because formula and bacterial exposure without asphyxia resulted in normal intestine and minimal mortality (12%) . Enteral bacterial colonization was not a significant determinant of NEC in this model . We conclude that the neonatal rat model is an excellent test system for the study of NEC . As in the human disease, asphyxia and formula feeding play an important role in the pathophysiology of experimental NEC. Mutagenesis, 1994 Nov, 9(6), 527 - 35 Resolution and conservation of mismatches in DNA end joining; Pfeiffer P et al.; DNA end joining is a major pathway for the elimination of double-strand breaks from chromosomal DNA of higher eucaryotic cells . Extracts of Xenopus laevis eggs rejoin such breaks even when their short single-stranded termini are expected to form imperfectly matched overlaps . However, end-joined products cloned in Escherichia coli, necessarily give rise to perfectly matched products . Therefore it has not been possible to determine whether the end joining process creates mismatched products, perfectly matched (resolved) products or both . To investigate whether mismatch resolution was the result of the X . laevis end joining process or of activities of the bacterial host we used denaturing gradient gel electrophoresis to analyse joined products . We found that the end joining process does include mismatch resolution, the degree of which varies with regard to the nature of the original overlap structure . Mismatches 3' to a gap are completely resolved, mismatches 3' to a nick and 5' to a nick or gap are resolved to some extent but are generally conserved . Mismatches between base matches are always conserved . These findings suggest competing processes of ligation, DNA fill-in synthesis or exonucleolytic excision of mismatched bases next to a gap or nick . At mismatches 3' to a nick the probability of ligation is greater than that of excision while at mismatches 3' to a gap the probability of excision is greater than elongation of a given mismatch . At mismatches 5' to nicks or gaps it appears that ligation or elongation and ligation, respectively, are the most probable pathways but products resulting from mismatch excision, elongation and ligation are also detected. Gen Comp Endocrinol, 1994 Nov, 96(2), 179 - 88 Cloning, expression, and characterization of a recombinant gilthead seabream growth hormone; Martinez-Barbera JP et al.; cDNA clones coding for the gilthead seabream (Sparus aurata) growth hormone (sbGH) were isolated from a pituitary expression library using a flounder cDNA probe . The nucleotide sequence of a GH cDNA clone containing an insert of 896 nucleotides was determined . The cDNA encoded a polypeptide of 204 amino acids including a signal peptide of 17 amino acids and contained a 5' and a 3' untranslated region of 48 and 233 nucleotides, respectively . The mRNA determined by Northern blot was approximately 1 kb . Amino acid sequence homologies of 97.1% with red seabream GH, 88.9% with the tuna GH, and 67% with the coho salmon GH was found . Transient expression of a sbGH cDNA was done in HeLa cells by induction with a vaccinia virus system, and the expressed GH was detected by immunofluorescence and immunoprecipitation with a specific antibody to the native sbGH . The sbGH cDNA was expressed in Escherichia coli by using the pGEX-3X and the pET-3a expression systems . The recombinant sbGH expressed in the pET-3a system was similar, if not identical, to the native hormone when analyzed by homologous radioimmunoassay and receptor binding assay. Cell Mol Biol (Noisy-le-grand), 1994 Nov, 40(7), 981 - 8 Expression of branching enzyme II of maize endosperm in Escherichia coli; Guan HP et al.; A cDNA clone encoding maize branching enzyme II (BEII) has been independently isolated from a maize endosperm cDNA library . The deduced protein sequence of maize BEII was compared with that of BE from diverse sources . The gene encoding mature BEII of maize endosperm has been expressed in E . coli using the T7 promoter . The expressed BEII was purified to near homogeneity so that amylolytic activity and bacterial BE could be completely eliminated from the BE preparation . The expressed enzyme showed very similar properties to those of BEII purified from developing maize endosperm . This result confirmed our earlier report that BEII had a lower rate of branching amylose and the rate of branching amylopectin was twice that of branching amylose . This study also showed a greater advantage of purifying BEII from the bacterial expression system than from developing maize endosperm . Most importantly, this study has established a useful tool to study the structure-function relationships of the maize BE using site-directed mutagenesis. Cell Mol Biol (Noisy-le-grand), 1994 Nov, 40(7), 899 - 905 A role for polyamines in the control of ppGpp levels in Escherichia coli; Goldemberg SH; As an approach to understand the involvement of polyamines in the variation of intracellular guanosine 5'-diphosphate 3'-diphosphate (ppGpp) levels, kinetic studies with several polyamine-requiring relA and spoT Escherichia coli mutants have been carried out . The accumulation and turnover of the nucleotide have been followed under conditions of aminoacid depletion or energy source starvation . The results obtained strongly suggest an important role of the polycations mainly in the degradation of ppGpp, and also in its synthesis mediated by ppGpp synthetase I (PSI). Cell Mol Biol (Noisy-le-grand), 1994 Nov, 40(7), 891 - 7 Protoporphyrinogen accumulation in cultured hepatocytes treated with the diphenyl ether herbicide, acifluorfen; Sinclair PR et al.; Diphenyl ether (DPE) herbicides such as acifluorfen inhibit both the plant and mammalian forms of protoporphyrinogen oxidase, a heme biosynthetic enzyme . Only small amounts of protoporphyrin accumulated in primary cultures of chick embryo and rat hepatocytes treated with acifluorfen and the porphyrin precursor, 5-aminolevulinic acid . However, there was a large accumulation of the porphyrin precursor, protoporphyrinogen, which was detected after oxidation to protoporphyrin by an E . coli membrane enzyme . In contrast, conventional methods of porphyrin analysis which depend on quantitative autoxidation of protoporphyrinogen failed to detect this accumulation of protoporphyrinogen . This is the first demonstration that protoporphyrinogen can accumulate to high levels and remain stable in liver cells . In addition, we found that the effect of a protoporphyrinogen oxidase inhibitor such as acifluorfen on the regulation of heme synthesis in hepatocyte cultures differed from that of an iron chelator. Biotechniques, 1994 Nov, 17(5), 974 - 80 Automated determination of beta-galactosidase specific activity; Bianco PR et al.; We describe a modification of an automated kinetic assay for beta-galactosidase (beta-gal) activity . This modification includes an assay to quantitate the amount of protein added to each assay . The determination of specific activity includes the amount of protein in the calculation which produces a specific activity with units of pmol product produced/minute/mg protein . In addition to this modification, we present a series of macros written in Microsoft Excel for either the Macintosh or Windows on the PC . These macros decrease the amount of time required to analyze the data from beta-gal assays. Biotechniques, 1994 Nov, 17(5), 922 - 6 Quantifying radiolabeled macromolecules and small molecules on a single gel; Morrison TB et al.; A protein phosphorylation cascade involved in chemotactic signaling in Escherichia coli was investigated with purified components in vitro . CheA, an auto-phosphorylating histidine kinase, was mixed with {gamma-32P}ATP, and the labeled protein was purified for use as a reagent in the assays . CheY, a response regulator protein, can acquire phosphate groups from CheA but then undergoes rapid hydrolysis, which releases inorganic phosphate . To follow the kinetics of the CheA-CheY phospho-transfer reaction and the subsequent dephosphorylation of phospho-CheY, we separated the reaction components by polyacrylamide gel electrophoresis and measured the amount of 32P label in the CheA . CheY and inorganic phosphate bands with phosphor storage screens . By reducing the time needed to separate and quantify the reaction products, we minimized diffusive spreading of the low molecular weight inorganic phosphate, which enabled us to measure it accurately on the same gel with the much larger proteins . In principle, any radiolabeled molecules that can be separated by relatively rapid means, such as acrylamide gel electrophoresis, and that are detectable with a phosphor storage screen, should be amenable to this technique. Bioessays, 1994 Nov, 16(11), 833 - 9 DNA damage tolerance, mismatch repair and genome instability; Karran P et al.; DNA mismatch repair is an important pathway of mutation avoidance . It also contributes to the cytotoxic effects of some kinds of DNA damage, and cells defective in mismatch repair are resistant, or tolerant, to the presence of some normally cytotoxic base analogues in their DNA . The absence of a particular mismatch binding function from some mammalian cells confers resistance to the base analogues O6-methylguanine and 6-thioguanine in DNA . Cells also acquire a spontaneous mutator phenotype as a consequence of this defect . Impaired mismatch binding can cause an instability in DNA microsatellite regions that comprise repeated dinucleotides . Microsatellite DNA instability is common in familial and sporadic colon carcinomas as well as in a number of other tumours . Several independent lines of investigation have identified defects in mismatch repair proteins that are causally related to these cancers. Neurosurgery, 1994 Nov, 35(5), 910 - 5; discussion 915-6 Stereotactic delivery of a recombinant adenovirus into a C6 glioma cell line in a rat brain tumor model; Badie B et al.; The dismal results of conventional therapy for primary malignant brain tumors has justified exploring gene therapy approaches for this disease . Transduction of animal brain tumor models in vivo has been reported previously with retroviruses and herpes viruses . Because adenoviruses have the advantage of transducing quiescent and actively dividing tumor cells, they may prove to be more effective in such therapy . We used a replication-deficient recombinant adenovirus bearing the Escherichia coli beta-galactosidase gene in a rat C6 glioma tumor model . Transduced cells were detected by X-5-bromo-4-chloro-3-indolyl beta-D-galactoside staining to reveal beta-galactosidase activity . Initial experiments in vitro showed 50% and 90% transduction at vector titers of approximately 10(7) and 10(8) plaque-forming units/ml, respectively . Although no cytopathic effects were seen at 10(7) plaque-forming units/ml, more than 50% reduction in tumor cell growth was noted at 10(8) plaque-forming units/ml both in vitro and in vivo . Stereotactic delivery of the recombinant adenovirus into the frontal lobe of normal rat brains resulted in intense staining of all cell types, that is, neurons, astrocytes, and ependymal cells . Stereotactic injection into C6 glioma brain tumors in rats stained 25 to 30% of the tumor cells . We conclude that adenovirus vectors can be used to transfer genes to central nervous system tumors in vivo . Using stereotactic delivery, adenovirus vectors can transfer genes into the central nervous system intended for tumor therapy. Free Radic Biol Med, 1994 Nov, 17(5), 379 - 88 Modulation of streptonigrin cytotoxicity by nitroxide SOD mimics; Krishna MC et al.; Nitroxides are cell-permeable, stable radicals that react readily with paramagnetic species such as transition metals or short-lived free radicals, though not generally with diamagnetic molecules . Nitroxides can undergo one-electron selective redox reactions and thereby potentially modify the activity of cytotoxic drugs . Streptonigrin (SN) toxicity requires bioreduction to yield the semiquinone radical, and the toxicity is reportedly mediated by transition metals and oxygen-derived reactive species via redox-cycling of the semiquinone intermediate . The present study shows that (1) nitroxides protected isolated DNA and also aerated or hypoxic bacterial cells from SN toxicity; (2) H2O2 potentiated the hypoxic cytotoxicity of the drug but inhibited the damage to aerated cells; (3) pretreatment of cells with H2O2 conferred some protection, but not when the drug alone was preexposed to H2O2; and (4) desferrioxamine and 2,2-dipyridyl, though neither diethylenetriamino pentaacetate, exogenous catalase, or superoxide dismutase, decreased SN-induced cell killing . The mechanisms by which nitroxides protect from SN toxicity involve both a selective radical-radical reaction with SN semiquinone and the reoxidation of reduced cellular transition metal ions . On the other hand, H2O2 appears to exert two opposing effects: (1) facilitation of cell killing by the Fenton reaction and (2) lowering the cellular level of reducing equivalents, thus inhibiting the bioreductive activation of SN. Surg Endosc, 1994 Nov, 8(11), 1332 - 4 Delayed gallstone abscess following laparoscopic cholecystectomy; Mellinger JD et al.; Delayed infectious complications following elective laparoscopic cholecystectomy have not been well delineated in the medical literature . Irretrievable spillage of gallbladder contents at the time of laparoscopic cholecystectomy is not rare, and has generally been felt to be of little consequence, particularly in the nonacute setting . The case presented documents an instance of delayed gallstone abscess formation after elective laparoscopic cholecystectomy . While rare, such cases highlight the need for refined techniques to prevent gallbladder, perforation during this procedure and to allow laparoscopic recovery of small gallstones spilled at the time of cholecystectomy. Kansenshogaku Zasshi, 1994 Nov, 68(11), 1381 - 9 {Studies on local immune response in mouse model of experimental Escherichia coli intrauterine infections}; Satoh T et al.; Studies were conducted to elucidate the immune cell response at infection sites by performing immunostaining of immune cells with a monoclonal antibody in an experimental Escherichia coli (E . coli) mouse uterine infection model . 1 . The incidence of uterine infection by E . coli decreased with the passage of time: 4/4 on Day 1, 4/6 on Day 3, 2/6 on Day 7, and 1/6 on each of Days 14 and 21 . It was surmised that clearance of the bacteria from the infection sites was being carried out by immune cells . 2 . Beginning from infection Day 1, the infected uterine tissue was observed to undergo a moderate degree of invasion by neutrophils, macrophages, CD4+ T cells, CD8+ T cells and IgA+ B cells . Then, beginning from infection Day 3, there was a mild degree of invasion of the infected uterine tissue by IgM+ B cells and IgG+ B cells . The number of neutrophils in the tissue decreased beginning from infection Day 14, but the degree of invasion of the infected tissue by the other kinds of immune cells remained almost constant through infection Day 21 . 3 . A comparison was made of the immune responses to local infection by E . coli, and Chlamydia trachomatis (C . trachomatis), an intracellular parasite . It was found that the invasion of the infection site by immune cells occurred earlier in the case of E . coli infection than C . trachomatis infection . In addition, the C . trachomatis infection site was observed to contain greater numbers of macrophages and CD8+ T cells play important roles in the immune defense at sites of infection by C . trachomatis. Kansenshogaku Zasshi, 1994 Nov, 68(11), 1330 - 7 {Molecular cloning of alpha antigen like protein gene of Mycobacterium leprae and its over production in Escherichia coli}; Yin Y; I have constructed the genomic library of M . leprae Thai 53 strain, and cloned the alpha antigen like protein gene by plaque hybridization method by using M . leprae alpha antigen DNA fragment as probe which was characterized in the previous study, I have termed it as alpha 2 antigen gene . The alpha 2 antigen gene has been characterized by sequencing . By comparing the deduced amino acid sequence of alpha and alpha 2 antigen with 85 complex antigen of other mycobacteria . I have found the higher homology between alpha 2 antigen and 85A antigen and between alpha antigen and 85B antigen . We have constructed the over expression system of M . leprae alpha and alpha 2 antigen gene in E . coli using vector pMALc-RI . Recombinant alpha and alpha 2 antigen has been purified by amylose column chromatography at the purity of more than 95% . More than 6 mg and more than 10 mg of recombinant alpha and alpha 2 antigen has been obtained from 200 ml of liquid culture, respectively . ELISA tests have been performed with the sera of leprosy patient and healthy control against the recombinant alpha and alpha 2 antigens . The antibody titers in sera of leprosy patient against the two kinds of antigens were all much higher than healthy controls . The antibody titer against the alpha 2 antigen was higher than that against alpha antigen . Recombinant alpha and alpha 2 antigens in this study could be used as a new specific antigen for serodiagnosis of leprosy. Gut, 1994 Nov, 35(11), 1613 - 6 Effect of Escherichia coli enterotoxins on macromolecular absorption; Verma M et al.; Macromolecular absorption of gliadin, a wheat protein and alpha lactalbumin, a milk protein was evaluated in control and Escherichia coli enterotoxin (heat-stable, heat-labile, and both heat-stable and heat-labile enterotoxin) treated mice . The peak concentration of gliadin and lactalbumin was two hours and three hours after their ingestion, respectively . There was also a significant increase (p < 0.01) in the absorption of both the proteins in all the three toxin treated groups compared with the control group . These results suggest that intestinal permeability and macromolecular absorption changes after E coli infection. J Exp Biol, 1994 Nov, 196, 443 - 56 Molecular physiology of the Na+/H+ antiporter in Escherichia coli; Padan E et al.; All living cells maintain an inwardly directed Na+ gradient and a constant intracellular pH . Na+/H+ antiporters have been assigned an essential role in these homeostatic mechanisms in all cells . In Escherichia coli, two Na+/H+ antiporter genes, nhaA and nhaB, have been cloned . Deletion of either one or both showed that NhaA is essential for adaptation to high salinity, for growth at alkaline pH in the presence of Na+ and for challenging Li+ toxicity . NhaB confers tolerance to low levels of Na+ and becomes essential when the activity of NhaA limits growth . The adaptive response to Na+ is mediated by the positive regulator nhaR, which transduces the signal (intracellular Na+) to expression of the nhaA gene . We have identified Glu-134 of NhaR as part of the 'Na+ sensor' of NhaA . In agreement with the role of NhaA in pH homeostasis, its Na(+)-dependent expression is enhanced at alkaline pH . Reconstitution of pure NhaA and NhaB in proteoliposomes demonstrates that, whereas both are electrogenic (the H+/Na+ stoichiometry of NhaA is 2), only NhaA is pH-dependent, increasing its activity 1000-fold between pH 7 and 8.5 . Mutating all the histidines of NhaA shows that His-226 is part of the 'pH sensor' of NhaA. J Exp Biol, 1994 Nov, 196, 213 - 28 Porters and neurotransmitter transporters; Nelson N et al.; Uptake of neurotransmitters involves multiple transporters acting in different brain locations under different physiological conditions . The vesicular transporters are driven by a proton-motive force generated by a V-ATPase and their substrates are taken up via proton/substrate exchange . The plasma membrane transporters are driven by an electrochemical gradient of sodium generated by a Na+/K(+)-ATPase . Two distinct families of transporters were identified in this group . One cotransports sodium with glutamate and other amino acids and requires additionally an outwardly directed potassium gradient . The second cotransports sodium, chloride and a variety of neurotransmitters, including gamma-aminobutyric acid (GABA), glycine and monoamines . Genes and cDNA encoding several members of the latter family have been cloned and studied in detail . The structure and function as well as the evolutionary relationships among these neurotransmitter transporters are discussed. J Exp Biol, 1994 Nov, 196, 183 - 95 The lactose permease meets Frankenstein; Kaback HR et al.; The lactose permease (lac) of Escherichia coli is a paradigm for membrane transport proteins . Encoded by the lacY gene, the permease has been solubilized, purified to homogeneity, reconstituted into phospholipid vesicles and shown to catalyse the coupled translocation of beta-galactosides and H+ with a stoichiometry of unity . Circular dichroism and other spectroscopic approaches demonstrate that the purified permease is about 80% helical . Based on hydropathy analysis of the primary amino-acid sequence, a secondary structure has been proposed in which the protein has 12 hydrophobic domains in alpha-helical conformation that traverse the membrane in zigzag fashion connected by hydrophilic loops . A variety of other approaches are consistent with the model and demonstrate that both the N and C termini are on the inner surface of the membrane, and studies on an extensive series of lac permease/alkaline phosphatase fusion proteins provide exclusive support for the topological predictions of the 12-helix motif . This presentation concentrates on the use of site-directed fluorescence spectroscopy to study structure-function relationships in the permease. Arch Dis Child Fetal Neonatal Ed, 1994 Nov, 71(3), F192 - 7 IgA antibodies in human milk: epidemiological markers of previous infections? Nathavitharana KA, Catty D, McNeish AS. The concept of an enteromammary link in secretory IgA (SIgA) antibody production was tested by hypothesising that specific SIgA antibody profiles in human milk might be an epidemiological marker for enteropathogens in a community . Milk from three subject groups was studied: 64 Sri Lankan women living in poor suburbs of Colombo, 20 Asian immigrant women domiciled in Birmingham, for a median period of five years (range 14 days-16 years), and 75 white women living in Birmingham . An enzyme linked immunosorbent assay (ELISA) was developed for the detection and measurement of SIgA antibodies to a panel of 14 crude O and 10 pure lipopolysaccharide antigens of diarrhoeagenic Escherichia coli strains well known to be endemic in the Indian subcontinent . The number of Sri Lankan and Asian immigrant women with SIgA antibodies to all 14 diarrhoeagenic E coli antigens (except O127 in Asian women) was significantly higher than in the white controls . The amount of E coli O antigen specific SIgA antibody activity as a percentage of total SIgA also gave significantly higher median values in Sri Lankan (6%) and in Asian immigrant (4%) women than in white controls (0.7%) . SIgA antibodies were highly O serogroup specific and showed excellent concordance between crude O and the corresponding purified lipopolysaccharide antigens . These results suggest that milk antibody profiles represent an epidemiological marker of exposure to enteral pathogens . The continuing specific milk antibody response in Asian women who have been domiciled in the United Kingdom for many years may indicate 'memory' in the human secretory immune system. Br J Haematol, 1994 Nov, 88(3), 520 - 6 Impaired TCR signal transduction, but normal antigen presentation, in a patient with common variable immunodeficiency; Fischer MB et al.; We describe a 27-year-old white man with common variable immunodeficiency (CVID) who has two healthy histoidentical brothers and one IgA-deficient sister who shares one HLA haplotype with the patient . T cells from the patient with CVID showed an impaired response to recall antigens (tetanus toxoid, E . coli), whereas his IgA-deficient sister and his two healthy histoidentical brothers responded normally . Cross-mixing experiments using isolated monocytes and T cells from the CVID patient and one histoidentical brother revealed that the patient's monocytes were fully functional in processing and presenting antigen to resting T cells of his brother, and provided normal accessory cell function for superantigen-induced activation of his brother's resting T cells . In contrast, the patient's T cells were unable to respond to antigen presented by the brother's monocytes and failed to respond with an increase in intracellular free Ca++ to stimulation with superantigen, which is known to bind to the TCR V beta-chain outside the antigen-binding groove . However, stimulation with a combination of PMA and IM, directly activating protein kinase C and increasing intracellular free Ca++ by bypassing membrane receptors, induced normal Ca++ flux . These data indicate that the patient with CVID has a defect in TCR-mediated signalling at the level of the T cells which is not present in his histoidentical healthy brothers or in his haploidentical IgA-deficient sister. Mol Gen Genet, 1994 Nov 1, 245(3), 390 - 6 Gene density and organization in a small region of the Arabidopsis thaliana genome; Le Guen L et al.; We have characterized a 6.4 kb genomic fragment from Arabidopsis thaliana ecotype Columbia overlapping the 5' end of the AKin10 gene which encodes a protein Ser/Thr kinase . Using, as probes, various restriction fragments located upstream of AKin10, two cDNA clones have been isolated from a cDNA library prepared from young shoot tissue . A comparison between the cDNA and the above genomic sequences allowed us to locate two novel genes, Atcys1 and Athyp1 (for Arabidopsis thaliana cystathionine gamma-synthase 1 and hypothetical protein 1) . The coding sequences of both genes are interrupted by introns and the exons match the sequences of the corresponding cDNAs . Further analysis of the genomic fragment revealed the presence of an open reading frame (ORF) of 609 nucleotides situated between the two genes . Atcys1, Athyp1, AKin10 and the ORF are very close to each other and organized in the same polarity; hence, the intergenic regions probably contain, within less than 0.5 kb, all the regulatory elements necessary to govern initiation and termination of transcription . The deduced protein sequence of Atcys1 shows a high degree of similarity with the cystathionine gamma-synthase from Escherichia coli . The putative product of the Athyp1 gene contains seven hydrophobic regions flanked by hydrophilic domains, reminiscent of membrane-spanning proteins . Southern blot hybridization experiments suggest the presence of one copy of Atcys1, Athyp1 and AKin10 per haploid genome, and Northern blot analysis demonstrates that the three genes are differentially expressed in roots, shoots and leaves. Mol Gen Genet, 1994 Nov 1, 245(3), 294 - 300 Recombination between repeats in Escherichia coli by a recA-independent, proximity-sensitive mechanism; Lovett ST et al.; We have examined the influence of proximity on the efficiency of recombination between repeated DNA sequences in Escherichia coli . Our experiments have employed a plasmid-based assay to detect deletions between direct repeats of 100 bp . The rate of deletion of the juxtaposed direct repeats was reasonably high at 6 x 10(-5) per cell . A comparison of recA+ and recA mutant strains showed that these deletion events are primarily the result of recA-independent recombination at these homologous sequences . Random restriction fragments of yeast or E . coli genomic DNA were used to separate the two repeats . Deletion rates decreased over two orders of magnitude with increasing separation of up to 7 kb . There was a surprisingly strong effect of even short sequence separations, with insertions of a few hundred base pairs exhibiting 10-fold reductions of deletion rates . No effect of recA on the efficiency of deletion was observed at any distance between repeats. Mol Gen Genet, 1994 Nov 1, 245(3), 279 - 85 MucAB but not UmuDC proteins enhance -2 frameshift mutagenesis induced by N-2-acetylaminofluorene at alternating GC sequences; Janel-Bintz R et al.; N-2-acetylaminofluorene has been shown efficiently to induce both -1 and -2 frameshift mutations in Escherichia coli as well as in mammalian cells . In E . coli, the genetic characteristics of -1 and -2 frameshift mutations were found to be distinct . The -1 frameshift mutation pathway occurs at monotonous runs of G residues (i.e . GGG-->GG) . This pathway exhibits the same genetic requirements as UV light-induced base substitution mutagenesis . Indeed, optimal mutagenesis requires the expression of both UmuDC and the activated form of RecA . The -2 frameshift mutation pathway operates at short alternating GpC sequences, such as the NarI sequence (i.e . GGCGCC-->GGCC) . In contrast to the -1 frameshift mutation pathway, optimal induction does not require the UmuDC and RecA proteins . This pathway involves a LexA-repressed function tentatively called Npf (for NarI processing factor) . In this paper, we show that MucAB efficiently stimulates the -2 frameshift mutation pathway . However, unlike the Npf pathway, MucAB-mediated stimulation requires expression of the RecA protein. Microbiology, 1994 Nov, 140 ( Pt 11), 2981 - 90 Regulation of transfer genes of promiscuous IncP alpha plasmid RK2: repression of Tra1 region transcription both by relaxosome proteins and by the Tra2 regulator TrbA; Zatyka M et al.; The Tra1 region of broad host range IncP alpha plasmid RK2 encodes proteins essential for its promiscuous conjugative transfer and includes oriT, the site at which nicking occurs to initiate transfer replication . Unregulated expression of the Tra1 region genes would be likely to place a major burden on the host . To investigate the control of these genes the three transcriptional promoters from this region were cloned by PCR and inserted into xylE promoter probe vectors . The strength of traJp and traKp was estimated to be six to eightfold less than the strong trfA promoter which is required for expression of genes for vegetative replication of RK2 . The traG promoter was about one-tenth the strength of the other two . These promoters are not repressed by products of the central control operon of RK2 . However, traJp and traKp, which are arranged as back to back divergent promoters in the oriT region, are repressed by TraK which constitutes part of the relaxosome necessary for nicking at oriT . A second relaxosome protein, TraJ, represses traJp . traGp is not repressed by any relaxosome proteins . All three promoters are repressed by TrbA, which is encoded at the start of the trb operon containing the rest of the transfer genes (the Tra2 region) . These circuits provide: (i) an autoregulatory way of ensuring production of enough relaxosome proteins without overburdening the host; and (ii) a means of coordinating expression of both blocks of transfer genes. Anal Chem, 1994 Nov 1, 66(21), 3840 - 7 Engineering the maltose binding protein for reagentless fluorescence sensing; Gilardi G et al.; This paper describes a mutant of the maltose binding protein (MBP) in which the serine residue at position 337 is replaced by a cysteine residue using site-directed mutagenesis . The mutant MBP has an approximately 2-fold lower affinity for maltose, and the cysteine residue can be modified with 4-{N-(2-(iodoacetoxy)ethyl)-N-methylamino}-7-nitrobenz-2-oxa-1,3-diazole (IANBD) and 6-acryloyl-2-(dimethylamino)-naphthalene (acrylodan) . This combined genetic and chemical modification places the fluorophores close to the maltose binding site such that when the ligand is added the fluorescence intensity of the labels increases by 60-180% over that of the ligand-free form . This change is consistent with the fluorophores being buried when the conformation of the protein changes with maltose binding . Titration of the labeled mutant proteins yields dissociation constants for maltose of 62 +/- 0.2 and 0.8 +/- 0.01 microM respectively for the IANBD and acrylodan modifications . The application of this strategy of combined genetic and chemical modification to the development of reagentless fluorescence sensing is discussed. Parasitology, 1994 Nov, 109 ( Pt 4), 525 - 30 Excretion of host immunoglobulin in tick saliva and detection of IgG-binding proteins in tick haemolymph and salivary glands; Wang H et al.; Host immunoglobulin G (IgG) crossed the gut wall into the haemocoel of adult Rhipicephalus appendiculatus female ticks when they fed on guinea-pigs . Guinea-pig IgG was also found in saliva of the feeding ticks . The concentration and antibody activity of IgG in haemolymph, salivary gland extract (SGE) and saliva at different stages of tick feeding were detected by enzyme-linked immunoassay . Specific activity of the IgG in tick samples was determined by feeding ticks on guinea-pigs which were immunized with killed Escherichia coli: 35-42% of the antibody activity in guinea-pig immune serum remained in the tick samples . The high relative concentration of IgG in tick saliva at later stages of feeding suggests that the tick may have a mechanism for getting rid of foreign proteins via the salivary gland . Such a mechanism could involve IgG binding proteins (IGBPs) which were found in both haemolymph and SGE of female ticks at day 6 of feeding using a guinea-pig IgG-agarose affinity column . In female ticks, the M(r) of IGBPs in SGE (23 and 57 kDa) were less than those in haemolymph (78 and > 100 kDa) . The existence of IGBPs in both the tick salivary gland and haemolymph indicate that haemolymph and salivary gland cooperate to remove foreign proteins, e.g . host immunoglobulin, from the body during feeding . This mechanism may be a part of the tick self-defence system. Appl Microbiol Biotechnol, 1994 Nov, 42(2-3), 313 - 8 Overproduction and purification of Lon protease from Escherichia coli using a maltose-binding protein fusion system; Sonezaki S et al.; Lon protease, which plays a major role in degradation of abnormal proteins in Escherichia coli, was overproduced and efficiently purified using the maltose-binding protein (MBP) fusion vector . The MBP-Lon fusion protein was expressed in a soluble form in E . coli and purified to homogeneity by amylose resin in a single step . Lon protease was split from MBP by cleaving a fusion point between MBP and Lon with factor Xa and purified by amylose resin and subsequent gel filtration . In this simple method, Lon protease was purified to homogeneity . Purified MBP-Lon fusion protein and Lon protease showed similar breakdown activities with a peptide (succinyl-L-phenylalanyl-L-leucyl-phenylalanyl-beta-D-methoxynaphthyl amide) and protein (alpha-casein) in the presence of ATP . Therefore, the gene-fusion approach described in this study is useful for the production of functional Lon protease . MBP-Lon fusion protein, which both binds to the amylose resin and has ATP-dependent protease activity, should be especially valuable for its application in the degradation of abnormal proteins by immobilized enzymes. Appl Microbiol Biotechnol, 1994 Nov, 42(2-3), 304 - 8 Sequence of the region downstream of the Vitreoscilla hemoglobin gene: vgb is not part of a multigene operon; Liu SC et al.; The 1668 base pairs (bp) downstream of the Vitreoscilla hemoglobin gene were sequenced in the hope of finding related genes that might be part of an operon . Instead, a sequence was found that constituted an open reading frame (ORF) of 569 amino acids (apparently the carboxy-terminal part of a larger ORF), in the direction opposite to the hemoglobin gene . This sequence was found to have 64% similarity with the 1685 bp at the 3' end of the Escherichia coli uvrA gene . The inferred amino acid sequence of the Vitreoscilla DNA has 69% similarity with the corresponding sequence of the E . coli uvrA protein, with similarities of 90, 100, and 85% in the helix-turn-helix, C-terminal ATP binding, and C-terminal zinc finger domains, respectively . The distance between the 3' ends of the Vitreoscilla hemoglobin and uvrA genes is 63 bp. Biotechnol Prog, 1994 Nov-Dec, 10(6), 648 - 51 Ammonium-mediated reduction of plasmid copy number and recombinant gene expression in Escherichia coli; Vila P et al.; The effect of ammonium as a medium supplement on plasmid-encoded recombinant beta-galactosidase synthesis was explored in Escherichia coli cells during aerobic growth in complex medium . After induction, only doses of ammonium chloride below 1 g/L are able to transiently enhance the yield . However, the presence of nontoxic ammonium chloride concentrations of up to 10 g/L results in lower values of beta-galactosidase in a concentration-dependent fashion . A significant reduction in plasmid DNA content explains the decrease in the yield by a gene-dosage-involving mechanism. Trends Biotechnol, 1994 Nov, 12(11), 456 - 63 Recent developments in heterologous protein production in Escherichia coli; Hockney RC; During the past three to four years, remarkable progress has been made in our understanding of protein folding, protein translocation across biological membranes, and the role of molecular chaperones in these processes . In conjunction with recent developments in Escherichia coli expression systems, this understanding has led to an improved capability to accumulate proteins in a soluble form, secrete proteins from the cell cytoplasm, accumulate proteins in the cytoplasmic membrane, and direct proteins to the outer membrane of the cell for surface display . These advances suggest that E . coli should now be considered seriously for applications that, only a few years ago, would have been thought beyond the scope of this organism. Lett Appl Microbiol, 1994 Nov, 19(5), 312 - 6 Loss of salt-tolerance and transformation efficiency in Escherichia coli associated with sub-lethal injury by centrifugation; Wyber JA et al.; Sub-lethal injury of Escherichia coli has been detected following centrifugation at g-forces between 5 and 30 kg . The extent of injury was measured either as a reduction in colony forming ability when plated onto NaCl-containing plates (2% w/v), or as a reduction in transformation efficiency associated with plasmid pBR322 encoding ampicillin resistance . In both cases, the extent of sub-lethal injury was found to increase with increasing centrifugal force and probably reflects structural damage to the cell envelope. Bioorg Med Chem, 1994 Nov, 2(11), 1119 - 32 Synthetic carbohydrate vaccines: synthesis and immunogenicity of Tn antigen conjugates; Toyokuni T et al.; A tumor-associated carbohydrate antigen, Tn antigen (GalNAc alpha 1-->O-Ser), was synthesized with a spacer arm, and assembled to dimeric and trimeric structures using N-tert-butyloxycarbonyl-O-(2-acetamido-3,4,6-tri-O-acetyl-2-deoxy-alpha- D-galactopyranosyl)-L-serine as a key building block . The synthetic antigens were conjugated with OSA and their immunogenicity examined in mice . Mice immunized with dimeric or trimeric Tn antigen showed a stronger antibody (IgM) response to a Tn-glycoprotein (asialo-ovine submaxillary mucin) than mice immunized with monomeric Tn antigen . The dimeric and trimeric Tn antigens also induced measurable IgG responses . The dimeric Tn antigen was further coupled to a Starburst dendrimer (5th generation) and to tripalmitoyl-S-glycerylcysteinyl-serine, a synthetic lipopeptide of the active moiety of a major lipoprotein of Escherichia coli . Unexpectedly, the Starburst dendrimer conjugate did not stimulate any immune response specific to Tn antigen . On the other hand, immunization of mice with the lipopeptide conjugate produced not only a high IgM response but also significant IgG anti-Tn response without any carrier molecules or additional adjuvants . The production of IgG antibody is quite significant since carbohydrate antigens are in general known to produce only IgM antibody response . Being a totally synthetic, low-molecular weight, and carrier-free immunogen, the lipopeptide conjugate could be a prototype of synthetic carbohydrate vaccines. Res Microbiol, 1994 Nov-Dec, 145(9), 699 - 709 The role of the adsorption complex in the termination of filamentous phage assembly; Gailus V et al.; The adsorption complex of filamentous phage fd consists of two minor coat proteins, g3p and g6p, and is considered to be not only a structural entity, but also a functional unit to terminate phage assembly . Cells were infected with phage M13am8H1, which cannot assemble because it lacks the major coat protein g8p, although producing all of the other minor coat proteins . The membranes of infected cells were solubilized and analysed by non-denaturing PAGE and gel filtration . The data suggest the presence of the adsorption complex in these membranes . Furthermore, the non-polar gene 3 amber-mutant phage R171 was shown to lack g6p in the phage coat as well . The termination of assembly of this phage is disturbed, resulting in synthesis of polyphages . Electron micrographs and transient electrical birefringence show that these polyphages are eight times longer as compared to unit length phage . From these results, we conclude that the formation of the g3p-g6p complex is essential for correct termination of filamentous phage assembly. Shock, 1994 Nov, 2(5), 344 - 50 Pulmonary capillary wedge pressure estimates of left ventricular preload are inaccurate in endotoxin shock: contribution of Starling resistor forces to septic pulmonary hypertension; Krahmer RL et al.; We tested the hypothesis that Starling resistor forces play a significant role in the increase in pulmonary vascular resistance during endotoxin shock . Anesthetized pigs (n = 9) were given Escherichia coli endotoxin (ETX; .5 mg/kg intravenously over 30 min) . Mean pulmonary arterial pressure (MPAP) and pulmonary capillary wedge pressure (PCWP) were recorded through a Swan-Ganz catheter . Pulmonary capillary pressure (Pc) was obtained from the analysis of the transient pulmonary artery pressure decay curve upon balloon inflation . Both proximal (Ra) and distal (Rv) pulmonary vascular resistance were calculated from cardiac output (CO), MPAP, Pc, and PCWP . Left atrial pressure (LAP) was measured directly via a left atrial catheter . Left ventricular end-diastolic wall thickness (LV-EDWT) was monitored by sonomicrometry, and used as an index of left ventricular preload . The results at baseline (t = 0) and t = 60 (30 min after the cessation of endotoxin infusion) were compared with saline control animals (n = 6) . Data were analyzed with a two-way ANOVA followed by contrast of residuals (p < or = .05) . After endotoxin, arterial blood pressure and CO fell significantly, an effect not seen in control pigs . In the control group neither LAP nor PCWP changed significantly over time, and remained equivalent to each other . In the septic shock group there was no difference between LAP and PCWP at t = 0 . However, by t = 60 LAP dropped and PCWP rose significantly . This fall in LAP and increase in PCWP were significantly different from the time-matched control values, and from each other.(ABSTRACT TRUNCATED AT 250 WORDS) Shock, 1994 Nov, 2(5), 336 - 43 The impact of infection on gluconeogenesis in the conscious dog; McGuinness OP; The effect of infection on gluconeogenesis was assessed in the chronically catheterized conscious dog . Dogs were studied 42 h after implantation of a sterile (n = 7) or an Escherichia coli containing (n = 7) fibrinogen clot into the peritoneum (54 h fasted) . Infection increased arterial plasma glucagon and cortisol (4.2- and 2.1-fold, respectively), but it did not alter arterial plasma insulin, catecholamines, or glucose concentrations . Infection increased tracer ({3-3H}glucose) determined glucose production and utilization and net hepatic glucose output by 35% . Net hepatic alanine and lactate uptake were also increased by 34 and 54%, respectively, without an alteration in their net hepatic fractional extraction . The intrahepatic efficiency of conversion of {14C}alanine to {14C}glucose was not decreased in the septic dog (.77 +/- .08) vs . .95 +/- .10 in noninfected and infected, respectively) . Intestinal glucose uptake and lactate release were increased approximately twofold . The increase in intestinal lactate release accounted for 35% of the increase in net hepatic lactate delivery seen in response to infection . In conclusion, a good model of hypermetabolic infection was developed in which the characteristic increases in hepatic glucose production and gluconeogenesis were observed in the fasted state . The increase in gluconeogenesis was due to an increase in hepatic gluconeogenic precursor uptake with no impairment in the net fractional hepatic extraction of gluconeogenic precursors or the efficiency of gluconeogenesis . In addition, the intestine is a significant contributor to the increase in gluconeogenic precursor supply seen in response to infection. Shock, 1994 Nov, 2(5), 332 - 5 Generation of a CD11b/c upregulating and chemotactic factor by hepatocytes of endotoxic rats--nonidentity with interleukin-8; Zhang P et al.; To further clarify the mechanism of polymorphonuclear leukocyte (PMN) recruitment into the liver associated with short term endotoxin infusion (1), we investigated the effect of a noval factor generated by hepatocytes of such endotoxic rats on the expression of PMN adhesion molecules CD11b/c and chemotactic activity . Conditioned medium of hepatocytes from endotoxin-infused rats shows a fast induction and dose-dependent activity for upregulating CD11b/c expression in and chemotactic activity for blood PMN of naive rats . Supernatants of naive control rats cultured in the presence of endotoxin and Kupffer cells and liver PMNs of endotoxic rats also produce activation, but to a much lesser extent . The upregulating activity can be reduced significantly by heat inactivation at 100 degrees C for 10 min and by pronase hydrolysis at 37 degrees C for 60 min . Generation of the activity does not depend on cyclooxygenase products or phospholipase A2 activity, and it does not seem to be associated with the complement pathway . The activity is associated with molecular masses of 9-12 and 27-32 kDa and cannot be reduced by antiserum to rat interleukin-8 in serial dilutions ranging from 1:50 to 1:25,600 . The results show that hepatocytes from acutely endotoxin infused rats generate a small molecular weight protein factor (or factors) that is capable of upregulating PMN 11b/c expression and chemotactic activity and is seemingly different from rat interleukin-8 . Thus, hepatocytes in endotoxemia may play an important role in modulating neutrophil function and contributing to the mechanism of neutrophil sequestration into the liver. Agents Actions, 1994 Nov, 43(1-2), 13 - 6 In vitro effect of cetirizine on PGE2 release by rat peritoneal macrophages and human monocytes; Roch-Arveiller M et al.; Cetirizine was first described as a specific anti-H1 molecule displaying potent antiallergic activity . It was later found that its pharmacological properties extended to cellular actions as on eosinophil recruitment at inflammatory sites in allergic patients . Monocytes and macrophages participate in allergic mechanisms, particularly through high affinity H1 and H2 membrane receptors and generation of pro- and anti-inflammatory agents; among them histamine-induced factors, IL-1 and prostanoids are of importance . The aim of this work was to investigate the effect exerted by various concentrations of cetirizine (0.1-10 micrograms/ml) applied in vitro to human monocytes and peritoneal rat macrophages cultured for 24 h . Peritoneal macrophages were collected either from normal or experimentally inflamed rats . Human monocytes, isolated from peripheral blood, were studied either in a resting state or after stimulation by LPS from Escherichia coli (1 and 10 micrograms/ml) . Cetirizine (10 micrograms/ml) significantly enhanced IL-1 release by human monocytes stimulated by a weak LPS concentration (1 microgram/ml) but could not modify the maximal increase of IL-1 release induced by 10 micrograms/ml of LPS . It did not exert any effect on resting cells . Cetirizine (0.1-10 micrograms/ml) enhanced PGE2 release by resting human monocytes . Concentrations of 1 and 10 micrograms/ml enhanced PGE2 release by LPS-stimulated monocytes, and by healthy and inflamed rat macrophages . This effect was concentration-dependent . Our findings point to an anti-inflammatory action of cetirizine via PGE2 release and histamine H2 interactions . Cetirizine did not directly modify IL-1 generation by resting monocytes but the IL-1 production observed after LPS stimulation could promote the mechanisms by which PGE2 is released. Mol Gen Mikrobiol Virusol, 1994 Nov-Dec, (6), 8 - 12 {In vitro and in vivo study of the activity of secreted alkaline phosphatase mRNA ribozyme gene}; Zakharchuk AN et al.; A ribozyme was constructed of the catalytic domain of tobacco ringspot virus satellite RNA and flanking sequences complementary to the target, secreted alkaline phosphatase (SEAP) mRNA . The ribozyme specifically cleaved the substrate in vitro; compared with that constant temperature . The relationship between specific endoribonuclease activity of the ribozyme and Mg2+ concentration was shown . The ribozyme was active in 293 cell line which was cotransfected with plasmids carrying the SEAP gene under control of RSV LTR promoter and the ribozyme gene under early HCMV promoter: SEAP activity reduced by half. Eur Cytokine Netw, 1994 Nov-Dec, 5(6), 533 - 8 Molecular studies on interleukin-1 alpha; Furutani Y; When rabbit alveolar macrophages were treated with phorbol-12-myristate-13-acetate (PMA) and lipopolysaccharide (LPS), the synthesis of interleukin-1 (IL-1), as well as tumor necrosis factor (TNF), was greatly increased . These inducible cytokines were subjected to cloning by the differential colony hybridization method and the subsequent mRNA hybridization-translation assay . Cloned rabbit IL-1 cDNA was disclosed to encode the sequence of the counterpart of the mouse IL-1 alpha . This cDNA was used as a hybridization probe to screen a human cDNA library which was constructed from induced HL-60 cells, a human promyelocytic leukemia cell line . Isolated human IL-1 alpha cDNA was shown to direct the synthesis of a polypeptide with IL-1 activity in E . coli expression system . The chromosomal gene for human IL-1 alpha was isolated and characterized to elucidate the structural organization of this gene . To identify the region that is essential for regulating IL-1 alpha gene expression, various CAT (chloramphenicol acetyltransferase) fusion plasmids were constructed and analysed for their ability to direct CAT synthesis in a transient expression system . The unpublished results obtained in the early stages of these experiments are also presented and discussed in this review. Microb Pathog, 1994 Nov, 17(5), 313 - 22 Immune response against the L-lactate dehydrogenase of Mycoplasma hyopneumoniae in enzootic pneumonia of swine; Frey J et al.; The L-lactate dehydrogenase (LDH) of Mycoplasma hyopneumoniae, formerly named protein P36, belongs to the predominant immunogenic proteins in pigs which were naturally or experimentally infected with M . hyopneumoniae . The antigenic reaction against M . hyopneumoniae LDH has been shown to be species specific . Recombinant M . hyopneumoniae LDH, which was genetically engineered to contain six histidine residues at its C-terminal end, was expressed in E . coli and purified to a high degree using Ni-chelate affinity chromatography . The genetically engineered LDH still showed the same biochemical activity and immunological specificity as the wild-type LDH and was used as an antigen for a M . hyopneumoniae LDH ELISA . Using this assay, we showed that pigs experimentally infected with M . hyopneumoniae raised antibodies against LDH in two steps . An early, relatively weak anti-LDH response was detected between 5 to 10 weeks post-infection when clinical signs and lung lesions occur . This first minor raise of anti-LDH antibodies occurred simultaneously with the strong appearance of antibodies against an antigen consisting of membrane proteins of M . hyopneumoniae prepared with Tween 20 extraction . A second, strong raise in anti-LDH antibodies was observed from the twelfth week after infection, at a time when the disease signs and the infectious agent disappeared . The high anti-LDH titer persisted until 21 weeks post-infection, in contrast to the antibody titer against the membrane proteins which started to decrease after its peak at 12 weeks post-infection . A LDH-ELISA may also be useful for detecting past infections. Development, 1994 Nov, 120(11), 3173 - 83 The mouse tissue plasminogen activator gene 5' flanking region directs appropriate expression in development and a seizure-enhanced response in the CNS; Carroll PM et al.; Tissue plasminogen activator (t-PA) is a secreted serine protease implicated in multiple aspects of development . In the adult rat brain, transcription of t-PA is an immediate-early response in the hippocampus following treatments that induce neuronal plasticity . To study the sequence elements that govern transcription of this gene, in situ analysis was used to define t-PA's temporal and spatial expression pattern in midgestation embryos . Transgenic mice were then generated carrying t-PA 5' flanking sequences linked to the E . coli lacZ gene . Constructs containing 4 kb of the flanking sequences (4.0TAMGAL) confer beta-galactosidase activity mostly to the same tissues that exhibit high levels of t-PA mRNA by in situ analysis . In 4.0TAMGAL embryos from embryonic day 8.5 (E8.5) to 13.5 (E13.5), the majority of expression observed is localized to neural ectoderm-derived tissues . beta-galactosidase activity is first detected in restricted neuromeres in the midbrain and diencephalon, at E8.5 and E9.5 respectively . At E10.5, transgene expression is observed in neural crest-derived cranial nerves and dorsal root ganglia, but not placode-derived cranial nerves . From E10.5 to E13.5, beta-galactosidase activity is observed in postmitotic neurons of the midbrain, spinal cord, neural retina and the developing olfactory system . beta-galactosidase activity is also detected in areas undergoing tissue remodeling such as the pinna of the ear, whisker follicles and the limbs . In adult mice, lacZ is expressed in the hippocampus and this expression was found to be enhanced upon seizure in the giant pyramidal neurons of CA3 . These results reinforce the concept that t-PA plays a role in neurogenesis and morphogenesis, and identifies the promoter region that directs its transcriptional regulation both in development and in the CNS. Biochem Mol Biol Int, 1994 Nov, 34(5), 955 - 61 Specific interaction of the Escherichia coli chaperone GroEL (60-KDA heat shock protein) with the liganded form of the galactose binding protein; Richarme G et al.; The Escherichia coli chaperone GroEL interacts more strongly with the liganded form of the galactose binding protein (the galactose binding protein-galactose complex), than with its unliganded form . This specific interaction is reflected by the stimulation of the ATPase activity of GroEL by the liganded galactose binding protein . Interactions between native proteins and chaperones could be more frequent than generally suspected, and may help to detect protein conformational changes. Protein Sci, 1994 Nov, 3(11), 2055 - 63 Protein structural similarities predicted by a sequence-structure compatibility method; Matsuo Y et al.; A method for protein structure prediction has been developed, which evaluates the compatibility of an amino acid sequence with known 3-dimensional structures and identifies the most likely structure . The method was applied to a large number of sequences in a database, and the structures of the following proteins were predicted: (1) shikimate kinase (SKase), (2) the hydrophilic subunit of mannose permease (IIABMan), (3) rat tyrosine aminotransferase (Tyr AT), and (4) threonine dehydratase (TDH) . The functional and evolutionary implications of the predictions are discussed . (1) The structural similarity between SKase and adenylate kinase was predicted . Alignment of their sequences reveals that the ATP-binding type A sequence motif and 2 ATP-binding arginine residues are conserved . The prediction suggests a similarity in their functional mechanisms as well as an evolutionary relationship . (2) The structural similarity between IIABMan and galactose/glucose-binding protein (GGBP) was predicted . The IIA and IIB domains are aligned with the N- and C-terminal domains of GGBP, respectively . The 2 phosphorylated residues, His 10 and His 175, of IIABMan are threaded onto loops located in the substrate-binding cleft of GGBP . The prediction accounts for the phosphoryl transfer from His 10 to His 175, and to the sugar substrate . (3) The structural similarity between rat Tyr AT and Escherichia coli aspartate AT was predicted, as well as (4) the structural similarity between TDH and the tryptophan synthase beta subunit . Predictions (3) and (4) support the previous predictions based on observations of the functional similarities between the proteins. Protein Sci, 1994 Nov, 3(11), 2005 - 14 Kinetics and crystal structure of a mutant Escherichia coli alkaline phosphatase (Asp-369-->Asn): a mechanism involving one zinc per active site; Tibbitts TT et al.; Using site-directed mutagenesis, an aspartate side chain involved in binding metal ions in the active site of Escherichia coli alkaline phosphatase (Asp-369) was replaced, alternately, by asparagine (D369N) and by alanine (D369A) . The purified mutant enzymes showed reduced turnover rates (kcat) and increased Michaelis constants (Km) . The kcat for the D369A enzyme was 5,000-fold lower than the value for the wild-type enzyme . The D369N enzyme required Zn2+ in millimolar concentrations to become fully active; even under these conditions the kcat measured for hydrolysis of p-nitrophenol phosphate was 2 orders of magnitude lower than for the wild-type enzyme . Thus the kcat/Km ratios showed that catalysis is 50 times less efficient when the carboxylate side chain of Asp-369 is replaced by the corresponding amide; and activity is reduced to near nonenzymic levels when the carboxylate is replaced by a methyl group . The crystal structure of D369N, solved to 2.5 A resolution with an R-factor of 0.189, showed vacancies at 2 of the 3 metal binding sites . On the basis of the kinetic results and the refined X-ray coordinates, a reaction mechanism is proposed for phosphate ester hydrolysis by the D369N enzyme involving only 1 metal with the possible assistance of a histidine side chain. Protein Sci, 1994 Nov, 3(11), 1998 - 2004 Glu-50 in the catalytic chain of Escherichia coli aspartate transcarbamoylase plays a crucial role in the stability of the R quaternary structure; Tauc P et al.; Glu-50 of aspartate transcarbamoylase from Escherichia coli forms a set of interdomain bridging interactions between the 2 domains of the catalytic chain; these interactions are critical for stabilization of the high-activity high-affinity form of the enzyme . The mutant enzyme with an alanine substituted for Glu-50 (Glu-50-->Ala) exhibits significantly reduced activity, little cooperativity, and altered regulatory behavior (Newton CJ, Kantrowitz ER, 1990, Biochemistry 29:1444-1451) . A study of the structural consequences of replacing Glu-50 by alanine using solution X-ray scattering is reported here . Correspondingly, in the absence of substrates, the mutant enzyme is in the same, so-called T quaternary conformation as is the wild-type enzyme . In the presence of a saturating concentration of the bisubstrate analog N-phosphonacetyl-L-aspartate (PALA), the mutant enzyme is in the same, so-called R quaternary conformation as the wild-type enzyme . However, the Glu-50-->Ala enzyme differs from the wild-type enzyme, in that its scattering pattern is hardly altered by a combination of carbamoyl phosphate and succinate . Addition of ATP under these conditions does result in a slight shift toward the R structure . Steady-state kinetic studies indicate that, in contrast to the wild-type enzyme, the Glu-50-->Ala enzyme is activated by PALA at saturating concentrations of carbamoyl phosphate and aspartate, and that PALA increases the affinity of the mutant enzyme for aspartate . These data suggest that the enzyme does not undergo the normal T to R transition upon binding of the physiological substrates and verifies the previous suggestion that the interdomain bridging interactions involving Glu-50 are critical for the creation of the high-activity, high-affinity R state of the enzyme. Protein Eng, 1994 Nov, 7(11), 1401 - 6 The effects of induction conditions on production of a soluble anti-tumor sFv in Escherichia coli; Sawyer JR et al.; CC49 is a second generation monoclonal antibody (mAb) with high affinity to a pancarcinoma antigen, TAG-72 . A single-chain Fv (sFv) of CC49 may have a role in managing human carcinomas . Most reported sFvs have been expressed as insoluble products that must be solubilized and renatured . Soluble sFv expression is advantageous as activity can be assayed directly from the periplasmic fraction . Also, gene-level immunoconjugates may not be amenable to refolding protocols . Using a vector that carries the tac promoter and omp A signal, we have examined the effects of four variables on the expression and accumulation of soluble CC49 sFv: (i) linker sequence joining VL and VH, (ii) isopropylthio-beta-D-galactoside concentration for induction, (iii) temperature, and (iv) the addition of non-metabolizable sugars to the medium . We have been able to demonstrate, using rapidly prepared periplasmic extracts, that the yield of soluble sFv improves by the addition of 0.4 M sucrose to the medium and by inducing expression with a very low concentration of IPTG (0.02-0.03 mM) . Under these induction conditions periplasmic extracts demonstrate increased expression of the sFv, as shown by the larger amount of a 27 kDa band on SDS-polyacrylamide gel, and an increased ability to inhibit binding of the mAb CC49 to immobilized tumor extracts. Protein Eng, 1994 Nov, 7(11), 1395 - 9 The role of the sequence extensions in beta-crystallin assembly; Kroone RC et al.; The modular construction of the eye lens beta gamma-crystallins makes them good candidates for protein engineering to ascertain the rules of assembly of oligomers . X-ray studies have shown that although the polypeptide chains of beta B2-crystallin and gamma-crystallins fold to form similar N- and C-terminal domains, the conformation of the connecting peptides are such that the gamma-crystallins are monomers and the beta-crystallin is a dimer . Unlike gamma-crystallins, the numerous beta-crystallins have extensions of variable sequence from the globular domains . We have tested the effect of removing the N- and C-terminal extensions from rat beta B2-crystallin using a bacterial expression system . Abundant proteins were produced in Escherichia coli using the pET or pQE vectors . Full-length and truncated proteins were purified and checked for refolding using circular dichroism . Sizing of the truncated proteins using gel filtration chromatography showed that the absence of either the N- or C-terminal extension does not affect dimerization of beta B2-crystallin. Protein Eng, 1994 Nov, 7(11), 1387 - 93 Human aldolase B: liver-specific properties of the isozyme depend on type B isozyme group-specific sequences; Kusakabe T et al.; A series of chimeric enzymes between two human aldolases A, B or C were constructed to identify the molecular regions responsible for isozyme-specific functions . Chimeras constructed between aldolases A and B were AB34 (an AB chimera connected at position 34), ABA34-306 and ABA212-306 (the ABA chimeras) . Those between aldolases B and C are BC243, BC263 and BC306 (the BC chimeras connected at positions as indicated), as well as CB55, CB243, CB263 and CB306 (the CB chimeras connected at positions as indicated), CBC55-263 (a CBC chimera), and BCB55-193, BCB55-306, BCB79-193 and BCB79-306 (the BCB chimeric enzymes) . Through the analysis of the properties of these chimeras, it was found that for aldolase B, isozyme B group-specific sequences (IGSs)-1 and -4 were required for exerting type B-specific functions, while the IGSs-2 and -3 enhanced, in collaboration with the IGS-1, the catalytic activity of aldolase B . In addition, the alpha/beta-barrel and the restricted stretches, which were not specified but occupied two distinct regions spanning the amino acid positions 108-137 (designated connector 1) and 243-306 (designated connector 2), were found to be indispensable for showing full catalytic activity of aldolase B. Chem Res Toxicol, 1994 Nov-Dec, 7(6), 823 - 8 Reductive metabolism of 1-nitropyrene accompanies deamination of cytosine; Malia SA et al.; 1-Nitropyrene (1-NP), a common environmental pollutant, is a mutagen and tumorigen . Nitroreduction is a major pathway by which 1-NP is metabolized . In order to study the mutational specificity of reductively activated 1-NP, single-stranded M13mp18 DNA was treated with tritium-labeled 1-nitrosopyrene in the presence of ascorbic acid to generate N-hydroxy-1-aminopyrene in situ . HPLC analysis of the treated DNA, following enzymatic digestion, showed that > 95% of tritium was located in one major adduct, N-(deoxyguanosin-8-yl)-1-aminopyrene . Transfection of these adducted M13 DNA in Escherichia coli indicated a dose-dependent reduction in viability with concomitant enhancement in mutagenesis in the lacZ gene fragment . Without SOS functions, the major type of mutation was C-->T transition (48%) . Further studies have shown that cytosine deamination occurred during ascorbic acid-induced nitroreduction, which was likely responsible for the C-->T transitions . Deamination of cytosine alco occurred at a significant frequency when nitroreduction of either 1-NP or 1-nitrosopyrene was catalyzed by xanthine oxidase, a mammalian nitroreductase. Arch Oral Biol, 1994 Nov, 39(11), 995 - 1000 Increased prostaglandin I2 and thromboxane A2 production by rat dental pulp after intravenous administration of endotoxin; Hirafuji M et al.; The effect of systemic endotoxin (lipopolysaccharide from Escherichia coli 0111:B4) on prostaglandin I2 (PGI2) and thromboxane A2 (TXA2) production by rat dental pulp was investigated . Intravenous injection of endotoxin increased ex vivo production of both PGI2 and TXA2 by the pulp tissue, when determined by radioimmunoassay . A significant effect on PGI2 and TXA2 production was observed with endotoxin doses of greater than 2 and 0.4 mg/kg, respectively . A significant increase was also observed at 30 min after injection of 10 mg/kg endotoxin, reaching a maximum after 60 min for both PG and TX production . Endotoxin (10 mg/kg for 60 min) also increased TXA2 but not PGI2 production in lung tissue, but had no effect in jejunal tissue . Indomethacin (10 microM) completely inhibited PGI2 and TXA2 production by the pulp of physiological saline- and endotoxin-treated rats . Further, arachidonic acids (10 microM) significantly increased PG and TX production by the pulp of saline- but not of endotoxin-treated rats . Endotoxin (100 micrograms/ml) had no in vitro effect on PG or TX production when incubated with isolated pulp, lung and jejunal tissues, suggesting that the endotoxin-induced increases in PG and TX production are an indirect effect . The endotoxin-induced increase in TXA2 production, but not in PGI2 production, by the pulp tissue was significantly suppressed by WEB 2170, a platelet-activating factor (PAF) antagonist . These results indicate that arachidonate metabolism in pulp tissue is susceptible to endotoxaemia in comparison with the lung and jejunum, and further suggest that the endotoxin-induced increase in, at least, TXA2 production by the pulp is mediated by PAF. J Endod, 1994 Nov, 20(11), 558 - 9 Hazards of laser smoke during endodontic therapy; McKinley IB Jr et al.; The purpose of this study was to evaluate the potential for spreading bacterial contamination from the root canal to the patient and the dental team via the smoke produced by the laser . Five extracted teeth were deliberately inoculated with a specific strain of Escherichia coli . The canals were subjected to an agron laser . The smoke plume was captured and cultured . All of the cultures were positive for growth of the E . coli used . It was concluded that the laser smoke does present a hazard of bacterial dissemination and that precautions must be taken to protect against spreading infections when using lasers in the root canal. Nat Struct Biol, 1994 Nov, 1(11), 765 - 70 Critical elements in proteasome assembly; Zwickl P et al.; Coexpression of both subunits of the Thermoplasma proteasome in Escherichia coli yields fully assembled and proteolytically active proteasomes . Post-translational processing of the beta-subunit occurs in E . coli as it does in Thermoplasma . Coexpression of the alpha-subunit and the beta delta pro-subunit, a mutant beta-subunit lacking the propeptide, also yields fully assembled and active proteasomes . This indicates that the beta-propeptide is not essential for the folding and assembly of Thermoplasma proteasomes . Separately expressed alpha-subunits assemble into heptameric rings indistinguishable from the terminal rings of a proteasome . Mutational analysis shows that the amino terminus, which is highly conserved in all proteasomal alpha-type proteins, is essential for assembly . In the absence of alpha-subunits the beta-subunits are monomeric and post-translational processing of the beta-propeptide does not occur. Circ Shock, 1994 Nov, 44(3), 154 - 9 Effect of estradiol on endotoxin-induced changes in steroid hormone levels and lethality in male rats; Christeff N et al.; We examined the effect of exogenous estradiol on the changes in serum steroid hormone levels induced by a nonlethal dose of Escherichia coli endotoxin in male rats and the deaths due to nonlethal and lethal doses of endotoxin . Injection of estradiol 5 min before a nonlethal dose of endotoxin changed the serum sex steroid hormone response of male rats to endotoxin . The serum estrogen concentrations of estradiol + endotoxin-treated rats decreased by 50% (P < 0.001), while those of the endotoxin-treated rats increased (2- to 5-fold) . The serum androgen concentrations of estradiol + endotoxin-treated rats did not change significantly, while those of endotoxin-treated rats dropped to 30-40%, P < 0.001 . Exogenous estradiol also appeared to influence the percentage of endotoxin-induced deaths in a dose-dependent manner . It reduced the number of deaths induced by nonlethal (2 mg/kg) dose of endotoxin but increased the number of deaths induced by a highly lethal dose (8 mg/kg) . These results, together with the known relationships between estrogen and the immune response, suggest that estrogens affect the course of septic shock in a complex fashion and may have either protective or deleterious effect. Circ Shock, 1994 Nov, 44(3), 148 - 53 Modulation of lung injury by platelet-activating factor antagonism in nonhypotensive porcine endotoxemia; Abu-Zidan FM et al.; Endotoxemia was induced by intravenous infusion of Escherichia coli endotoxin in 18 anesthetized pigs in a dose of 36 micrograms/kg/hr . Nine pigs were pretreated with BB-882, a novel platelet-activating factor (PAF) antagonist, 33 mg/kg/hr, starting 30 min before endotoxin, and nine pigs received a similar volume of vehicle . Normotension was maintained with intravenous crystalloid resuscitation . Six pigs received only BB-882 and served as controls . Endotoxemia induced an acute transient 300% increase in pulmonary vascular resistance, identical in both groups . The initial increase was followed by a second, more gradual, rise in resistance, which was significantly attenuated by BB-882 (P < 0.01, repeated measurements ANOVA) . Endotoxin-induced arterial deoxygenation and fall in lung/thorax compliance was not significantly altered by BB-882 . Hematocrit was less in endotoxic pigs receiving BB-882 (P < 0.02) . There were no significant changes compared to baseline in the control group . The results indicate that PAF is a minor determinant of early pulmonary dysfunction in nonhypotensive porcine endotoxemia. Braz J Med Biol Res, 1994 Nov, 27(11), 2551 - 5 Lethal interaction between hydrogen peroxide and o-phenanthroline in Escherichia coli; Asad NR et al.; The iron chelator o-phenanthroline enhances the lethal effect of H2O2 about four hundred times in Escherichia coli when both substances are added simultaneously to the culture medium . If o-phenanthroline is added for increasing periods of time prior to the addition of H2O2, there is a shift from this lethal interaction to protection by the chelator about seven hundred times . It is known that the Fe(2+)-o-phenanthroline(I) and Fe(2+)-o-phenanthroline(II) complexes are formed quickly whereas the final and more stable Fe(2+)-o-phenanthroline(III) complex is formed slowly . Moreover, the mono and bis complexes react with H2O2 to produce OH., whereas the tris complex is stable towards H2O2 . Therefore, the lethal effect could be explained by the kinetics of reaction of o-phenanthroline with intracellular Fe2+, i.e., the mono and bis complexes are more reactive than intracellular Fe2+. Can J Physiol Pharmacol, 1994 Nov, 72(11), 1308 - 12 Nitric oxide synthase induction and cytoprotection of rat gastric mucosa from injury by ethanol; Tepperman BL et al.; The present study determined the effects of nitric oxide (NO) synthase induction on ethanol-mediated damage to rat gastric mucosa . NO synthase activity was determined by {14C}arginine conversion to radiolabeled citrulline . Ca(2+)-independent NO synthase activity was determined by citrulline formation in the presence of EGTA (1 mM) in the incubation mixture . Intraluminal ethanol administration (2 mL; 40% w/v) to control rats resulted in an increase in mucosal damage characterized as vasocongestion and hemorrhagic necrosis and a reduction in Ca(2+)-dependent NO synthase activity . Administration of Escherichia coli lipopolysaccharide (LPS; 3 mg/kg i.v.) augmented Ca(2+)-independent NO synthase activity (determined 4 h later) and reduced damage in response to intraluminal ethanol instillation . Ethanol treatment did not significantly affect induction of NO synthase activity . Dexamethasone pretreatment (1 mg/kg i.v . 2 h before LPS administration) reduced both Ca(2+)-independent NO synthase activity and the gastroprotective effect of LPS against ethanol-mediated mucosal injury . Likewise, concurrent administration of the NO synthase inhibitor NG-nitro-L-arginine methyl ester (10 mg/kg s.c.) inhibited the gastroprotection associated with LPS treatment, an effect abolished by pretreatment with the NO substrate L-arginine (300 mg/kg s.c.) . Indomethacin (5 mg/kg i.v.) was ineffective in suppressing LPS-mediated gastroprotection . These results suggest that while Ca(2+)-dependent NO formation is inhibited by ethanol treatment, the inducible Ca(2+)-independent NO synthase plays a role in LPS-mediated gastroprotection against ethanol-mediated damage to the gastric mucosa. Zentralbl Bakteriol, 1994 Nov, 281(4), 433 - 41 rRNA gene restriction patterns as possible taxonomic tools for the genus actinomyces; Barsotti O et al.; Species delineation in the genus Actinomyces remains unclear, particularly regarding the two taxa, A . naeslundii and A . viscosus . The ribotyping patterns of 64 strains of Actinomyces, representing 8 species and comprising different serotypes, were studied as possible taxonomic tools, using an acetyl-aminofluorene (AAF)-labelled E . coli 16S + 23S rRNA probe . Similarities between patterns were assessed using Jaccard's coefficient and clustering achieved using the unweighted pair-group method with average linkage (UPGMA) on a Macintosh II (Apple, Cupertino, USA) computer . The dendrogram obtained from the ribotypes gave results which were in reasonable agreement with many previous reports: A . bovis, A . gerensceriae, A . israelii, A . meyerii, A . odontolyticus and A . pyogenes were found to be distinct species but the two taxa A . naeslundii and A . viscosus remained unclear . Further investigations, using a larger number of A . naeslundii and A . viscosus strains and other endonucleases, need to be carried out to provide more information concerning the relatedness of these two taxa . Nevertheless, these preliminary results suggested that the Actinomyces chromosome contains multiple rRNA operons which may be used as an epidemiological and taxonomical tool. Biochem Mol Biol Int, 1994 Nov, 34(5), 1035 - 48 Amino acid functional groups involved in the binding of Escherichia coli ribosomal protein S1 to ribosomes and nucleic acids; Ravi K et al.; Histidine, arginine, tyrosine, lysine and cysteine residues of protein S1 were modified with diethyl pyrocarbonate & rose bengal, 2,3-butanedione (diacetyl), tetranitromethane, pyridoxal 5-phosphate, and N-ethyl-maleimide, respectively . Modification of the residues and the number of modified residues were determined by either fluorescence or UV spectroscopy . The effect of chemical modification on the function of protein S1 was studied with respect to nucleic acid (poly U and M13 ssDNA) binding and ribosome binding properties of the protein . We tested S1 binding to these two types of polynucleotides because of their reported (Draper et al . (1977) PNAS 74, 4786-4790) binding to two different sites on S1 . The results indicate that histidine and lysine residues of S1 play an important role in the binding of S1 to both types of nucleic acids and that histidine and to some extent tyrosine residues are involved in the binding of S1 to ribosomes . The Data indicate the need for a re-evaluation of the two nucleic acid binding-site model. Mol Microbiol, 1994 Nov, 14(4), 731 - 41 mRNA degradation in Escherichia coli: a novel factor which impedes the exoribonucleolytic activity of PNPase at stem-loop structures; Causton H et al.; Stem-loop structures can protect upstream mRNA from degradation by impeding the processive activities of 3'-5' exoribonucleases . The ability of such structures to impede exonuclease activity in vitro is insufficient to account for the stability they can confer on mRNA in vivo . In this study we identify a factor from Escherichia coli which specifically impedes the processive activity of the 3'-5' exonuclease PNPase at stem-loop structures in vitro . This factor can, potentially, reconcile the apparent discrepancy between the ability of 3' stem-loop structures to stabilize upstream mRNA in vitro and in vivo . Its mechanism of action, and possible role in regulating mRNA degradation, is discussed. Mol Microbiol, 1994 Nov, 14(3), 427 - 36 The FinO protein of IncF plasmids binds FinP antisense RNA and its target, traJ mRNA, and promotes duplex formation; van Biesen T et al.; Most of the genes required for the conjugative transfer of DNA are encoded by the 33 kb transfer (tra) operon of F-like conjugative plasmids . Transcription of the tra operon is positively regulated by the TraJ transcriptional activator which, in turn, is negatively regulated by the FinOP fertility inhibition system . The FinOP system consists of an antisense RNA, FinP, and a 21.2 kDa protein, FinO, which together inhibit TraJ expression . Previously, it has been demonstrated that FinO increases the in vivo stability of the FinP RNA in the absence of the traJ mRNA target . Using electrophoretic mobility shift assays, we have shown that FinO is an RNA-binding protein that binds to one of the two stem-loops in FinP and to its complementary structure in traJ mRNA . This interaction presumably protects FinP RNA from degradation in vivo and increases the rate of formation of the FinP-traJ mRNA duplex fivefold . Thus, TraJ expression appears to be influenced by a unique RNA-protein interaction that precedes duplex formation between the FinP antisense RNA and its target traJ mRNA. FEMS Immunol Med Microbiol, 1994 Nov, 10(1), 19 - 24 Determination of specificities of rabbit antisera against the O-antigenic polysaccharide from Escherichia coli O126; Bhattacharyya T et al.; Rabbit polyclonal antibodies against the lipopolysaccharide of Escherichia coli O126 were serologically characterized by ELISA . The antibody specificities were determined by studying the inhibitory effects of the methyl glycosides of both anomeric configurations of the constituent monosaccharides and the oligosaccharides derived from the O-antigenic polysaccharide of E . coli O126 . It was found that, amongst the monosaccharides, beta-D-N-acetyl glucosamine was the most effective inhibitory sugar in the O126 polysaccharide and the major specificity of the polyclonal antibodies was found to be directed against the trisaccharide having the structure alpha-D-Galp (1-->3)-beta-D-GlcpNAc(1-->2)-D-Manp. Mamm Genome, 1994 Nov, 5(11), 717 - 22 Genomic structure of the mouse apurinic/apyrimidinic endonuclease gene; Takiguchi Y et al.; A mammalian apurinic/apyrimidinic endonuclease (AP endonuclease) is known to have two distinct functional domains . One domain is responsible for regulating the activity of Fos/Jun proto-oncogene products to bind to DNA at specific recognition sites . The other domain, which is highly conserved from bacteria to mammals, is responsible for repairing DNA damage caused by ionizing radiation, oxidative damage, and alkylating agents . This study reports on the isolation and characterization of the genomic structure of the mouse AP endonuclease gene (Apex) . The genomic sequence of the Apex gene was 2.14 kb in length and contained four exons . Exon 1 contained a 0.24-kb untranslated 5' region upstream of the initiation codon . Consensus sequences for two CAAT boxes and a GC box were found upstream of the end of exon 1 . A polymorphism was noted in the untranslated region of exon 1 in a comparison of a number of mouse strains . These data indicate that the 5' end of the mouse gene (Apex) differs from the previously isolated human gene (Ape), which has five exons and an untranslated region between exons 1 and 2 . Data are also presented that suggest the presence of two pseudogenes in the mouse. Differentiation, 1994 Nov, 58(1), 53 - 64 Sequences 5' of the bovine keratin 5 gene direct tissue- and cell-type-specific expression of a lacZ gene in the adult and during development; Ramirez A et al.; Expression of keratin K5 (and K14) in multilayered epithelia occurs predominantly in the basal layer of proliferating keratinocytes . When a keratinocyte becomes committed to terminal differentiation, it moves out of the basal layer towards the epithelial surface . As part of this program of terminal differentiation, the expression of K5 (and K14) is downregulated in suprabasal cells, and new pairs of differentiation-specific keratins are expressed . To define the cis-acting DNA sequences required for K5 cell-type- and differentiation-specific expression, chimeric gene fusions between portions of the bovine keratin K5 locus and the Escherichia coli lacZ gene were used to generate transgenic mice . In the genomic fragment consisting of 5.3 kb of 5' flanking sequences, 6.1 kb corresponding to the body of the gene and 4.5 kb of 3' flanking sequences, the subfragment extending from -5300 bp to +138 bp was the smaller region that directed lacZ expression to stratified epithelia in a manner analogous to the endogenous keratin K5 . Proximal sequences from -1300 bp to +138 bp were inactive . We also determined the expression pattern of keratin K5 during mouse development using an antiserum specific for mouse keratin K5 . Expression was first detected in ectodermal cells of 11.5 days postcoitum embryos, and from day 13.5 postcoitum onwards K5 was detected in the precursors of most epithelia and organs which express K5 at adult stages . This pattern was reproduced, with few differences, by the construct with sequences from -5300 bp to +138 bp fused to the lacZ gene . These findings identify sequences between -5.3 kb and -1.3 kb of the bovine K5 gene as being important for cell-type- and differentiation-specific gene expression both during mouse development and in the adult. Eur J Pharmacol, 1994 Nov 1, 292(1), 111 - 4 Lipopolysaccharide induces Ca(2+)-independent nitric oxide synthase activity in rat gastric mucosal cells; Brown JF et al.; Ca(2+)-independent nitric oxide synthase was detected in gastric mucosal cells isolated from rats injected 4 h previously with Escherichia coli lipopolysaccharide (3 mg/kg i.v.) . Induced nitric oxide synthase was located in an elutriated cell fraction of intermediate size which contained epithelial cells, but was absent from the parietal cell fraction . Administration of dexamethasone (2 mg/kg i.p.) 1 h before lipopolysaccharide inhibited the appearance of Ca(2+)-independent nitric oxide synthase, and prevented the observed reduction in cell viability (trypan blue exclusion) . Ca(2+)-independent nitric oxide synthase activity can thus be induced in certain cells of the gastric mucosa, and may contribute to gastric pathologies where there is activation of the immune system. Cell Mol Biol (Noisy-le-grand), 1994 Nov, 40(7), 933 - 44 Interactions between polyamine analogs with antiproliferative effects and tRNA: a 15N NMR analysis; Fernandez CO et al.; N-Bisalkylpolyamine analogs have been shown to exert antiproliferative effects in many tumor models, with the bisethyl derivatives exerting the greatest activities . 15N NMR spectroscopy was used to explore the interactions between these analogs and tRNA . When tRNA was added to solutions of 15N-enriched homospermine (4-4-4), bisethylhomospermine (BE-4-4-4), bismethylhomospermine (BM-4-4-4), bisethylspermine (BE-3-4-3) and 1,19-bis(ethylamino)-5,10,15-triazanonadecane (BE-4-4-4-4), the spin-lattice relaxation times T1 of the nitrogens were strongly reduced . From the temperature dependence of these T1's we calculated the rotational activation energies (Ea) of the correlation times of the amino groups in the presence and absence of tRNA . These data indicate that: i) the N-bisethyl derivatives bind strongly to tRNA through their-NH2(+)-groups (most likely, through hydrogen bonding); ii) the binding is weakest in the N-bismethyl derivative and iii) homospermine binds very weakly and mainly through its -NH3(+)-group (most likely, through electrostatic binding) . The binding of the polyamine analogs to tRNA was also estimated by the increase of the half-line widths (D1/2) of the -NH2(+)-groups, derived from the effects that tRNA has on the spin-spin relaxation time T2 . The decrease of the V1/2 values of the -NH2(+)-groups in the (15N-polyamine)-tRNA complexes when the analogs were chased away by an excess of spermine confirmed the stronger binding of the bisethyl- with respect to the bismethyl derivatives, as well as the weak binding of homospermine to tRNA . A correlation was also found between the binding strengths of the analyzed polyamine analogs and their antiproliferative activities. Mol Gen Genet, 1994 Nov 1, 245(3), 355 - 62 Role of guanosine tetraphosphate in gene expression and the survival of glucose or seryl-tRNA starved cells of Escherichia coli K12; Nystom T; The concentration of guanosine 3',5'-bispyrophosphate (ppGpp) increases in bacteria in response to amino acid or carbon/energy source starvation . An Escherichia coli K12 delta relA delta spoT mutant lacking the ability to synthesize ppGpp lost viability at an increased rate during both glucose and seryl-tRNA starvation . Also, the deleterious effect of chloramphenicol on starved wild-type cells could be overcome by inducing expression of RelA from a plasmid carrying the relA gene transcribed from a tac promoter, prior to starvation and chloramphenicol treatment . As demonstrated by two dimensional gel electrophoresis, this induction of the RelA protein resulted in global alterations in gene expression including increased synthesis of some rpoS-dependent proteins . The delta relA delta spoT mutant maintained high expression of several ribosomal proteins during starvation and appeared to exhibit significantly decreased translational fidelity, as demonstrated by an unusual heterogeneity in the isoelectric point of several proteins and the failure to express higher molecular weight proteins during starvation . Moreover, both rpoS-dependent and independent genes failed to exhibit increased expression in the mutant . It is suggested that the deleterious effects on the cells of the relA, spoT deletions are not due solely to the inability of these cells to induce the sigma factor sigma s, but also to deficiencies in translational fidelity and failure to exert classical stringent regulation. Mol Gen Genet, 1994 Nov 1, 245(3), 265 - 71 Expression of the linear DNA plasmid pRS64 in the plant pathogenic fungus Rhizoctonia solani; Hongo M et al.; The plant pathogenic isolate RI-64 of anastomosis group 4 of Rhizoctonia solani possesses three linear DNA plasmids (pRS64-1, -2, and -3) . Unique poly(A)- RNA, 0.5 kb in length and hybridizable with the pRS64 DNAs was found in mycelial cells of the isolate RI-64 . The overall homology at the nucleotide level between pRS64-1, -2, and -3, and the cDNA prepared from the poly(A)- RNA was 100%, 73%, and 84%, respectively . The open reading frames found in pRS64-1, -2, and -3 (ORF1-1, ORF2-1, and ORF3-1) are 68 amino acids long . The amino acids sequence showed no significant homology with known proteins . Extracts from Escherichia coli cells expressing ORF1-1 contain a specific protein of 7 kDa . Antisera raised against the ORF1-1 product obtained from E . coli cells cross-reacted with the specific proteins found in the mycelia . The results indicate that the DNA plasmids found in R . solani contain a sequence that encodes a specific protein which may be involved in determination of plant pathogenicity. J Chem Technol Biotechnol, 1994 Nov, 61(3), 219 - 24 Liquid-liquid extraction of a recombinant protein with reverse micelles; Pires MJ et al.; Recombinant cytochrome b5 was extracted into the reversed micelle phase of an anionic surfactant (AOT) in octane and back-extracted to a final aqueous phase . The extraction of the protein was controlled by an electrostatic mechanism, since it was dependent on the global charge of the protein . This was directly demonstrated by experiments with native and mutant cytochromes obtained by site directed mutagenesis . The back-extraction of cytochrome b5 to a fresh aqueous phase was decreased by factors that reduced the size of the water pool of the organic phase, such as high salt concentrations (1-2 mol dm-3 NaCl) and low temperatures (4 degrees C), probably because of an increase in a favourable interaction of this protein with the surfactant at closer distances. Mutat Res, 1994 Nov, 315(3), 229 - 37 The XPA protein is a zinc metalloprotein with an ability to recognize various kinds of DNA damage; Asahina H et al.; The XPA (xeroderma pigmentosum group A) gene encodes a protein of 273 amino acids with a zinc finger motif . The human XPA cDNA was placed in an Escherichia coli expression vector for the synthesis of the recombinant XPA protein . The molecular weight of the wild-type protein was about 40 kDa in SDS-PAGE . Microinjection of the wild-type protein specifically restored the defect of UV-induced unscheduled DNA synthesis in XP-A cells . Thus, the bacterially expressed XPA protein retains biochemical properties identical to those of natural sources . The wild-type protein binds preferentially to UV-, cis-diamminedichloroplatinum(II) (cisplatin)- or osmium tetroxide (OsO4)-damaged DNA as assayed by retention on nitrocellulose filters . In addition, the data from atomic absorption and UV-CD spectra revealed that the wild-type protein is a zinc metalloprotein with secondary structure . Furthermore, the mutant protein, of which the cysteine-103 residue in the zinc finger motif was replaced with serine, has a vastly different protein conformation resulting in a loss of XP-A correcting and DNA-binding activities . These findings indicate that the XPA protein is a zinc-binding protein with affinity for various DNA damages, and a cysteine residue in the C4-type zinc finger motif is indispensable for normal protein conformation. Mutat Res, 1994 Nov 1, 311(1), 57 - 67 Spontaneous mutations in lacI-containing lambda lysogens derived from transgenic mice: the observed patterns differ in liver and spleen; Knoll A et al.; The pattern of somatic mutation observed in tumor suppressor genes, such as the p53 gene, vary dramatically with tumor type . Some of the observed differences are due to tissue specific effects of mutagens, but it is also possible that some differences may reflect the tissue/cell type specificity of spontaneous mutation . Transgenic mouse models with recombinant shuttle vectors containing the lacI or lacZ target genes may shed light on the extent to which spontaneous mutation displays tissue specificity . Herein we utilize a recently described selectable system to obtain spontaneous mutants for analysis of the molecular lesions . Spontaneous mutations were isolated in the lacI gene recovered from five transgenic mice carrying a lambda shuttle vector . Seventy-three and 67 independent mutations derived from liver and spleen DNA, respectively, were defined in the amino terminal region of lacI . Although technical barriers preclude a direct assessment of the E . coli derived pattern of mutation in this system, five pieces of circumstantial evidence suggest that many of the mutations arose in mouse rather than in E . coli . In DNA from both liver and spleen, mutations at CpG dinucleotides predominate (58% and 51%, respectively) . In spleen, most of the mutations at CpG are transitions, while in liver most are transversions . In addition, liver has a higher frequency of GC-->TA transversions at non-CpG dinucleotides while spleen had a higher frequency of deletions and insertions . The data provide evidence that the spontaneous pattern of mutation is tissue specific . In addition, the high frequency of transversions at CpG suggests the need to reevaluate the mechanisms by which mutations occur at this methylated dinucleotide. Mutat Res, 1994 Nov 1, 311(1), 103 - 9 Ruv and recG genes and the induced precise excision of Tn10 in Escherichia coli; Nagel R et al.; Induction of precise excision of Tn10 by UV or mitomycin C (MMC) is dependent on the expression of the SOS system . Ruv mutants of Escherichia coli, which are defective in DNA repair and recombination, showed diminished frequencies of both spontaneous and UV- or MMC-induced excision of Tn10 inserted in gal . RecG mutants, which are also defective in DNA repair and recombination, showed decreased induction of Tn10 excision with MMC, but not after UV treatment . A recG ruv double mutant showed a greater decrease in induction of excision with MMC than either single mutant . One can speculate that the Ruv proteins, which are known to be involved in the resolution of Holliday junctions, might also be involved in the resolution of putative intermediates generated during the precise excision of Tn10 . RecG protein, whose function partially overlaps those of Ruv proteins, might also have some role in this process. J Gen Virol, 1994 Nov, 75 ( Pt 11), 3177 - 84 Localization of functional regions of the cucumber mosaic virus RNA replicase using monoclonal and polyclonal antibodies; Hayes RJ et al.; Monoclonal antibodies were produced using a purified cucumber mosaic virus (CMV) replicase complex, and Escherichia coli-expressed CMV 1a and 2a proteins, as immunogens . Five out of eight monoclonal antibodies, which bound to the 1a and 2a proteins in immunoblots, inhibited the RNA-dependent RNA polymerase (RdRp) activity of the purified replicase complex in vitro . Epitope mapping showed that two of the inhibitory antibodies interacted with regions of the 1a protein containing putative helicase and methyltransferase domains respectively . Two other inhibitory antibodies mapped to a region of the 2a protein containing the GDD motif which is highly conserved in RdRps . Prior interaction of the latter antibodies with a peptide containing the GDD motif prevented the antibody-mediated inhibition of the replicase . Polyclonal antibodies which inhibited the RdRp activity of the replicase complex were also produced using peptides corresponding to conserved helicase and polymerase motifs in the 1a and 2a proteins . The greatest inhibition was shown by antibodies to a peptide containing the GDD motif . These results demonstrate the functional importance of the identified sequence motifs in CMV RNA replication and indicate that the motifs are located in the replicase complex at positions accessible to antibodies, consistent with roles in interacting with the RNA template, RNA primer and enzyme substrates. J Exp Med, 1994 Nov 1, 180(5), 1949 - 54 Apolipoprotein E (ApoE), a novel heparin-binding protein inhibits the development of Kaposi's sarcoma-like lesions in BALB/c nu/nu mice; Browning PJ et al.; Recombinant apolipoprotein E-3 (ApoE-3), expressed in Escherichia coli, was purified and used in an in vitro and an in vivo model system for acquired immunodeficiency syndrome-associated Kaposi's sarcoma (AIDS-KS) . This protein blocked cell proliferatio