Infection, 1993 May-Jun, 21(3), 179 - 86 Cross-reactivity of monoclonal antibodies and sera directed against lipid A and lipopolysaccharides; Kuhn HM; The cross-reactive capacity of monoclonal and polyclonal lipid A antibodies was tested with rough and smooth lipopolysaccharides (LPS) . The antibodies represented different specificities recognizing epitopes in the hydrophilic lipid A backbone . In none of the various assay systems applied did the antibodies react with complete rough or smooth-form LPS . Cross-reactions, in general, were only detected with the most rudimentary rough LPS tested, i.e . Re-LPS . A variety of reactivities with other LPS was shown not to be related to lipid A antibodies; such reactivities were present in rabbit sera as well as in crude ascites . These results underline the need for careful checks on the origin of reactivities observed . In addition, rabbit antisera raised with R- and S-LPS were screened for lipid A reactivity using synthetic lipid A and partial structures as antigens . No cross-reactivity of LPS antibodies with lipid A was detected in these sera. Biol Pharm Bull, 1993 May, 16(5), 437 - 43 Proton NMR study on a histone-like protein, HU alpha, from Escherichia coli and its complex with oligo DNAs; Shindo H et al.; It was confirmed that the flexible arm region of HU alpha forms an antiparallel beta-sheet and that all of the residues of phenylalanines, together with some of leucines and/or valines, form a hydrophobic core within the dimer of HU alpha . HU alpha protein alone is thermally labile and melts at 38 degrees C, but it becomes remarkably stabilized and melts at 59 degrees C in the presence of DNA . Several resonances from both HU alpha and DNA perturbed by their complex formation, notably those of His C-2 and C-4 protons, downfield shifted C alpha protons in the antiparallel beta-sheet, as well as Arg C delta and Lys C epsilon protons . The results indicated that a beta-sheet region of HU alpha binds to DNA, and also showed that rapid equilibrium occurs on the NMR time scale between bound and unbound states of HU alpha . A few intermolecular nuclear Overhauser effects (NOEs) were also observed between the protein and H1' protons of DNA in the complex, suggesting that HU alpha binds primarily to the minor groove of DNA. Vet Microbiol, 1993 May, 35(1-2), 179 - 85 Species-specific recombinant DNA probes for Mycoplasma meleagridis; Zhao S et al.; Two recombinant DNA probes (pMM-2 and pMM-13) were isolated from a Mycoplasma meleagridis strain 17529 genomic library prepared in plasmid pUC8, and Escherichia coli strain JM83 . In dot blot assays, 32P-labeled pMM-13 with a DNA insert of 3.5 kbp, hybridized with 18 isolates of M . meleagridis but not with 16 other known species of avian mycoplasmas . Except for weaker signals on hybridization with the M . meleagridis cultures, pMM-2 with an DNA insert of 0.85 kbp, showed a similar reaction pattern . The minimal concentration of M . meleagridis strain 17529 chromosomal DNA that pMM-13 and pMM-2 detected were 1 and 8 ng, respectively . Neither probe hybridized with chromosomal DNA of M . gallisepticum strain S6, M . synoviae strain WVU-1853, or M . iowae strain I-695 at concentration of 256 ng. J Surg Res, 1993 May, 54(5), 474 - 9 Effect of anti-lipid A monoclonal antibody (E5) on microcirculatory function during lipopolysaccharide shock; Schwartz RW et al.; Early septic shock is characterized by fever, increased cardiac output, decreased systemic vascular resistance, and dilation of higher-order arterioles in peripheral tissues, such as skeletal muscle . We used a rat model of low-dose lipopolysaccharide (LPS) "septic" shock to investigate the potential benefit of an anti-lipid A monoclonal antibody preparation (E5) on macro- and microcirculatory function . Twenty-five male Sprague-Dawley rats were anesthetized and instrumented for measurement of arterial pressure (AP), heart rate (HR), and cardiac output (CO) . The left cremaster muscle of each rat was prepared for in vivo video microscopic examination of changes in third-order arteriolar (A3) diameter and erythrocyte velocity . Rats were randomly assigned to two groups: Group I (n = 13) received E5 vehicle and 200 micrograms/kg Escherichia coli LPS; Group II (n = 12) received 2 mg/kg E5 iv prior to LPS administration . All variables were recorded at 15-min intervals for 30 min prior to and 150 min following LPS . Microcirculatory recordings were restricted to those rats where arteriolar diameters were 20-40 microns and vessels displayed obvious vasomotion (n = 7/group) . Infusion of LPS caused no significant change in AP, an increase in CO by 105 min, an increase in HR by 75 min, an increase in diameter by 75 min, and a decrease in velocity by 165 min (P < 0.01) . Pretreatment with E5 inhibited the A3 vasodilation but did not affect the macrocirculatory changes . These data suggest a potential therapeutic role for E5 in ameliorating LPS-induced changes in skeletal muscle microcirculation. J Surg Res, 1993 May, 54(5), 418 - 25 In vivo hepatocyte transduction with retrovirus during in-flow occlusion; Rettinger SD et al.; Gene therapy research would be facilitated by a technically simple procedure for transducing hepatocytes in vivo . Previously reported methods have employed partial hepatectomy followed 24 hr later by asanguineous perfusion of the regenerating liver with retrovirus . We have developed a simpler method of in vivo transduction in which we deliver an intraportal bolus of retrovirus to the regenerating rodent liver during a brief period of hepatic in-flow occlusion . On Day 0, adult male Sprague-Dawley rats (N = 19) underwent 70% hepatectomy to induce hepatocyte replication . On Day 1, retroviral supernatant was harvested from an amphotropic retroviral packaging cell line that packaged an LNL6-derived vector containing the cytomegalovirus promoter driving expression of the Escherichia coli beta-galactosidase (beta gal) gene . Twenty-four hours after partial hepatectomy, experimental rats (N = 17) received 6 x 10(5) colony-forming units of retrovirus by intraportal injection during a 3-min occlusion of the hepatic artery and portal vein . Control rats (N = 2) received intraportal medium (without retrovirus), also during in-flow occlusion . The procedure required 20-25 min, and the survival rate was 84% . Cryostat sections were prepared from liver biopsies obtained on Post-transduction Days 8 and 15 and stained with 5-bromo-4-chloro-3-indolyl- beta-D-galactopyranoside to detect beta gal expression . Light microscopic examination of Day 8 sections from surviving experimental rats (N = 14) revealed 0.10-1.00% blue (i.e., transduced) hepatocytes per low power field, while sections from control rats (N = 2) exhibited no blue cells . Day 15 sections from experimental rats revealed a somewhat lower frequency of hepatocytes expressing beta gal.(ABSTRACT TRUNCATED AT 250 WORDS) J Surg Res, 1993 May, 54(5), 393 - 400 Endotoxin increases hepatic glutamine transport activity; Inoue Y et al.; Glutamine uptake by the liver is accelerated during endotoxemia, but little is known regarding the influence of sepsis on the plasma membrane transport systems catalyzing hepatic glutamine uptake . We hypothesized that this augmented uptake was due to an increase in hepatocyte plasma membrane transport activity . We investigated the activities of the Na(+)-dependent transport System N (transports glutamine into the hepatocyte) and the Na(+)-independent System n (transports glutamine out of the cell) in hepatocyte plasma membrane vesicles (HPMVs) prepared from livers of rats treated with Escherichia coli endotoxin (LPS) in vivo . HPMVs were prepared by differential centrifugation and the transport of {3H}glutamine was assayed by a rapid mixing/filtration technique in the presence and absence of sodium . Vesicle integrity and functionality were confirmed by enzyme marker enrichments and classic "overshoots" in the presence of sodium . Carrier-mediated Na(+)-independent glutamine transport activity was not altered by LPS administration . In contrast, endotoxemia resulted in a time- and dose-dependent two- to threefold increase in Na(+)-dependent glutamine transport activity in HPMVs secondary to an increase in the transport Vmax, consistent with the appearance of increased numbers of corresponding transporter proteins in the hepatocyte plasma membrane . The Km (affinity for glutamine) of the System N transporter was not affected by LPS treatment . Maximal increases in transport were observed 4 hr after exposure to endotoxin . System N transport activity returned to basal levels by 12 hr . This increase in transport activity represents an important mechanism regulating the accelerated hepatic glutamine uptake that occurs during severe infection. Plasmid, 1993 May, 29(3), 236 - 40 Overproduction and purification of the colicin E1 immunity protein; Shirabe K et al.; Colicin E1 immunity protein encoded by the plasmid colicin E1 was overproduced in Escherichia coli from an expression plasmid which was constructed by placing the immunity protein-encoding sequence downstream of the tac promoter and by properly positioning the ribosome binding site on a run-away replication vector . The immunity protein was solubilized with 0.5% Brij 58 from the membrane fraction and purified to homogeneity by a simple batch procedure with hydroxyapatite gel and reverse-phase chromatography . A 15-residue NH2-terminal amino acid sequence was determined to be the same as that deduced from the DNA sequence . The effect of the purified immunity protein on membranes was tested in vitro using solute-loaded liposomes . The immunity protein added to the liposomes showed a small but significant channel or lytic activity that is an indicator of its hydrophobic nature. Plasmid, 1993 May, 29(3), 222 - 32 A natural A/T-rich sequence from the yeast FBP1 gene exists as a cruciform in Escherichia coli cells; del Olmo M et al.; Palindromic or semipalindromic sequences can adopt cruciform structures in DNA in vitro . It has been demonstrated in some cases that A/T-rich cruciforms exist also in vivo in Escherichia coli . The biological function of those structures is not understood although putative cruciforms have been found in interesting locations on replication origins, operators, or transcriptional termination regions . Here we show by means of the use of structure-dependent nucleases that the 3' end of the yeast FBP1 gene contains a stable cruciform both in vitro and in E . coli cells and that in both cases, its extrusion depends on the DNA supercoiling state. Mol Microbiol, 1993 May, 8(5), 903 - 14 Sequence, genetic, and lacZ fusion analyses of a nifR3-ntrB-ntrC operon in Rhodobacter capsulatus; Foster-Hartnett D et al.; Transcription of Rhodobacter capsulatus genes encoding the nitrogenase polypeptides (nifHDK) is repressed by fixed nitrogen and oxygen . Regulatory genes required to sense and relay the nitrogen status of the cell are glnB, ntrB (nifR2), and ntrC (nifR1) . R . capsulatus nifA1 and nifA2 require ntrC for activation when fixed nitrogen is limiting . The polypeptides encoded by nifA1 and nifA2 along with the alternate sigma factor RpoN activate nifHDK and the remaining nif genes in the absence of both fixed nitrogen and oxygen . In this study we report the sequence and genetic analysis of the previously identified nifR3-ntrB-ntrC regulatory locus . nifR3 is predicted to encode a 324-amino-acid protein with significant homology to an upstream open reading frame cotranscribed with the Escherichia coli regulatory gene, fis . Analysis of ntrC-lacZ fusions and complementation data indicate that nifR3 ntrBC constitute a single operon . nifR3-lacZ fusions are expressed only when lacZ is in the proper reading frame with the predicted nifR3 gene product . Tn5, a kanamycin-resistance cassette, and miniMu insertions in nifR3 are polar on ntrBC (required for nif transcription) . This gene organization suggests that the nifR3 gene product may be involved in nitrogen regulation, although nifR3 is not stringently required for nitrogen fixation when ntrBC are present on a multicopy plasmid . In addition, a R . capsulatus strain with a 22-nucleotide insert in the chromosomal nifR3 gene was constructed . This nifR3 strain is able to fix nitrogen and activate nifA1 and nifA2 genes, again supporting the hypothesis that nifR3 is not stringently required for ntrC-dependent gene activation in R . capsulatus. Mol Microbiol, 1993 May, 8(5), 875 - 89 Synthesis of the Escherichia coli K-12 nucleoid-associated DNA-binding protein H-NS is subjected to growth-phase control and autoregulation; Dersch P et al.; Mutations in the structural gene (hns) for the Escherichia coli nucleoid-associated DNA-binding protein H-NS cause highly pleiotropic effects on gene expression, site-specific recombination, transposition of phage Mu, the stability of the genetic material and the topological state of the DNA . We have investigated the regulation of hns expression and found that hns transcription is subjected to stationary phase induction and negative autoregulation . A set of hns-lacZ protein and operon fusions was constructed in vitro and integrated in single copy into the attB site of the bacterial genome . Quantification of beta-galactosidase activity along the bacterial growth curve showed that hns expression increases approximately 10-fold in stationary phase compared with exponentially growing cells . Immunological detection of the H-NS protein in growing and stationary phase cells supported the genetic data and showed that H-NS synthesis varies with growth phase . In addition, primer extension experiments demonstrated that the amount of hns mRNA is elevated in stationary phase cultures and that hns transcription is directed by a unique promoter functioning in both log and stationary phase . Disruption of the hns gene by an insertion mutation led to the derepression (approximately fourfold) of the expression of an hns-lacZ operon fusion integrated at the attB site, showing that hns transcription is subjected to negative regulation by its own gene product . Autoregulation of hns expression is particularly pronounced in log phase . Both stationary phase control and autoregulation of hns transcription are associated with a 130 bp fragment that contains the hns promoter . In order to study the interaction of H-NS with its own regulatory region, we developed an efficient overproduction procedure and a simple purification scheme for H-NS . DNA gel retardation assays showed that the H-NS protein can preferentially interact with a restriction fragment carrying the hns promoter . This restriction fragment showed features of curved DNA as judged by two-dimensional polyacrylamide gel electrophoresis performed at 4 degrees C and 60 degrees C. Mol Gen Mikrobiol Virusol, 1993 May-Jun, (3), 26 - 9 A method for isolation of sequences missing in one of two related genomes; Lisitsyn NA et al.; We describe a novel technique for isolation of sequences that are present in one genome (tracer), but absent in another (driver) . Tracer DNA, cleaved with Sau 3A and capped with a single stranded PCR adapter, is allowed to hybridize with an excess of sheared biotinylated driver; biotinylated DNA and its hybrids with the tracer are removed by phenol/chloroform extraction after incubation with streptavidin . After several rounds of subtraction the ends of self-annealed tracer molecules from the nonextractable fraction are filled-in with Tag polymerase and amplified, using the single stranded PCR adapter as a primer . The method has been applied to purification of fragments from a 2.9 kb plasmid added to E . coli DNA at equimolar quantity . Plasmid derived fragments (250-1000 bp), initially comprising 1/1400th part of tracer DNA, were purified to homogeneity after two rounds of subtraction followed by PCR. J Photochem Photobiol B, 1993 May, 18(2-3), 205 - 10 Inhibition of dimer excision in repeatedly UV-irradiated Escherichia coli: its requirement for RecA protein and de novo protein synthesis; Fridrichova I et al.; In UV-irradiated Escherichia coli dimer excision was found to be inhibited by predamage (M . Sedliakova, F . Masek and J . Brozmanova, FEBS Lett., 23 (1972) 325-326) or overproduction of RecA protein, which suggests that the coating of the dimers by this protein may make them inaccessible to the excision nuclease (M . Sedliakova, K . Kleibl and F . Masek, Mutat . Res., 191 (1987) 13-16) . We measured the levels of RecA protein and dimer excision in cells irradiated with (i) a single dose of 50 J m-2, (ii) two separate doses of 30 and 50 J m-2, post-incubated with chloramphenicol; (iii) two separate doses of 30 and 50 J m-2, post-incubated without chloramphenicol . Dimer excision was complete in the first two cases, but in the latter it was inhibited by 40% . At the time of active dimer excision, there were marked differences in RecA protein content between the cells irradiated with a single dose and cells irradiated with two separate doses (both post-incubated without chloramphenicol), which might account for the differences in dimer excision . However, relatively small differences in RecA protein content were found in cells irradiated with two doses and post-incubated with or without chloramphenicol, which could therefore not account for the differences in dimer excision . The data suggest that the inhibition of dimer excision involves some short-lived component(s) other than RecA protein. J Photochem Photobiol B, 1993 May, 18(2-3), 191 - 6 Biochemical and morphological changes in Escherichia coli irradiated by coherent and non-coherent 632.8 nm light; Bertoloni G et al.; Irradiation of Escherichia coli cells with either coherent or non-coherent 632.8 nm light (4 J cm-2) causes a transient acceleration of cell proliferation, which is maximal about 60 min after the end of the phototreatment . The stimulatory effect is dose dependent and is especially evident in the case of defective E . coli strains which are in the logarithmic phase of growth, while it becomes less important when cells are exposed to non-coherent 600-700 nm light . Stimulated cells exhibit biochemical and morphological changes, such as an intensified synthesis of cytoplasmic membrane proteins, increased cell volume and ribosomal content, which are suggestive of an enhanced cell metabolism. J Biochem (Tokyo), 1993 May, 113(5), 568 - 72 Properties of DNA-binding of HU heterotypic and homotypic dimers from Escherichia coli; Tanaka H et al.; The interactions of three forms of HU dimer from Escherichia coli with DNA were compared . The complexes formed between HU and short DNA fragments (35 to 132 bp), uncurved oligodeoxyribonucleotide (oligo) with and without 5-bp deoxyriboadenosine (dA) stretches and curved oligo with 5-bp dA stretches, were analyzed by the gel retardation technique . The binding of HU homodimers to the DNA was less efficient than that of HU heterodimer, and the HU-1 homodimer had lower DNA-binding capacity than the HU-2 homodimer . The equilibrium dissociation constant (KD) for DNA of the HU-1 homodimer was 3 times that of the HU heterodimer . The HU dimers had two- to fourfold higher affinity for DNA duplexes containing 5-bp dA stretches, particularly for curved DNA . The binding of HU heterodimer to the DNA was inhibited more by the curved DNA competitor than the uncurved DNA competitor without dA stretches. Biotech Histochem, 1993 May, 68(3), 169 - 74 In situ hybridization at the electron microscopic level using a bromodeoxyuridine labeled DNA probe; Yao CH et al.; A bromodeoxyuridine (BrdU) labeled DNA probe was used for in situ hybridization at the electron microscopic (EM) level . A BrdU labeled DNA probe was hybridized in situ to cryostat sections of paraformaldehyde fixed OCT compound embedded cultured HL-60 cells . After hybridization, some sections were incubated with FITC-conjugated anti-BrdU monoclonal antibody for fluorescence microscopy (FM), and others were embedded in Quetol for electron microscopy (EM) . The ultrathin sections of Quetol-embedded specimens were incubated with the anti-BrdU monoclonal antibody and the immunoglobulin: gold colloid . In both FM and EM studies, the signals were concentrated in the rough endoplasmic reticulum . Moreover, some label was arranged from the nucleus to the cytoplasm at the EM level . Relatively simple methods using the BrdU labeled DNA probe for the detection of the defined nucleic acid sequence with reasonable tissue preservation and high resolution are described here . This method may be useful for developmental and disease related studies of specific mRNA in cells and tissues. Res Vet Sci, 1993 May, 54(3), 290 - 8 Experimental infection of neonatal pigs with CNF toxin-producing strains of Escherichia coli; Wray C et al.; To examine and compare the pathogenicity of cytotoxic necrotising factor (CNF)-producing Escherichia coli, two litters of piglets were infected orally with 10(10) E coli O88 or 10(10) E coli O32 strains . Of the six piglets infected with E coli O88, two died within 24 hours and three developed blood-stained diarrhoea . The other piglets were killed one, five, six and eight days after infection, when bacterial cultures indicated an overwhelming bacteraemic infection with E coli O88 in the early stages followed by clearance through the large intestine . The pathological changes consisted of an early enteritis, progressing to enterocolitis and a bacteraemic spread to the lungs . The histopathological changes were characteristic of toxaemic effects in brain, heart, liver and kidney, and characterised by congestion, oedema and exudation . Infection with E coli O32 produced a milder but similar enterocolitis, also with bacterial colonisation in the lungs . The histopathological findings again reflected a toxaemia . The enteritis was more persistent after E coli O32 infection and the strain persisted in large numbers in the intestine . No evidence of bacterial adherence to the intestinal mucosa was found with either strain . Enteroinvasion was only evident in one E coli O88-infected piglet, but the consistent occurrence of interstitial pneumonia showed the predilection of these organisms for the lung . The results confirm the toxigenic properties of CNF+ E coli and suggest an important role for this organism in enteric infection of young pigs. Photochem Photobiol, 1993 May, 57(5), 895 - 904 Photochemistry, photophysics, and mechanism of pyrimidine dimer repair by DNA photolyase; Kim ST et al.; DNA photolyases photorepair pyrimidine dimers (Pyr < > Pyr) in DNA as well as RNA and thus reverse the harmful effects of UV-A (320-400 nm) and UV-B (280-320 nm) radiations . Photolyases from various organisms have been found to contain two noncovalently bound cofactors; one is a fully reduced flavin adenine dinucleotide (FADH-) and the other, commonly known as second chromophore, is either methenyltetrahydrofolate (MTHF) or 8-hydroxydeazaflavin (8-HDF) . The second chromophore in photolyase is a light-harvesting molecule that absorbs mostly in the near-UV and visible wavelengths (300-500 nm) with its high extinction coefficient . The second chromophore then transfers its excitation energy to the FADH- . Subsequently, the photoexcited FADH- transfers an electron to the Pyr < > Pyr generating a dimer radical anion (Pyr < > Pyr.-) and a neutral flavin radical (FADH.) . The Pyr < > Pyr.- is very unstable and undergoes spontaneous splitting followed by a back electron transfer to the FADH. . In addition to the main catalytic cofactor FADH-, a Trp (Trp277 in Escherichia coli) in apophotolyase, independent of other chromophores, also functions as a sensitizer to repair Pyr < > Pyr by direct electron transfer. J Gen Microbiol, 1993 May, 139 ( Pt 5), 1083 - 92 Expression of a plasmid gene of Chlamydia trachomatis encoding a novel 28 kDa antigen; Comanducci M et al.; Plasmid pCT is present in essentially all isolates of Chlamydia trachomatis and may encode factors important for survival in the natural environment . However, no pCT-associated phenotype has been described so far . With the purpose of investigating the possibility of a role of pCT in C . trachomatis pathogenicity we examined the expression of an ORF (ORF3), potentially encoding a 28 kDa polypeptide (pgp3) . Analysis of RNA extracted from chlamydia-infected Vero cells detected ORF3-specific transcripts, from 20 h post-infection onwards, mainly as discrete RNA species of 1390 nucleotides comprising the downstream ORF4 sequence . ORF3 DNA was cloned and expressed in Escherichia coli as a 39 kDa fusion protein (MS2/pgp3) . Antibodies raised against purified MS2/pgp3, specifically recognized a 28 kDa protein on Western blots of protein from purified chlamydial elementary bodies (EBs) . The same antibodies detected chlamydial inclusions in methanol-fixed infected cells by immunofluorescence . Western blot analysis of EBs extracted with 2% Sarkosyl, showed that a large proportion of the 28 kDa antigen is associated with the detergent-insoluble ('membrane') fraction . Antibodies recognizing pgp3 epitopes were detected in sera from patients with chlamydial infections, but not in sero-negative control sera . The finding support the hypothesis that pCT may provide a function related to chlamydial cell physiology. J Appl Physiol, 1993 May, 74(5), 2155 - 60 Attenuation of acute lung injury and oxygen radical production by the 21-aminosteroid, U-78518F; Tanigaki T et al.; Oxygen radicals play an important role in the mechanism of acute lung injury . The 21-aminosteroid lazaroid, U-78518F, is a potent antioxidant . We examined the effect of intravenous U-78518F on acute lung injury in septic guinea pigs over 8 h . The experimental groups (n = 6) were 1) saline control, 2) Escherichia coli (2 x 10(9)/kg i.v.), 3) pretreatment (U-78518F 5 mg/kg bolus + 1 mg.kg-1 x h-1, 15 min before E . coli injection), and 4) posttreatment (U-78518F 30 min after E . coli injection) . We measured wet-to-dry weight ratio (W/D) as an index of pulmonary edema and concentration ratios of 125I-labeled albumin in lung tissue and bronchoalveolar lavage fluid compared with plasma (L/P and BAL/P, respectively) as indexes of lung protein fluxes . In septic guinea pigs, pretreatment with U-78518F attenuated W/D, L/P, and BAL/P and posttreatment attenuated W/D and BAL/P (P < 0.05 for each) . Furthermore, we studied the effect of U-78518F on human neutrophil oxygen radical production (ORP) by using flow cytometry to assess intracellular ORP and lucigenin-dependent chemiluminescence to assess extracellular ORP . Neutrophils (5 x 10(5) were stimulated with 0.5 micrograms/ml of phorbol myristate acetate . With flow cytometry, we measured intracellular ORP, cross-sectional cell area, and degranulation in neutrophils . U-78518F (minimum concn 1.0 microM) decreased intracellular ORP (n = 4; P < 0.05) when the dihydrorhodamine 123 assay was used . U-78518F (minimum concn 1.0 microM) inhibited phorbol myristate acetate-induced neutrophil chemiluminescence (n = 4; P < 0.05).(ABSTRACT TRUNCATED AT 250 WORDS) Genetika, 1993 May, 29(5), 748 - 59 {Increased frequency of unequal crossing over in recBC mutant cells in conjugational crosses in Escherichia coli K-12}; Sukhodolets VV; The formation of heterozygous tandem duplications was studied in conjugational crosses of Escherichia coli using mutations for deo-operon genes . The frequency of tandem duplications in recF::Tn3 and recBC sbcB mutants was not substantially changed from that of the wild type strain . In contrast, the frequency of duplications was increased about ten-fold when using recBC (sbcB+) mutants as recipients . This result can be explained by a participation of the RecBCD protein in the pairing of chromosomes during recombination . We also found that HfrH thyA recBC (sbcB+) bacteria grow very poorly on rich medium and that good growth is restored by conversion from the Hfr to the F+ state, as well as by mutations suppressing the RecBC phenotype; such F+ strains retain sensitivity to UV light and to mitomycin C, which are characteristic of the recBC phenotype. Toxicon, 1993 May, 31(5), 615 - 26 Immunization against Echis ocellatus (carpet viper) venom using liposomes incorporating immunostimulants: role of lipopolysaccharide in conferring protection in a mouse model; Laing GD et al.; Varying doses of whole West African Echis ocellatus venom were incorporated, with or without immunostimulants, into membrane-stabilized reverse phase evaporation (REV) liposomes . Preparations were given either subcutaneously (s.c.) or intravenously (i.v.) to mice and the immune responses compared . Addition of lipopolysaccharide (LPS) significantly increased the venom antibody response . Lipid A produced a less pronounced and less sustained effect and the addition of muramyl dipeptide analogues made no significant contribution to the antibody response . The protective ability of circulating venom antibodies was assessed by challenging the immunized mice with a minimum lethal dose of whole venom after 175 days . A dose of 250 micrograms E . ocellatus venom + 300 micrograms LPS in REVs injected s.c . conferred the highest protection against lethal venom effects . Orally administered venom/liposomes incorporated with the mucosal adjuvant avridine primed the antibody response and produced a classic secondary response following a sublethal boost of whole venom . Single injections of venom or venom fraction/liposome preparations which produce a high and sustained immune response have potential in commercial antivenom production and in active immunization of man in areas of high snakebite incidence and mortality. Nippon Geka Gakkai Zasshi, 1993 May, 94(5), 466 - 74 {Experimental studies on the relation of platelet activating factor (PAF) to high mortality in sublethal endotoxemia after massive hepatectomy and effects of its antagonist}; Onuki Y et al.; The effects of platelet activating factor antagonist (PAF-A) for sublethal endotoxemia after 70% or 84% hepatectomy in rats were studied . Endotoxin of 1mg/kg b.w . was intravenously given once on the first, third or fifth day hepatectomy . One week survival rates of 84%-hepatectomized rats with endotoxin injection on the third postoperative day was the lowest at 20%, and the lowest survival rate improved to 80% with PAF-A administration . Pathophysiology of PAF-A treated and nontreated rats with endotoxemia was investigated on the third day after 84% hepatectomy . There was no significant difference in SGOT, SGPT and blood glucose level between the two groups . PAF-A nontreated rats' prothrombin time was prolonged (p < 0.05) and serum fibrinogen level significantly decreased (p < 0.01), compared to treated rats . Platelet count distinctly decreased in both groups . Fibrin was pathologically proved in the glomeruli of the kidney in both groups . However, single cell necrosis of hepatocytes was significantly reduced by PAF-A administration . Rats without PAF-A had bloody ascites in 75% of cases and showed shock and pathologically pulmonary congestion . PAF may be related to DIC, shock, bloody ascites and high mortality rate in sublethal endotoxemia after massive hepatectomy . PAF-A was significantly effective for endotoxemia after hepatectomy. Mol Microbiol, 1993 May, 8(4), 697 - 706 Phosphate starvation and low temperature as well as ultraviolet irradiation transcriptionally induce the Escherichia coli LexA-controlled gene sfiA; Dri AM et al.; The LexA repressor controls the expression of several SOS genes, such as lexA, recA and sfiA, which are induced by DNA damage . Induction results from the activation of the RecA protein that favours the cleavage and thus the inactivation of LexA . It has been shown that the activation of RecA results from its binding to damaged DNA . It is therefore believed that in growing bacteria, in the absence of any DNA-damaging treatment, the intracellular level of LexA remains stable at a high basal level and, hence, SOS genes are expressed at relatively low basal levels . In contrast, we show here that the intracellular level of LexA and the rate of transcription of the sfiA gene may vary markedly throughout the growth cycle of wild-type Escherichia coli . We provide evidence that such changes result from two superimposed processes: proteolytic cleavage of LexA upon dilution of stationary phase bacteria, and increase in strength of the promoters of the lexA and sfiA genes when bacteria approach the stationary phase . We show that a signal which strongly increases the strength of the sfiA gene promoter is starvation for phosphate . Such induction was not significantly affected by mutations either in phoB (encoding the transcriptional regulator for the phosphate regulon) or rpoS (encoding a putative stationary phase-specific sigma factor) . However, sfiA induction by phosphate starvation appeared to be markedly inhibited by the presence of the osmZ205 mutation which alters the histone-like protein H-NS, suggesting that changes in the DNA structure may play a role in signal transduction during phosphate starvation . As previously shown for several processes which are controlled by H-NS, induction of sfiA was modulated by growth temperature. J Am Soc Nephrol, 1993 May, 3(11), 1783 - 91 Cytokine formation within rat glomeruli during experimental endotoxemia; Fouqueray B et al.; Increasing evidence supports a role of cytokines, tumor necrosis factor alpha (TNF alpha), interleukin-1 (IL-1), and IL-6 in the development of endotoxin-induced acute renal failure . Several activities of these cytokines require a local rather than a systemic production and function . Thus, this study investigates the chronology of cytokine expression in glomeruli isolated from normal rats or rats given iv lipopolysaccharide injections . Detectable levels of TNF alpha could be found in glomeruli isolated from normal rats as assessed by L-929 fibroblast lytic assay and ELISA . Glomeruli isolated from rats given lipopolysaccharide transiently released increased amounts of TNF alpha in relation to the dose of lipopolysaccharide (10 to 500 micrograms/kg body wt) and the lag period between lipopolysaccharide injection and glomerular isolation (20 to 120 min) . TNF alpha was released in similar amounts by glomeruli from normal rats that were exposed in vitro to lipopolysaccharide challenge (0.01 to 10 micrograms/mL), indicating that lipopolysaccharide had direct effects on the release of TNF alpha from glomerular cells . These cells consisted mainly of resident cells because reduction of glomerular infiltration by bone marrow-derived cells after the irradiation of normal rats did not affect TNF alpha release . Glomerular IL-1 and IL-6 production was evaluated by specific bioassays under identical conditions . No IL-1 activity could be detected in the medium or within the glomerular cells at any time within 120 min after lipopolysaccharide injection . By contrast, glomerular IL-6 production was induced after lipopolysaccharide challenge both in vivo and in vitro.(ABSTRACT TRUNCATED AT 250 WORDS) J Am Soc Nephrol, 1993 May, 3(11), 1753 - 64 Dysfunction of glomerular fibrinolysis in experimental antiglomerular basement membrane antibody glomerulonephritis; Feng L et al.; Glomerular plasminogen activator inhibitor-1 (PAI-1) steady-state mRNA and bioactivity were increased after the induction of an augmented form of antiglomerular basement membrane (GBM) antibody glomerulonephritis . PAI-1 mRNA expression was noted at 6 h, peaking at 1 day, and although falling thereafter, remained higher than that of the control group through Day 17 . PAI-1 mRNA expression correlated with glomerular PAI-1 bioactivity as determined by a functional tissue type plasminogen activator (t-PA) binding assay . Glomerular PAI-1 bioactivity, not detected in controls, increased to 1.4 +/- 0.3 ng/mg of glomerular lysate at 6 h and then decreased to 0.7 +/- 0.1 ng/mg of glomerular lysate by Day 6 . The mRNA of the plasminogen activators (urokinase plasminogen activator), t-PA) either remained unchanged or declined through Day 1, with a slight increase in t-PA mRNA at Day 6 . Interleukin-1 beta mRNA expression was maximal at 6 h, declining by Day 3 . Transforming growth factor beta 1 (TGF-beta 1) mRNA began to increase at Day 1, was maximal at Day 6, and fell only slightly by Day 17 . Epidermal growth factor mRNA decreased . The increase in PAI-1 mRNA and bioactivity, possibly induced early by the interleukin-1 beta response and perhaps later by the TGF-beta 1 response, was associated with striking glomerular capillary lumen fibrin accumulations on Day 1, which decreased and appeared to recanalize as the PAI-1 mRNA and bioactivity fell . The glomerular lesion continued to have some fibrin deposits even on Day 17 and, in addition, had changes of thickened GBM, suggestive of the early stages of diffuse glomerulosclerosis . The latter had a temporal relationship with the persisting increase in TGF-beta 1 and PAI-1 mRNA levels . These observations suggest the possibility that inhibition of enzymes capable of remodeling excessive extracellular matrix production may have contributed to the thickened GBM. Br J Nutr, 1993 May, 69(3), 757 - 66 The effect of Escherichia coli heat-stable (STA) enterotoxin on in vitro uptake of amino acids by small intestine of sucking rats during fluid accumulation; Tin-Aung et al.; In vitro uptake of 14C-labelled amino acids by segments of small intestine was determined in sucking (2-4-d-old) Wistar rats . Intragastric injections of heat-stable (ST) toxin of enterotoxigenic Escherichia coli (ETEC) were given to produce fluid accumulation, defined as a gut weight: carcass weight value of > 0.085 . Continued active uptake of the prototypic amino acids, leucine (by active transport system 1 for monoamino monocarboxylic (neutral) amino acids), lysine (by active transport system 2 for dibasic amino acids), and proline (by active transport system 3 for N-substituted amino acids), persisted during the active fluid accumulation response to ETEC ST toxin . The mean Kt and mean Vmax of the amino acid transport systems were similar in control (non-injected) and ST toxin-injected rats . The present study provides a scientific basis for the use of amino acids in oral rehydration solutions utilizing amino acid transport systems which are linked to absorption of Na (and water) so that reduction in diarrhoeal stools can be achieved, and emphasizes the importance of maintaining feeding during acute diarrhoea to prevent development of malnutrition. Mol Microbiol, 1993 May, 8(3), 525 - 33 Charged amino-terminal amino acids affect the lethal capacity of Lambda lysis proteins S107 and S105; Steiner M et al.; The lysis inhibitor protein S107 and the lysis effector protein S105 start at Met codons 1 and 3 of the Lambda S gene, respectively . The antagonistic action of both proteins precisely schedules lysis by formation of a non-specific lesion in the inner membrane through which the Lambda-encoded murein transglycosylase can pass . Here, we show that the main difference between lysis-effector and lysis-inhibitor is the degree by which an energized membrane inhibits either protein from hole formation . To dissect the structural parameters responsible for intrinsic inhibition of both proteins, charged amino acids were replaced proximal to the first putative membrane-spanning region in both S proteins . Our results show that the distribution of amino-terminal charged amino acids as well as the total amino-terminal net charge of S107 and S105 influence their lethal potential . The data are interpreted in terms of a model in which the electrostatic status of the amino-terminus of both S107 and S105 is an important feature affecting their conformational change required for formation of the S-dependent hole. Mol Microbiol, 1993 May, 8(3), 435 - 41 Intermolecular complementation of the kinase activity of CheA; Swanson RV et al.; CheA is a dimeric autophosphorylating protein kinase that plays a critical role in the signal transduction network controlling chemotaxis in Escherichia coli . The autophosphorylation reaction was analysed using mutant proteins defective in kinase and regulatory functions . Proteins in which the site of autophosphorylation was mutated (CheA48HQ) or missing (CheAs) were found to phosphorylate the kinase-defective mutant, CheA470GK . The kinetics of this reaction support the hypothesis that autophosphorylation is the result of trans-phosphorylation within a dimer . The carboxy-terminal portion of CheA was previously shown to be dispensable for autophosphorylation, but required for regulation in response to environmental signals transmitted through a transducer and CheW . Mixing of CheA48HQ or CheA470GK with a truncated protein lacking this regulatory domain demonstrated that regulated autophosphorylation requires the presence of both carboxy-terminal portions in a CheA dimer . These results indicate that the dimeric form of CheA plays an integral role in signal transduction in bacterial chemotaxis. Dev Comp Immunol, 1993 May-Jun, 17(3), 195 - 200 Specific immune recognition of insect hemolin; Schmidt O et al.; Bacteria entering the body cavities of insects are recognized by hemolymph components and subsequently inactivated by phagocytosis and nodule formation . A hemolymph component called hemolin is apparently involved in the recognition process . It binds to a bacterial surface molecule and forms a stable complex with other hemolymph proteins . Hemolin binding is independent of bacterial lipopolysaccharide (LPS) structure, whereas the complex formation is dependent on the presence of the carbohydrate moiety of LPS molecules . The specificity of immune recognition in insects is discussed. PCR Methods Appl, 1993 May, 2(4), 305 - 12 Importance of different variables for enhancing in situ detection of PCR-amplified DNA; Nuovo GJ et al.; This study determined the effects of several variables on the in situ signal after PCR amplification in fixed cells . A signal was evident in all human peripheral blood monocytes fixed in buffered formalin using primers for the human proto-oncogene bcl-2 with in situ PCR only after prolonged fixation and protease digestion . A much lower detection rate was noted after ethanol or acetone fixation due to loss of amplified product out of the nucleus into amplifying solution . This observation demonstrates the importance of cross-linking fixatives for retention of amplified DNA at the site of origin . The increased amount of target-specific DNA synthesis evident with the manual hot start modification to in situ PCR was also seen with chemical hot start mediated by the Escherichia coli single-stranded DNA-binding protein . The manual hot start method strongly suppressed in situ unwanted DNA synthesis dictated by nonsense primers; residual nonspecific synthesis was influenced by annealing temperatures and post-fixation protease digestion conditions. Acta Anaesthesiol Scand, 1993 May, 37(4), 396 - 403 Effects of pentoxifylline on hemodynamics, gas exchange and multiple organ platelet sequestration in experimental endotoxic shock; Sigurdsson GH et al.; In an intensive care setting we studied the effects of pentoxifylline on hemodynamics, gas-exchange and platelet sequestration in multiple organs in three groups of sheep exposed to endotoxin shock (n = 7 in each) . Group P-E was given pentoxifylline before and group E-P after E . coli endotoxin infusion, while group E received normal saline (controls) . The endotoxin infusion caused a three-fold increase in pulmonary artery pressure (PAP) and a significant decrease in mean arterial pressure (25-30%; MAP), respiratory compliance (CT; 60%) and arterial oxygen tension (65-70%; Pao2) in all groups after 30 min . After 4 h MAP had improved significantly in the pretreated animals (group P-E) and arterial pH, Pao2 and CT improved in both pentoxifylline-treated groups compared with the controls . On the other hand, the effects of endotoxin on PAP and cardiac index were not significantly influenced by pentoxifylline treatment . In addition, there was a pronounced platelet sequestration in the lungs and in the liver in groups E and E-P during the 4 h study, but in the pretreated group (group P-E) the changes were significantly less marked (P < 0.01) . The wet-to-dry weight ratios of the lungs were significantly lower in both pentoxifylline-treated groups compared with the controls (P < 0.01) . It was concluded that pentoxifylline modified the effects of endotoxin on hemodynamics, gas exchange and platelet sequestration in the lungs and liver in sheep when it was given prior to endotoxin . However, when it was given after hemodynamic and respiratory signs of shock had appeared, the effects were more moderate. J Cell Biochem, 1993 May, 52(1), 78 - 83 Unphosphorylated alpha-PKC exhibits phorbol ester binding but lacks protein kinase activity in vitro; Filipuzzi I et al.; Expression of the alpha-isoform of protein kinase C (alpha-PKC) in E . coli yielded the unphosphorylated 74 kD precursor molecule . This precursor form exhibited phospholipid- and calcium-dependent phorbol ester binding but lacked, in contrast to the phosphorylated enzyme, protein kinase activity . In addition, the precursor molecule was found to interact with both threonine and an ATP analogon, which demonstrates that phosphorylation of alpha-PKC is not required for binding of substrates, cofactors, or activators . These results, therefore, suggest that posttranslational phosphorylation of alpha-PKC is not needed for the formation of a functional enzyme-substrate complex but is necessary for the catalytic transfer of phosphate residues from ATP to protein substrates. Virus Res, 1993 May, 28(2), 153 - 70 Non-homologous recombination between adenovirus and AcNPV DNA fragments in cell-free extracts from insect Spodoptera frugiperda nuclei; Schorr J et al.; In previous work, we have developed a cell-free system from nuclear extracts of hamster cells to study the mechanism of integrative recombination between adenovirus type 12 (Ad12) DNA and hamster cell DNA (Jessberger et al., 1989; Tatzelt et al., 1992) . We have also demonstrated that in insect cells the left terminal fragment of Ad2 DNA can insert by non-homologous recombination into the 32.6 to 34.5 map unit (EcoRI-O) fragment and into other segments of Autographa californica nuclear polyhedrosis virus (AcNPV) DNA (Xiong et al., 1991) . We have now imitated this recombination event in vitro by incubating the E1 fragment of Ad2 DNA and the EcoRI-O fragment of AcNPV DNA, both in the plasmid-cloned circular forms, with partly purified nuclear extracts from Spodoptera frugiperda insect cells . Proteins from these extracts have been fractionated by gel filtration . After the reextraction of DNA from the incubation mixture, recombinants generated in this cell-free system have been identified directly with the polymerase chain reaction (PCR) by using Taq polymerase and appropriate primers which are unique to either of the two reaction partners . The recombinants identified are all different . The results of control experiments argue against the possibility that unspecific reaction products might have been generated during PCR . Nucleotide sequence determinations in some of the recombinants localize the sites of genetic exchange between the two partners and assess the non-homologous nature of the reaction . The recombinants are characterized by the presence of short patch homologies at or close to the sites of linkage between the reaction partners, as described earlier in the hamster cell system (Tatzelt et al., 1992) . The occurrence of recombinants in the cell-free system can also be demonstrated by a biological test in which potential recombinants are isolated by transfection into recA- strains of Escherichia coli. Mol Biol (Mosk), 1993 May-Jun, 27(3), 589 - 607 {Stable maintenance of dicentric mini-chromosomes in CHL4 mutants in yeast}; Kuprina NIu et al.; Earlier we have identified the chl4-1 mutation in a screen for yeast mutants with increased loss of chromosome III and circular artificial minichromosome in mitosis . Mutation in the CHL4 gene leads to a 50-100-fold promotion in the rate of chromosome loss per cell division compared to the isogenic wild type strain . Detailed analysis of behaviour of the circular minichromosome marked by the CUP1 gene has shown that minichromosome nondisjunction (2:0 segregation) leading to an increase in the copy number of minichromosome in part of a cell population is the main reason of minichromosome instability in the mutant . The unique peculiarity of chl4-1 mutation is the ability of the strains carrying this mutation to stably maintain circular dicentric minichromosomes without any rearrangement during many generations . (In the wild type strains dicentric minichromosomes are extremely unstable . As a consequence of that there is a strong selection for cells harboring monocentric derivatives in a population of cells derived from a cell containing a dicentric plasmid) . Introduction of the second centromere into one of the natural chromosomes (chromosomes II or III) in the chl4-1 mutant leads to the same dramatic consequences as that in the wild type strain (mitotic lag of cells harboring dicentric chromosomes and, as a result of that, selective pressure for cells harboring monocentric derivatives of dicentric chromosome) . A genomic clone of CHL4 was isolated by complementation of the chl4-1 mutation . Nucleotide sequence analysis of CHL4 revealed a 1.4-kb open reading frame with a predicted 53-kDa protein sequence . Analyzing the sequence of the CHL4 protein we have found a region meeting the necessary requirements for the helix-turn-helix (HTH) structure . This region of the CHL4 protein has about 40% homology with the repressor of tryptophane operon (TrpR) of E . coli . A strain containing a null allele of CHL4 was viable under standard growth conditions, but had temperature-sensitive phenotype (conditional lethality at 34 degrees C) . We suggest that the CHL4 gene product is one of the components of the segregation cell machinery. Zhonghua Yu Fang Yi Xue Za Zhi, 1993 May, 27(3), 141 - 3 {The genotoxicity of air particles tested by SOS chromotest}; Lei XF et al.; Samples of variously-sized total suspended particles in the air of one sampling site of TaiYuan city were collected . The samples were extracted with acid and a simulated lung fluid (SLF) respectively . Both extracts mainly consisted of metallic elements . Using SOS chromotest as a means to test the gentoxity of the extracts and five metallic compounds, namely Cr6+, Ni2+, Pb2+, Mn2+ and Cd2+ were tested first . All the compounds tested could induce the SOS response to various extent, showing that the method was sensitive to metallic compounds . For the extracts of air particles, both the extracts of acid and of SLF of the smaller-sized particles could induce SOS response . This indicated the existence of metaklic genotoxicants in the smaller-sized particles . Being convenient to use fast and precise, the SOS Chromotest has its unique advantage for detecting carcinogenic metallic compounds. Zhonghua Yu Fang Yi Xue Za Zhi, 1993 May, 27(3), 135 - 8 {Inhibitory effects of garlicin and cinnamaldehyde on SOS response and their mechanisms in Escherichia coli}; Jin ZC; Inhibitory effects of garlicin and cinnamaldehyde on SOS response and their mechanisms in Escherichia coli were investigated . Garlicin and cinnamaldehyde suppressed the lambda cI depended SOS response induced by 4-nitroquinoline-N-oxide (4NQO), mytomycin (MMC) and methyl methane-sulfonate (MMS), and the lexA-depended SOS response induced by 4NQO and UV to various extent, respectively . They also diminished the rexA 441-depended SOS response induced by temperature (at 42 degrees C) However, they did not show any effect on the constitution of SOS functions . It suggested that this inhibitory effect could be via RecA protease which regulates the cleavage of lexA repressor, and perhaps, lambda cI repressor. J Hered, 1993 May-Jun, 84(3), 157 - 65 Studies in swine on inheritance and variation in expression of small intestinal receptors mediating adhesion of the K88 enteropathogenic Escherichia coli variants; Hu ZL et al.; Small intestinal enterocyte preparations from 368 pigs were phenotyped by an in vitro adhesion test using six strains of K88 Escherichia (E.) coli, each expressing one of three K88 fimbrial antigenic variants: K88ab, K88ac, or K88ad . All pigs tested were classified into one of four adhesion brush border phenotypes: I (K88ab-, K88ac-, K88ad-); II (K88ab-, K88ac-, K88ad+); III (K88ab+, K88ac+, K88ad-); or IV (K88ab+, K88ac+, K88ad+) . The segregation and adhesion affinity data suggest that there are two adhesion affinity receptors for K88ad+ E . coli: a high affinity (adH) and a low affinity (adL) receptor . The high affinity receptor cosegregates with receptors for K88ab and K88ac fimbrial antigens forming together the phenotype IV; the low affinity receptor is associated with the adhesion phenotype II, and its physiological expression is terminated by 16 weeks of age . In contrast, the K88adH receptor is expressed during the entire life cycle . The presence of a mixed adhesion phenotype, K88adM, assumed to be determined by K88ab(-),ac(-),adL(+)/K88ab(+),ac(+),adH(+) heterozygous genotype, is interpreted as an indication that each of the two types of brush border adhesion for the K88ad antigen is expressed on independent enterocytes. Bull Cancer, 1993 May, 80(5), 418 - 30 {Phase I trial of recombinant human granulocyte-macrophage colony stimulating factor . Results in patients with advanced tumors}; Berthaud P et al.; Fourteen patients with advanced solid tumors were included in a phase I trial of recombinant human E coli derived granulocyte-macrophage colony-stimulating factor (GM-CSF) given daily subcutaneously for 10 consecutive days . Dose levels were increased from 250 micrograms/m2 to 500, 750 and 1,000 micrograms/m2 . Adverse effects were mainly fever, local irritation, lethargia, arthalgia . Three patients did not complete the 10-day cycle: one patient died due to progressive disease without toxic effects related to GM-CSF, one was withdrawn because of suspicion of pulmonary embolism (not confirmed), one patient had hypotension, not recurring after treatment with GM-CSF . Although the maximum tolerated dose was not reached, the trial was stopped at 1,000 micrograms/m2, considering the satisfactory response and the high white blood cell counts observed with lower dose levels . N-fold increases of leucocyte count ranged between 4.2 and 8.2 for the first dose level (250 micrograms/m2), 4 and 10.1 for 500 micrograms/m2, 8.5 and 12.3 for 750 micrograms/m2 and 5.6 and 8.3 for 1,000 micrograms/m2 . Increases of granulocyte, neutrophil and eosinophil counts had a similar pattern with a weaker response at 1,000 micrograms/m2 (two patients who completed the cycle) . In contrast, even for the first three levels, no dose response relationship was shown for increases of monocytes (between 2.8 and 12 n-fold whatever the dose), or lymphocytes (between 1.7 and 10.7 n-fold whatever the dose) . Decreases of platelets (between 6 and 55%) were observed, followed by a rebound after stopping treatment . No modifications of erythrocyte count were observed . Subcutaneous GM-CSF was well-tolerated up to 1,000 micrograms/m2 during a 10-day course . Hematological effects were observed from the first dose level of 250 micrograms/m2. Tsitol Genet, 1993 May-Jun, 27(3), 68 - 71 {The ultrastructural characteristics of the changes in the rat liver in the dynamics of experimental endotoxemia}; Kharlanova NG et al.; Electron microscopic study was performed in the experiments on the rat liver while intravenously administering E . coli endotoxin . The dynamics of ultrastructural disorders during endotoxemia has been established and the role of "Kupffer cell-hepatocyte" microsystem in the liver detoxification function is shown. Gen Pharmacol, 1993 May, 24(3), 681 - 5 Changes in pre- or postsynaptic adrenergic mechanisms modify the thermoregulatory responses produced by pyrogen in rabbits; Gagalo IT et al.; 1 . Thermoregulatory responses to BHT 920, prazosin (PRA) and 6-hydroxydopamine (6-OHDA) were investigated in pyrogen (lipopolysaccharide Escherichia coli, LPS) treated rabbits . 2 . All the compounds in question, despite their different selectivity for pre- or postsynaptic adrenergic structures, significantly reduced pyrogen fever . Antipyresis was associated with inhibition of the metabolic rate . 3 . The role of adrenergic mechanisms in fever, with particular respect to those of postsynaptic alpha-2, is discussed. Mol Microbiol, 1993 May, 8(5), 865 - 73 Ligand-based histochemical localization and capture of cells expressing heat-stable enterotoxin receptors; Almenoff JS et al.; The heat stable enterotoxins (ST) of enterotoxigenic Escherichia coli (ETEC) cause diarrhoea by binding specific intestinal receptors . Precise histochemical localization of ST receptors could provide more information about the pathophysiology of secretory diarrhoea and the role of ST receptors in normal biology . To accomplish this, we quantitatively coupled biotin to the N-terminus of ST1b using biotin-X-X-N-hydroxysuccinimide ester . The derivatized toxin (BST) has an apparent Kd of 11.7 +/- 10 nM for rat brush border receptors . We used BST in an affinity panning cell-capture system, to validate its ability to discriminate between receptor-positive and receptor-negative cells . Cell lines expressing ST receptors (human colon carcinoma T84, and COS cells transfected with guanylyl cyclase-C (GC-C) ST receptor cDNA) were captured to streptavidin and anti-biotin-coated plates with high efficiency and specificity . This system provides a novel approach to screening cells for the presence of unique ST-binding proteins . BST was then used with streptavidin-gold to demonstrate the cellular topography of ST receptors at the light microscopic level . Villus enterocytes were intensely stained, but only a faint signal was observed in upper crypts of rat small intestine . Thus, a gradient of increasing receptor density was seen as upper crypt cells matured into villus enterocytes . Higher magnification revealed that ST receptors are concentrated at the apical aspect of villus enterocytes . Recently, guanylin, a putative endogenous ligand for ST receptors, has been localized to Paneth cells, at the base of intestinal crypts . Thus, ST receptors are concentrated in villus enterocytes, while guanylin appears to be produced at the base of the crypts.(ABSTRACT TRUNCATED AT 250 WORDS) Int J Radiat Biol, 1993 May, 63(5), 597 - 607 Background and radiation-induced 8-hydroxy-2'-deoxyguanosine in gamma-irradiated Escherichia coli; Claycamp HG et al.; The DNA base damage product, 8-hydroxy-2'-deoxyguanosine (7-hydro-8-oxo-2'-deoxyguanosine, or 8-OHdG) was measured in 2'-deoxyribonucleoside digests of DNA from irradiated E . coli or model DNA using HPLC separation and electrochemical detection (LCED) . The LCED technique enabled the specific quantitation of 25 fmol of 8-OHdG (signal: noise ratio = 4:1) among a variety of DNA base lesions within the sample . A radiation dose-yield relationship for 8-OHdG was observable in cells that were rapidly lysed after irradiation using a chloroform-saturated, sarkosyl and EDTA solution at 4 degrees C (Tilby and Loverock 1983) . The observed yield of 8-OHdG was 7.05 +/- 0.27 pmol J-1 and was against a background of 120 8-OHdG per E . coli genome . This yield translates to an average of 12 additional 8-OHdG lesions per genome at the mean lethal dose . The low radiochemical yield and high background suggests that factors other than the toxicity of 8-OHdG per se--such as its participation in clusters of base lesions (e.g . Ward 1985)--must contribute significantly to cell killing . Although exposure of DNA to phenol mixtures has often been reported to increase 8-OHdG, we found that a more significant effect of phenol mixtures was the potentiation of the radiochemical yields of 8-OHdG in model DNA from 5.26 +/- 0.48 to 158 +/- 12.2 nmol J-1 . Other factors, such as the exposure of dried 2'-deoxyribonucleoside digests to ambient humidity in the presence of buffer components, were shown to contribute more significantly to the background of 8-OHdG. J Clin Microbiol, 1993 May, 31(5), 1194 - 9 A novel mu-capture enzyme-linked immunosorbent assay based on recombinant proteins for sensitive and specific diagnosis of hemorrhagic fever with renal syndrome; Zoller LG et al.; Hantavirus nucleocapsid protein has recently been identified as a major antigen inducing an early and long-lasting humoral immune response in patients with hemorrhagic fever with renal syndrome . A mu-capture enzyme-linked immunosorbent assay utilizing recombinant nucleocapsid proteins of Hantavirus strains Hantaan 76-118 (Hantaan serotype) and CG 18-20 (Puumala serotype) as diagnostic antigens and specific monoclonal antibodies as the detection system has been developed . Histidine-tailed recombinant proteins were expressed in Escherichia coli and purified in a single step by affinity chromatography on a nickel-chelate resin . The assay was evaluated with a panel of sera from patients with hemorrhagic fever with renal syndrome originating from various geographic regions . The overall sensitivity of the mu-capture enzyme-linked immunosorbent assay (both recombinant antigens) was 100%, and its specificity was also found to be 100% . Immunoglobulin M antibodies were detected as early as on day 3, and maximum titers were obtained between days 8 and 25 after onset of the disease . The assay was regularly found to be positive within 3 to 4 months but in some cases up to 2 years after the acute phase of the disease. J Clin Microbiol, 1993 May, 31(5), 1185 - 8 Clonal analysis of Escherichia coli of serogroups O9, O20, and O101 isolated from Australian pigs with neonatal diarrhea; Woodward JM et al.; The genetic diversity of 87 isolates of Escherichia coli recovered from Australian pigs with neonatal diarrhea was examined by multilocus enzyme electrophoresis . The isolates were of serogroups O9, O20, and O101, and although most isolates lacked K88(F4), K99(F5), 987P(F6), and F41 fimbriae, they were considered to be involved in the etiology of the diarrhea . The isolates were extremely diverse, considering their origin from a single pathological condition in one country . There were estimated to be 18, 16, and 12 clones of the three respective serogroups in the collection, with serogroup diversities of 0.387, 0.448, and 0.275, respectively . Comparison with the results previously obtained for isolates from piglets with postweaning diarrhea suggested that bacteria from piglets with these two conditions did not come from any particular common genetic background . The overall genetic diversity for the combined collection was the same as that reported by others for representative isolates selected from throughout the species (0.47) . The current results indicate that if isolates of these O groups are involved in porcine diarrhea, their pathogenicity is directly linked to their O somatic antigen type and is not simply due to the wide distribution of a small number of virulent clones. Protein Sci, 1993 May, 2(5), 800 - 13 Time-resolved fluorescence and computational studies of adenylylated glutamine synthetase: analysis of intersubunit interactions; Atkins WM et al.; Adenylylation of Tyr-397 of each subunit of Escherichia coli glutamine synthetase (GS) down-regulates enzymatic activity in vivo . The overall structure of the enzyme consists of 12 subunits arranged as two hexamers, face to face . Research reported in this paper addresses the question of whether the covalently attached adenylyl group interacts with neighboring amino acid residues to produce the regulatory phenomenon . Wild-type GS has two Trp residues (positions 57 and 158) and the adenylylation site lies within 7-8 A of the Trp-57 loop in the adjacent subunit of the same hexameric ring; Trp-158 is about 35 A from the site of adenylylation . Fluorescence lifetimes and quantum yields have been determined for two fluorophores with wild-type and mutant GS . One fluorophore is epsilon-AMP adenylylated GS (at Tyr-397), and the other fluorophore is the intrinsic protein residue Trp-57 . These experiments were conducted in order to detect possible intersubunit interactions between adenylyl groups and the neighboring Trp-57 to search for a role for the Trp-57 loop in the regulation of GS . The fluorescence due to epsilon-AMP of two adenylylated enzymes, wild-type GS and the W158F mutant, exhibits heterogeneous decay kinetics; the data adequately fit to a double exponential decay model with recovered average lifetime values of 18.2 and 2.1 ns, respectively . The pre-exponential factors range from 0.66 to 0.73 for the long lifetime component, at five emission wavelengths . The W57L-epsilon-AMP enzyme yields longer average lifetime values of 19.5 and 2.4 ns, and the pre-exponential factors range from 0.82 to 0.85 for the long lifetime component . An additional residue in the Trp-57 loop, Lys-58, has been altered and the K58C mutant enzyme has been adenylylated with epsilon-AMP on Tyr-397 . Lys-58 is near the ATP binding site and may represent a link by which the adenylyl group controls the activity of GS . The fluorescence of epsilon-AMP-adenylylated K58C mutant GS is best described by a triple exponential decay with average recovered lifetime values of 19.9, 4.6, and 0.58 ns, with the largest fraction being the median lifetime component . Relative quantum yields of epsilon-AMP-Tyr-397 were measured in order to determine if static quenching occurs from adenine-indole stacking in the wild-type GS . The relative quantum yield of the epsilon-AMP-adenylylated W57L mutant is larger than the wild-type protein by the amount predicted from the difference in lifetime values: thus, no static quenching is evident.(ABSTRACT TRUNCATED AT 400 WORDS) J Bacteriol, 1993 May, 175(10), 3236 - 9 Change in direction of flagellar rotation in Escherichia coli mediated by acetate kinase; Dailey FE et al.; Strains of Escherichia coli lacking all cytoplasmic chemotaxis proteins except CheY swim smoothly under most conditions . However, they tumble when exposed to acetate . Acetate coenzyme A synthetase (EC 6.2.1.1) was thought to be essential for this response . New evidence suggests that the tumbling is mediated instead by acetate kinase (EC 2.7.2.1), which might phosphorylate CheY via acetyl phosphate . In strains that were wild type for chemotaxis, neither acetate coenzyme A synthetase, acetate kinase, nor phosphotransacetylase (EC 2.3.1.8) (and thus acetyl phosphate) was required for responses to aspartate, serine, or sugars sensed by the phosphotransferase system . Thus, acetate-induced tumbling does not appear to play an essential role in chemotaxis in wild-type cells. J Bacteriol, 1993 May, 175(10), 2970 - 9 The murI gene of Escherichia coli is an essential gene that encodes a glutamate racemase activity; Doublet P et al.; The murI gene of Escherichia coli was recently identified on the basis of its ability to complement the only mutant requiring D-glutamic acid for growth that had been described to date: strain WM335 of E . coli B/r (P . Doublet, J . van Heijenoort, and D . Mengin-Lecreulx, J . Bacteriol . 174:5772-5779, 1992) . We report experiments of insertional mutagenesis of the murI gene which demonstrate that this gene is essential for the biosynthesis of D-glutamic acid, one of the specific components of cell wall peptidoglycan . A special strategy was used for the construction of strains with a disrupted copy of murI, because of a limited capability of E . coli strains grown in rich medium to internalize D-glutamic acid . The murI gene product was overproduced and identified as a glutamate racemase activity . UDP-N-acetylmuramoyl-L-alanine (UDP-MurNAc-L-Ala), which is the nucleotide substrate of the D-glutamic-acid-adding enzyme (the murD gene product) catalyzing the subsequent step in the pathway for peptidoglycan synthesis, appears to be an effector of the racemase activity. Biochem J, 1993 May 1, 291 ( Pt 3), 927 - 32 {13C}propionate oxidation in wild-type and citrate synthase mutant Escherichia coli: evidence for multiple pathways of propionate utilization; Evans CT et al.; The metabolism of propionate was examined in wild-type Escherichia coli and cells lacking citrate synthase by high-resolution 13C n.m.r . Spectra of cell extracts from wild-type E . coli show that glutamate becomes highly enriched in 13C when 13C-enriched propionate is the sole carbon source . No glutamate labelling was detected when the tricarboxylic acid cycle was blocked either by deletion of citrate synthase or by inhibition of succinate dehydrogenase by malonate . The 13C fractional enrichment in glutamate C-2, C-3 and C-4 in wild-type cells was quantitatively and qualitatively different when {2-13C}propionate as opposed to {3-13C}propionate was supplied . Approximately equal labelling occurred in the C-2, C-3 and C-4 positions of glutamate when {3-13C}propionate was available, and multiplets due to carbon-carbon spin-spin coupling were observed . However, in cells supplied with {2-13C}propionate, very little 13C appeared in the glutamate C-4 position, and the remaining glutamate resonances all appeared as singlets . The unequal and non-identical labelling of glutamate in cells supplied with {2-13C}- as opposed to {3-13C}propionate is consistent with the utilization of propionate by E . coli via two pathways, oxidation of propionate to pyruvate and carboxylation of propionate to succinate . These intermediates are further metabolized to glutamate by the action of the tricarboxylic acid cycle . The existence of an organized tricarboxylic acid cycle is discussed as a consequence of the ability to block utilization of propionate in tricarboxylic acid-cycle-defective E . coli. Arch Biochem Biophys, 1993 May, 302(2), 455 - 60 Lysine241 of tyrosine hydroxylase is not required for binding of tetrahydrobiopterin substrate; Daubner SC et al.; The lysine residues at positions 194 and 198 in phenylalanine hydroxylase have been shown to react with a photoaffinity label which is an analog of phenyltetrahydropterin (Gibbs, B . S., and Benkovic, S . J . (1991) Biochemistry 30, 6795-6802), in a manner suggesting that these lysine residues are involved in tetrahydrobiopterin binding . The related enzyme tyrosine hydroxylase has a lysine at position 241 which, given the 75% identity between its C-terminal 330 amino acids and those of phenylalanine hydroxylase, corresponds to lysine194 of phenylalanine hydroxylase . Site-directed mutagenesis was used to alter lysine241 of tyrosine hydroxylase to alanine . Steady-state kinetic parameters were measured for wild-type and K241A tyrosine hydroxylase . No kinetic parameter differed between the wild-type and K241A enzymes, including Vmax values, Michaelis constants for tetrahydrobiopterin, 6-methyl-tetrahydropterin, and tyrosine, and the inhibition constants for norepinephrine . These results show that lysine241 is not required for tetrahydrobiopterin binding to tyrosine hydroxylase. Proc Natl Acad Sci U S A, 1993 May 1, 90(9), 3978 - 82 A polypeptide bound by the chaperonin groEL is localized within a central cavity; Braig K et al.; Chaperonins are oligomeric protein complexes that play an essential role in the cell, mediating ATP-dependent polypeptide chain folding in a variety of cellular compartments . They appear to bind early folding intermediates, preventing their aggregation; in the presence of MgATP and a cochaperonin, bound polypeptides are released in a stepwise manner, associated with folding to the native state . Chaperonin complexes appear in the electron microscope as cylindrical structures, usually composed of two stacked rings, each containing, by negative staining, an electron dense central "hole" approximately 6.0 nm in diameter . We sought to identify the site on the Escherichia coli chaperonin groEL, where the "molten globule"-like intermediate of dihydrofolate reductase (DHFR) becomes bound, by examining in the scanning transmission electron microscope complexes formed between groEL and DHFR molecules bearing covalently crosslinked 1.4-nm gold clusters . In top views of the groEL complexes, gold densities were observed in the central region; in side views, the densities were seen at the end portions of the cylinders, corresponding to positions within the individual rings . In some cases, two gold densities were observed in the same groEL complex . We conclude that folding intermediates are bound inside central cavities within individual chaperonin rings . In this potentially sequestered location, folding intermediates with a compact conformation can be bound at multiple sites by surrounding monomeric members of the ring; localization of folding within the cavity could also facilitate rebinding of structures that initially fail to incorporate properly into the folding protein. J Bacteriol, 1993 May, 175(9), 2692 - 701 Alterations of highly conserved residues in the regulatory domain of nitrogen regulator I (NtrC) of Escherichia coli; Moore JB et al.; Transcription of many nitrogen-regulated (Ntr) genes requires the phosphorylated form of nitrogen regulator I (NRI, or NtrC), which binds to sites that are analogous to eukaryotic enhancers . A highly conserved regulatory domain contains the site of phosphorylation and controls the function of NRI . We analyzed the effects of substitutions in highly conserved residues that are part of the active site of phosphorylation of NRI in Escherichia coli . Fourteen substitutions of aspartate 54, the site of phosphorylation, impaired the response to nitrogen deprivation . Only one of these variants, NRI D-54-->E (NRI-D54E), could significantly stimulate transcription from glnAp2, the major promoter of the glnALG operon . Cells with this variant grew with arginine as a nitrogen source . Experiments with purified components showed that unphosphorylated NRI-D54E stimulated transcription . In contrast, substitutions at aspartate 11 were not as deleterious as those at aspartate 54 . Finally, we showed that NRI-K103R, in which arginine replaces the absolutely conserved lysine, is functionally active and efficiently phosphorylated . This substitution appears to stabilize the phosphoaspartate of NRI . The differences between our results and those from study of homologous proteins suggest that there may be significant differences in the way highly conserved residues participate in the transition to the activated state. Infect Immun, 1993 May, 61(5), 2249 - 52 Identification of plasmid and chromosomal copies of 987P pilus genes in enterotoxigenic Escherichia coli 987; Casey TA et al.; A DNA probe derived from the subunit gene of a cloned 987P determinant was used to characterize the locations of the 987P genes in several Escherichia coli strains . We examined E . coli 987, a nonpiliated mutant of strain 987 (I36) that does not express 987P in vitro, and a variant of I36 that expressed 987P following growth in vivo . We found that plasmid and chromosomal copies of the 987P subunit gene could be differentiated in strain 987 by restriction endonuclease analysis and Southern blot hybridization . The nonpiliated mutant I36 had lost the plasmid copy but retained the chromosomal copy of the 987P gene . A 987P-piliated variant of I36, which did not contain the 987P plasmid, colonized and caused diarrhea in neonatal pigs similarly to wild-type 987 . The plasmid that hybridized with the 987P probe was transferred from strain 987 to an E . coli K-12 strain by conjugation . We were unable to demonstrate expression of 987P by these transconjugants . The data suggest that the chromosomal and plasmid copies of the 987P genes may interact to influence 987P expression. Infect Immun, 1993 May, 61(5), 2108 - 15 Specificity of S fimbriae on recombinant Escherichia coli: preferential binding to gangliosides expressing NeuGc alpha (2-3)Gal and NeuAc alpha (2-8)NeuAc; Hanisch FG et al.; The adhesins of Escherichia coli strains HB101(pANN801-13) and HB101(pAZZ50), which express S fimbriae encoded by a recombinant plasmid containing the sfaI and sfaII gene clusters, respectively, were characterized with regard to the detailed structural requirements of their binding to sialyloligosaccharides on (neo)glycoproteins and (neo)glycolipids . From binding and binding inhibition studies in solid-phase enzyme immunoassays with isolated S fimbriae, several major conclusions can be drawn . S fimbriae bind specifically to sialic acid on gangliosides . The most active structural variant of sialic acid on GM3 ganglioside is N-glycolylneuraminic acid (NeuGc) . In contrast to previous reports, high binding activities were measured also for b-series gangliosides expressing NeuAc alpha (2-8)NeuAc . In agreement with earlier studies, the site of sialic acid substitution to subterminal sugars strongly influences the binding to sialyloligosaccharides, i.e., alpha-6-linked sialic acid is only poorly recognized by the adhesin compared with alpha-3-linked sialic acid . C-8 and C-9 hydroxyl groups form essential structural elements of sialic acid in the binding event. Urol Nefrol (Mosk), 1993 May-Jun, (3), 13 - 6 {Fibronectin and its significance in pyelonephritis}; Rudenko AV et al.; Fibronectin levels were measured by enzyme immunoassay in 68 patients with pyelonephritis and 10 patients with chronic cystitis . The patients with chronic cystitis, acute and chronic non-obstructive pyelonephritis exhibited significantly high average blood fibronectin levels, whereas those with chronic obstructive pyelonephritis displayed the levels slightly different from those observed in healthy individuals . Hypofibronectinemia was detected in 23.3% of patients with chronic pyelonephritis . Preincubation of the neutrophils isolated from pyelonephritis patients with fibronectin increased the initially low phagocytic capacity without enhancing their metabolic activity . In experimental pyelonephritis, administration of exogenous fibronectin was demonstrated to contribute to a rapid bacterial elimination from the kidneys, to decrease the intensity of an inflammatory response, to prevent renal histostructural lesions, to enhance the functional activity of immunocompetent cells and to stabilize their membranes . The findings may serve the basis for using fibronectin in clinical practice as a promising agent to treat pyelonephritis. Biosci Biotechnol Biochem, 1993 May, 57(5), 760 - 5 Sequencing analysis of mutation points in the biotin operon of biotin-overproducing Escherichia coli mutants; Ifuku O et al.; We analyzed mutation points of the biotin operon from biotin-overproducing mutants of Escherichia coli resistant to two biotin analogs, actithiazic acid and 5-(2-thienyl)-valeric acid, by DNA sequencing . The biotin operons cloned from these mutants were classified into three groups . One point mutation, which was a GC-->AT change within the operator overlapping the -10 region of the rightward (bioB) promoter, was considered to result in disruption of operator structure and enhancement of promoter activity . Two other point mutations, which were both GC-->AT changes just before and after the initiation codon of the bioB gene, were considered to activate the translation efficiency . These mutations significantly accelerated the biotin-forming activity from dethiobiotin in cell-free extracts. Biosci Biotechnol Biochem, 1993 May, 57(5), 710 - 4 Cleavage of potato virus Y polyprotein in Escherichia coli depending on the proteolytic activity of viral protease; Hidaka M et al.; We have cloned a 5-kb cDNA corresponding to the 3'-half of genomic RNA of potato virus Y (PVY), the type member of plant potyvirus . Based on its nucleotide sequence, the cDNA was presumed to encode PVY protease responsible for the self-processing of polyprotein precursor expressed from PVY genome . To test its proteolytic activity, the cDNA was modified so as to express the protease-polymerase-coat protein (CP) portion of PVY polyprotein in Escherichia coli . By Western blotting analysis of the cell extract of E . coli expressing the polyprotein, mature CP was detected . A large deletion in the polymerase-coding region did not affect the emergence of mature CP, but a small deletion in the putative protease region resulted in disappearance of mature CP and appearance of the intact polyprotein . These results indicated that the polyprotein expressed in E . coli was cleaved depending on the proteolytic activity of PVY protease. Appl Microbiol Biotechnol, 1993 May, 39(2), 227 - 34 Biosafety investigations in an r-DNA production plant; Weibel EK et al.; Employees of a biotechnological production plant for recombinant interferon alpha-2a have been participating in a 1-year study on occupational hygiene and the resulting biosafety aspects . Most of the employees have been employed in the plant for more than 6 years . Weekly stool samples were analysed for tetracycline (used as selection marker)-resistant coliforms as well as for rDNA (IFN gene) (interferon gene) and for the production organism . Various analytical methods, including the polymerase chain reaction, were applied to show that neither rDNA nor the production organism could be found in any of the stool samples and that there was no change or trend in the gut flora with respect to tetracycline resistance . In addition it could be shown that the tetracycline-resistance gene, as well as the rDNA, are completely inactivated in the course of the production process and thus no further recombination can take place . Blood samples were taken to show that none of the employees had anti-product antibodies. Appl Microbiol Biotechnol, 1993 May, 39(2), 221 - 6 Cloning, expression, and characterization of a synthetic analog to the bioadhesive precursor protein of the sea mussel Mytilus edulis; Salerno AJ et al.; Repetitious gene cassettes that encode the consensus decapeptide repeat of Mytilus edulis bioadhesive protein were designed, constructed, and expressed in Escherichia coli . The bioadhesive precursor (BP) with a relative molecular mass of 25,000 was expressed from one 600-bp gene at levels approaching 60% of total cell protein in strains employing T7 RNA polymerase for induction and carrying a repetitious gene comprised of a 30-bp unit repeat that accounts for E . coli codon bias . BP forms intracellular inclusions and yet methionine was processed from the N-terminus of the purified protein, as shown by amino acid composition and N-terminal sequencing, to give an authentic consensus precursor protein. Biotechnology (N Y), 1993 May, 11(5), 601 - 5 Recombinant colorimetric antibodies: construction and characterization of a bifunctional F(ab)2/alkaline phosphatase conjugate produced in Escherichia coli; Ducancel F et al.; We have designed a vector which allows the synthesis in Escherichia coli of bifunctional F(ab)2-alkaline phosphatase conjugates . The vector contains a di-cistronic operon encoding truncated heavy chain (Fd or VH-CH1) of an IgG inserted between residues +6 and +7 of bacterial alkaline phosphatase (PhoA), and the light chain of the same IgG . We demonstrate the utility of this approach with the heavy and light chain domains of a snake toxin-specific monoclonal antibody, M alpha 2-3 . We show that the VH-CH1-PhoA hybrid and VL-CL are concomitantly expressed and exported to the periplasm of E . coli where they form a disulfide-linked chimeric protein . The hybrid has the same affinity as M alpha 2-3 for the snake toxin antigen and possesses PhoA enzymatic activity. Gastroenterol Jpn, 1993 May, 28 Suppl 5, 52 - 4 High detection rate of hepatitis C virus E2 antibody in patients with type C hepatitis; Yokosuka O et al.; Putative hepatitis C virus E2 protein was applied for detection of anti-E2 antibody in patients with type C hepatitis . The Putative E2 sequence was expressed in E . coli and approximately 38 KDa hepatitis C E2 protein fused with 42 KDa maltose binding protein was obtained . The HCV E2 antibody was detected in 2 of 7 acute hepatitis cases, in 8 of 12 chronic persistent hepatitis, in 17 of 25 chronic active hepatitis, and in 2 of 4 cirrhosis . It was not detected in 10 normal subjects . Anti-E2 antibody became undetectable after successful interferon treatment in patients with type C hepatitis . The High detection rate of E2 antibody in chronic hepatitis C and disappearance of the antibody after the interferon treatment indicate that the E2 antibody is related to viral replication and is unlikely to be a neutralizing antibody. Plasmid, 1993 May, 29(3), 241 - 4 Development of a native plasmid as a cloning vector in Clavibacter xyli subsp . cynodontis; Taylor J et al.; A 50-kilobase (kb) cryptic plasmid was found in three geographic isolates of Clavibacter xyli subsp . cynodontis (Cxc) . The DNA region essential for replication of the plasmid was cloned in an Escherichia coli vector . The resulting vector, which functions as a shuttle vector between E . coli and Cxc, was characterized further with respect to its stability and copy number. Parasitology, 1993 May, 106 ( Pt 4), 413 - 20 Cloning, over-expression and evaluation of a recombinant fusion protein of Wuchereria bancrofti towards its application as a diagnostic agent for bancroftian filariasis; Theodore JG et al.; A low molecular weight (15 kDa) surface antigen of the cattle filarial nematode, Setaria digitata, was earlier shown to be specifically recognized by the antibodies from human bancroftian filarial (Mf positive) patients' sera (Theodore & Kaliraj, 1990) . The filarial specific antibodies bound to a 15 kDa peptide in preparative Western blots were eluted and employed in screening of candidate antigens expressed in the genomic library of Wuchereria bancrofti at the IgG4 subclass antibody level . A recombinant clone (lambda WbG7) reacting strongly with filarial sera but poorly with sera from patients infected with non-filarial helminths was selected for further studies . The 2 kb DNA insert of the clone lambda WbG7 was recloned into a pMAL vector and the recombinant clone pGT7 thus obtained was over-expressed and affinity purified . The purified 105 kDa fusion protein of the clone pGT7 was specific and was not recognized by the non-filarial sera at the IgG4 level . All microfilaraemic individuals were positive by IgG4 assay . However, similar attempts to diagnose by filarial-specific IgE assays failed to recognize microfilaraemic individuals . Moreover, by filarial-specific IgG4 assays, the endemic normals were distinctly divided into two groups, showing higher and lower recognition for this antigen indicating current and past/no infection . Among the filarial-IgG4 (assay)-positive 'endemic normals', 14% showed 'microfilariae' during repeated peripheral night blood examination, confirming the validity of the recombinant antigen, pGT7 based assay. Mol Biol (Mosk), 1993 May-Jun, 27(3), 618 - 30 {Reverse transcriptase of the human immunodeficiency virus: isolation and substrate specificity}; Rozovskaia TA et al.; Human immunodeficiency virus (HIV-I) reverse transcriptase was expressed in E . coli and purified to homogeneity (E . coli strain RRI (pRC-RT, pRK 248cIts)) . We have investigated the substrate properties toward to DNA synthesis, catalyzed by this enzyme, of some nucleoside-5'-triphosphate analogues, previously studied in the same reactions, catalyzed by AMV and M-MLV reverse transcriptases . We have investigated substrate properties of new analogues of 2',3'-dideoxy-2',3'-didehydro- and 2',3'-dideoxytubercidin-5'-triphosphates . We have compared the relative efficiency of incorporation of different analogues tested in the DNA chain . It has been shown that expressed and purified HIV reverse transcriptase had the same specificity to analogues of 2'-deoxyribonucleoside-5'-triphosphates as was described for reverse transcriptases and natural HIV reverse transcriptase as well . These properties allow to apply the expressed HIV reverse transcriptase in different model systems. Mol Biol (Mosk), 1993 May-Jun, 27(3), 538 - 51 {Design of four-helix protein--a possible vaccine against human immunodeficiency virus (HIV-1)}; Eroshkin AM et al.; Successful approach to the development of safe and effective synthetic vaccines requires that different B- and T-cell epitopes of the infectious agent be included into the vaccine construction . It is suggested that vaccines should be constructed as proteins with both optimal epitope composition and predetermined tertiary structure . Based on analysis of B-cell and T-cell epitope properties, a possibility to use one well-known protein spatial motif--four-alpha-helix bundle--for vaccine construction is substantiated . Antigenic determinants of cellular immunity (amphipathic alpha-helices) and humoral immunity (flexible hydrophilic loop regions) can be used as blocks for vaccine design . Nonloop B-epitopes and nonhelical T-epitopes may be introduced in the protein N- and C-terminal regions . General principles of PTS-vaccine construction have been applied to anti-HIV-1 vaccine design . Experimentally studied T- and neutralizing B-cell epitopes from HIV-1 proteins were analyzed . The sequence of one possible four-alpha-helix protein vaccine has been constructed . Predicted secondary structure and T- and B-cell epitopes of this protein coincided with the planned ones . The amino acid composition of the protein was found to be consistent with the composition of globular water-soluble proteins . The gene of the protein with codon composition optimal for expression in E . coli has been synthesized . The advantages and limitations of this approach to vaccine design are discussed. J Neuroimmunol, 1993 May, 44(2), 157 - 62 Biological activity of recombinant human myelin basic protein; Oettinger HF et al.; Using an inducible expression vector in Escherichia coli, we have expressed, purified, and biologically characterized recombinant human myelin basic protein (r-MBP) . The recombinant protein binds in cation-exchange chromatography with similar affinity to purified, human MBP, and elutes as a single, 18.5-kDa species as judged by SDS-PAGE . The recombinant protein exhibits similar conformation to native MBP, as demonstrated by ELISA reactivity with a panel of six monoclonal antibodies directed against at least three different epitopes on human MBP . Additionally, recombinant MBP can trigger the proliferation of human T cell clones recognizing MBP, can induce experimental autoimmune encephalomyelitis (EAE) in the SJL mouse, and is capable of suppressing EAE following tolerization via oral administration . Most important, by using a novel method of purification, the recombinant protein preparation contains no detectable proteolytically derived fragments of MBP, a significant advantage over MBP purified from protease-rich central nervous system tissue . Purified recombinant MBP produced in E . coli will be useful as a biological reagent where highly purified protein devoid of degradation products is needed . Relevance to the study of antigen processing, interactions between MHC and the TCR, as well as for the investigation of antigen-driven immune tolerance are discussed. J Bacteriol, 1993 May, 175(10), 2895 - 906 Interaction between the antisense and target RNAs involved in the regulation of IncB plasmid replication; Siemering KR et al.; Physical analysis of RNA I, the small antisense RNA which regulates the replication of IncB miniplasmid pMU720, showed that it is a highly structured molecule containing an imperfectly paired stem closed by a 6-base hairpin loop . Mutational studies revealed that a 3-base sequence in the hairpin loop is critical to the interaction between RNA I and its complementary target in the RepA mRNA (RNA II) . Furthermore, a 2-base interior loop in the upper stem was found to play an important role in facilitating effective binding between RNA I and RNA II . From these analyses, a model describing the molecular mechanism of binding between RNA I and RNA II is proposed. Genes Dev, 1993 May, 7(5), 904 - 10 Phosphorylation of tobacco mosaic virus cell-to-cell movement protein by a developmentally regulated plant cell wall-associated protein kinase; Citovsky V et al.; In host plants, cell-to-cell spread of tobacco mosaic virus (TMV) presumably occurs through intercellular connections, the plasmodesmata . TMV movement is mediated by a specific virus-encoded single-strand nucleic acid-binding protein, P30 . The mechanism by which P30 operates is largely unknown . Here, we demonstrate that P30 expressed in transgenic plants is a phosphoprotein . We have developed an assay for in vitro phosphorylation of purified P30 by plant cell wall fractions and have localized the phosphorylation sites to amino acid residues Ser-258, Thr-261, and Ser-265 . Interestingly, the P30 phosphorylation sites do not correspond to any known consensus phosphorylation sites for protein kinases . While P30 binding to single-stranded DNA (ssDNA) was shown to involve Thr-261, phosphorylation of this residue does not appear to play a role in binding activity . The protein kinase activity contained in the cell wall fractions was developmentally regulated, expressed predominantly in leaves . Within a leaf, this protein kinase activity increased with leaf maturation and correlated with the reported development of secondary plasmodesmata, sites of P30 accumulation . We suggest that phosphorylation may represent a mechanism for the host plant to sequester P30 following its localization to cell walls. Biochem J, 1993 May 1, 291 ( Pt 3), 757 - 63 Identification of the domain recognized by anti-(ryanodine receptor) antibodies which affect Ca(2+)-induced Ca2+ release; Treves S et al.; In the present paper we have defined putative functional domains of the ryanodine receptor Ca2+ channel . cDNA fragments of the skeletal muscle ryanodine receptor were fused in-frame with the Escherichia coli trpe protein and the resulting fusion proteins were evaluated for their ability to react with anti-(ryanodine receptor) antibodies, which are known to block Ca(2+)-dependent activation of the Ca(2+)-release channel . Anti-(ryanodine receptor) antibodies react with epitopes lying within a 245-amino-acid-long polypeptide which is located in a region (residues 4380-4625) encompassing most of myoplasmic loop 2, the predicted transmembrane segment M5 and part of the next lumenal loop (45 residues) . Purification of the anti-(ryanodine receptor) antibodies by affinity chromatography led to the isolation of a population of antibodies which was capable of decreasing (by > 30%) the doxorubicin-induced Ca2+ release from isolated terminal cisternae . Polyclonal antibodies raised against a ryanodine receptor fusion encompassing part (198 out of 245 residues) of the immunopositive polypeptide decreased by 2-fold the first-order rate constant of Ca(2+)-induced 45Ca2+ efflux from isolated terminal cisternae . These results suggest strongly that the Ca(2+)-activating domain of the skeletal muscle Ca(2+)-release channel is close to, or associated with, myoplasmic loop 2. Mol Immunol, 1993 May, 30(7), 613 - 25 Identification of helper T cell epitopes of dengue virus E-protein; Leclerc C et al.; The T cell proliferative response to dengue 2 (Jamaica) E-glycoprotein (495 amino acids) was analyzed in vitro using either killed virus or E-protein fragments or synthetic peptides . Inactivated dengue virus stimulated dengue-specific lymph node (LN) CD4+T cell proliferation in BALB/c (H-2d), C3H (H-2k) and DBA/1 (H-2q) but not in C57BL/6 (H-2b) mice . Moreover, LN cells from dengue-virus primed BALB/c mice proliferated in vitro in response to three purified non-overlapping E-protein fragments expressed in E . coli as polypeptides fused to trpE (f22-205, f267-354, f366-424) . To further determine T cell epitopes in the E-protein, synthetic peptides were selected using prediction algorithms for T cell epitopes . Highest proliferative responses were obtained after in vitro exposure of virus-primed LN cells to peptides p135-157, p270-298, p295-307 and p337-359 . Peptide p59-78 was able to induce specific B and T cell responses in peptide-primed mice of H-2d, H-2q and H-2k haplotypes . Two peptides p59-78 corresponding to two dengue (Jamaica and Sri Lanka) isolates and differing only at position 71 cross-reacted at the B but not at the T cell level in H-2b mice . This analysis of murine T helper cell response to dengue E-protein may be of use in dengue subunit vaccine design. Clin Exp Immunol, 1993 May, 92(2), 308 - 16 Sera from patients with tuberculosis recognize the M2a-epitope (E2-subunit of pyruvate dehydrogenase) specific for primary biliary cirrhosis; Klein R et al.; Anti-M2 antibodies in primary biliary cirrhosis (PBC) have been shown to react with the alpha-ketoacid dehydrogenase complex of the inner mitochondrial membrane consisting of six epitopes (E2 subunit of the pyruvate dehydrogenase complex (PDC), 70 kD; protein X of the PDC, 56 kD; alpha-ketoglutarate dehydrogenase complex, 52 kD; branched-chain alpha-ketoacid dehydrogenase, 52 kD; E1 alpha subunit of PDC, 45 kD; and E1 beta-subunit of PDC, 36 kD) . These epitopes are also present in the M2 fraction which is a chloroform extract from beef heart mitochondria . The E2 subunit of the PDC at 70 kD (M2a), especially, is a major target epitope which is recognized by about 85% of all PBC sera . However, analysing sera from 28 patients with active pulmonary tuberculosis it became evident that 12 (43%) also recognized the PDC-E2 subunit (M2a), as shown by Western blotting using the M2 fraction, the purified PDC, and the recombinant PDC-E2 . In contrast, only two of 82 patients with other bacterial and viral infections including 25 patients with Escherichia coli infections reacted with the PBC-specific epitope at 70 kD . Naturally occurring mitochondrial antibodies (NOMA) were present in 54% of the patients with tuberculosis and in 50% of patients with other infectious disorders . They recognized either a determinant at 65 kD (epsilon) or at 60/55 kD (zeta/eta) . None of the sera from 100 blood donors had anti-M2 but 14 had NOMA . Testing anti-M2 and NOMA-positive marker sera by Western blotting against membrane fractions derived from mycobacteria and E . coli it could be shown that--like mammalian mitochondria--they contain both the PBC-specific M2 antigen as well as the non-PBC-specific naturally occurring mitochondrial antigen system (NOMAg) . The observation that PBC-specific antibodies were preferentially induced in patients suffering from a mycobacterial infection may provide some new clues to the still unknown etiology of PBC. Proc Natl Acad Sci U S A, 1993 May 1, 90(9), 4092 - 6 Evasion of protective immunity by Borrelia burgdorferi by truncation of outer surface protein B; Fikrig E et al.; We analyzed variability in outer surface protein B (OspB) from Borrelia burgdorferi (Bb), the causative agent of Lyme disease, to determine how Bb escapes immune destruction . We have shown that vaccination with OspB from Bb strain B31 protected mice from infection with Bb B31 but not against Bb N40 . The present study demonstrates that Bb N40 spirochetes which evade vaccination immunity to OspB have a truncated form of OspB, due to a TAA stop codon at nucleotide 577 . In contrast, Bb N40 spirochetes that express full-length OspB are unable to infect mice immunized with OspB, analogous to our previous studies with Bb B31 . Mapping of the OspB antibody response shows that epitopes in the C terminus of OspB are surface-exposed and bind protective monoclonal and polyclonal antibodies . This suggests that the C terminus of OspB is important for eliciting a protective immune response to OspB . Truncation or modification of outer surface proteins that do not bind protective antibody may be a means by which Bb evades host defenses. Proc Natl Acad Sci U S A, 1993 May 1, 90(9), 3938 - 42 Molecular cloning, sequencing, tissue distribution, and functional expression of a Na+/H+ exchanger (NHE-2); Collins JF et al.; The present studies demonstrate cloning, sequencing, tissue distribution, and functional expression of a Na+/H+ exchanger which was isolated from a rat intestinal cDNA library . The cloned cDNA recognizes two transcripts in poly(A)+ RNA from the stomach, jejunum, ileum, liver, large intestine, and uterus . Based on deduced amino acid sequences, this clone shares sequence homology with the other known Na+/H+ exchanger isoforms (NHE-1, NHE-3, and NHE-4) except for its 5' end . Overall, the protein exhibits 47.8%, 41.2%, and 56.2% amino acid sequence identity to NHE-1, NHE-3, and NHE-4, respectively . The hydropathy profile of the predicted protein shows 10 transmembrane domains, suggesting a protein with transport characteristics . The tissue distribution differs from that of the other Na+/H+ exchanger isoforms . The cDNA hybridizes to two closely related transcripts in the mRNA of these tissues, which suggests that the predominant transcript of this clone is alternatively spliced . Transfection of this cDNA into Na+/H+ exchanger-deficient mutant fibroblasts (PS120 cells) results in functional Na+/H+ exchange activity . These data suggest that we have cloned a member of the Na+/H+ exchanger family with tissue-specific expression . We suggest the designation of NHE-2 for this Na+/H+ exchanger. Dev Biol, 1993 May, 157(1), 214 - 23 Cloning and analysis of expression of a ubiquitin carboxyl terminal hydrolase expressed during oogenesis in Drosophila melanogaster; Zhang N et al.; Using the enhancer trap approach we have searched for genes with important functions in oogenesis . We selected a line of flies with a P insertion, carrying the Escherichia coli lac Z gene, which showed beta-galactosidase expression in the nurse cell nuclei during oogenesis . Surrounding the P insertion we discovered a cluster of transcription units with enriched expression in the ovary . One of these encodes a protein with extensive sequence similarity to the human and yeast ubiquitin carboxyl terminal hydrolase . Analysis of a fusion protein including the putative ubiquitin carboxyl terminal hydrolase indicated that this protein does have the appropriate enzyme activity, and the gene was assigned the name ubiquitin carboxyl terminal hydrolase uch-D . The expression of this gene is enriched in the nurse cells and transcripts are transported to the embryo . Transcripts are abundant for the first few hours of development . The transcripts are found to be enriched on the ventral side of the oocyte and nurse cells . Rather little is known about the ubiquitin pathway in Drosophila and the discovery of this gene enables us to make predictions as to the roles it may play during early embryogenesis. Oncogene, 1993 May, 8(5), 1241 - 7 Recombinant Csk expressed in Escherichia coli is autophosphorylated on tyrosine residue(s); Bougeret C et al.; The C-terminal src kinase (Csk) is responsible for the phosphorylation of the carboxy-terminal tyrosine of several tyrosine kinases of the Src family . This phosphorylation site has a negative regulatory function . Csk is unique among the members of the protein tyrosine kinase family because it lacks the conserved tyrosine autophosphorylation site and has been thought to be devoid of autophosphorylation activity . Using the glutathione S-transferase (GST) bacterial expression system, we have produced large amounts of a chimeric rat Csk protein . We have determined that the GST-Csk fusion protein isolated from bacteria is autophosphorylated on tyrosine residue(s) . GST-Csk and purified Csk are capable of undergoing autophosphorylation on tyrosine residue(s) in vitro . The GST-Csk fusion protein also phosphorylates exogenous substrates, including the heteropolymer poly-Glu/Tyr and enolase . This is the first indication that Csk is autophosphorylated on tyrosine residue(s) both in vivo in bacteria expressing Csk cDNA and in vitro . These findings suggest that the autophosphorylation of Csk might play a role in the regulation of its kinase activity as well as its binding to other cellular proteins. Mutat Res, 1993 May, 302(1), 53 - 9 The effect of non-mutagenic stress on liquid holding recovery in Escherichia coli AB2463; Fitt PS et al.; Previous work has shown that exposure to many non-mutagenic stresses causes greater UV resistance in Escherichia coli K12 via an error-free, excision repair-dependent process . Induction of the latter should enhance liquid holding recovery in the bacteria . The results in this paper show that this is the case and that the increased UV-resistance is due entirely to an increase in the capacity of the cells for DNA excision repair . The latter arises wholly or in part from an increase in the intracellular level of the key enzyme of the pathway, UvrABC endonuclease. Mutat Res, 1993 May, 302(1), 13 - 8 Mutation enhancing effect of o-vanillin in the lacZ gene of Escherichia coli: characterization of mutational spectrum; Watanabe K et al.; The enhancing effect of o-vanillin (2-hydroxy-3-methoxybenz-aldehyde) on mutations induced by N-methyl-N'-nitro-N-nitrosoguanidine (MNNG) was characterized with mutational specificity . The mutational spectrum of MNNG-induced mutations in the presence of o-vanillin was compared with that in the absence of o-vanillin by means of a series of mutant lacZ genes in E . coli that can detect each of the six types of base substitutions and five kinds of frameshift events . In the absence of o-vanillin, the mutational spectrum induced by MNNG consisted mainly of G.C-->A.T transitions and, to a lesser extent, -1(G.C) frameshift mutations . By adding o-vanillin at 75 micrograms/plate, a marked enhancement was observed in two transitions, G.C-->A.T and A.T-->G.C, and in two frameshift mutations, +1(G.C) and -1(G.C) . Only a small change was observed in the -2(C.G-G.C) fraction . Regarding the MNNG-induced frameshifts at the A.T base pair, the +1(A.T) frameshift was much more enhanced by o-vanillin than the -1(A.T) frameshift. Mutat Res, 1993 May, 299(3-4), 233 - 50 Double-strand breaks in plasmid DNA and the induction of deletions; Schulte-Frohlinde D et al.; Double-strand breaks (dsbs) have been produced in plasmid DNA by various restriction endonucleases and the survival and the deletion mutation incidence have been measured in E . coli . The deletion formation is known to depend upon the occurrence of short direct repeats within the DNA molecule . In order to study the role of these repeats we constructed plasmid molecules with repeats of various lengths or with a 10-base pair repeat at different distances from each other . Furthermore the influence of the location and the structure of the dsb was studied . Repair and deletion frequencies of the linearized plasmids were measured after transformation of E . coli . The yield of the specific deletion mutation (the one which occurs between the introduced repeats) increases nearly linearly with the square of the length of the repeat, while the yield of the correctly repaired DNA and the yield of all other deletion mutants remained constant . The slope of the linear increase of the yield of the specific deletion depends on the location and the structure of the dsb . The yield of the specific deletion mutation decreases with increasing distance between the repeats . A proposal for the rate-determining step of the deletion formation is made. Mutat Res, 1993 May, 299(3-4), 193 - 209 Multiple pathways of deletion formation in Escherichia coli; Balbinder E; We are investigating the mechanisms for deletion formation through the use of mutants which alter deletion frequency together with well characterized systems for deletion detection . We report here on three mutations which were isolated for their ability to stimulate deletions in plasmid pMC874 (dli mutations) . The mutation rec-2251 (formerly known as dli1) is a new allele of recBCD, a group of genes coding for the polypeptide components of the major recombination enzyme complex in E . coli; the second one, dli2 may be a new allele of uvrD, which codes for DNA helicase II; and the third one, dli3, has the phenotype of a mismatch repair mutation . Here we compare the effects of mutations in SOS-repair genes to those of the dli mutations on three different deletion events: (a) the deletion of short (60-100-bp) palindromic and non-palindromic inserts in derivatives of plasmid pBR325; (b) larger (600-800-bp) deletions in plasmid pMC874; and (c) the excision of the Tn10 transposon from chromosomal sites . Our results indicate that some form of SOS processing stimulates the loss of palindromes but not non-palindromes in plasmid pBR325 derivatives, and that RecA is necessary for UV-induced excision of Tn10 but this event is inhibited by UmuCD or its homolog MucAB . Each of the dli mutations showed unique effects on different classes of deletions . Mutation rec-2251 stimulated specifically deletions in pMC874 but had no effect on the deletion of non-palindromes in pBR325, and reduced the incidence of the other deletion events tested including loss of palindromic inserts in pBR325 as well as Tn10 excision . Mutation dli2, on the other hand, stimulated all deletions tested to varying extents, while dli3 did not affect markedly deletion formation in pBR325 plasmids but had a large stimulatory effect on both deletions in plasmid pMC874 and Tn10 excision . These results reveal that (a) some SOS-repair functions participate in deletion formation, (b) mutations selected for altering the incidence of one class of deletions may have totally different effects on other deletion events, and (c) the differences in mutant behavior may result in part from the ability of some pathways to discriminate among different deletion intermediates such as hairpins or cruciforms formed by palindromic sequences vs . transient secondary structures stabilized by direct repeats flanking non-palindromic sequences. Mutat Res, 1993 May, 299(3-4), 147 - 56 Thymine ring saturation and fragmentation products: lesion bypass, misinsertion and implications for mutagenesis; Evans J et al.; We have used thymine glycol and dihydrothymine as representative ring saturation products resulting from free-radical interaction with DNA pyrimidines, and urea glycosides and beta-ureidoisobutyric acid (UBA) as models for pyrimidine-ring fragmentation products . We have shown that thymine glycol and the ring-fragmentation products urea and beta-ureidoisobutyric acid, as well as abasic sites, are strong blocks to DNA polymerases in vitro . In contrast, dihydrothymine is not a block to any of the polymerases tested . For thymine glycol, termination sites were observed opposite the putative lesions, whereas for the ring-fragmentation products, the termination sites were primarily one base prior to the lesion . These and other data have suggested that thymine glycol codes for an A, and that a base is stably inserted opposite the damage, whereas when a base is inserted opposite the non-coding lesions, it is removed by the 3-->5 exonuclease activity of DNA polymerase I . Despite their efficiency as blocking lesions, thymine glycol, urea and UBA can be bypassed at low frequency in certain specific sequence contexts . When the model lesions were introduced individually into single-stranded biologically active DNA, we found that thymine glycol, urea, beta-ureidoisobutyric acid, and abasic sites were all lethal lesions having an activation efficiency of 1, whereas dihydrothymine was not . Thus the in vitro studies predicted the in vivo results . When the survival of biologically active single-stranded DNA was examined in UV-induced Escherichia coli cells where the block to replication was released, no increase in survival was observed for DNA containing urea or abasic sites, suggesting inefficient bypass of these lesions . In contrast, beta-ureidoisobutyric acid survival was slightly enhanced, and transfecting DNA containing thymine glycols was significantly reactivated . When mutation induction by unique lesions was measured using f1-K12 hybrid DNA containing an E . coli target gene, thymine glycols and dihydrothymine were found to be inefficient as premutagenic lesions, suggesting that in vivo, as in vitro, they primarily code for A . In contrast, urea and beta-ureidoisobutyric acid were efficient premutagenic lesions, with beta-ureidoisobutyric acid being about 4-5-fold more effective than urea glycosides, which have approximately the same rate of mutation induction as abasic sites from purines . Sequence analysis of the mutations resulting from these ring-fragmentation products shows that the mutations produced are both lesion and sequence context dependent . The possible roles that bypass efficiency and lesion-directed misinsertion might play in mutagenesis are discussed. Mutat Res, 1993 May, 299(3-4), 123 - 33 Site-directed mutagenesis in single cells: transitions produced by DNA carrying a single O6-alkylguanine residue; Chambers RW; Using a single burst assay based on a Poisson Distribution, I have determined the mutant virus frequency in single spheroplasts transfected with phi X174 form I' DNA carrying an O6-methyl-, ethyl-, n-propyl- or n-butylguanine residue at position 2401 of the minus strand . One set of experiments was performed with spheroplasts derived from Escherichia coli AB1157, which has normal DNA-repair systems . Of the cells examined after transfection with DNA carrying a methylguanine moiety, 30% produced mutant virus and 12