Can J Microbiol, 1995 Jan, 41(1), 92 - 8 Neither reduced uptake nor increased efflux is encoded by tellurite resistance determinants expressed in Escherichia coli; Turner RJ et al.; Rates of uptake of the TeO3(2-) oxyanion were investigated in Escherichia coli cells containing tellurite resistance determinants from both plasmid (RK2Ter, R478, pMER610, MIP233, pHH1508a, pMUR) and chromosomal (tehAB) sources . The uptake was investigated to determine whether or not reduced uptake or increased efflux is involved in the tellurite resistance mechanism . Reduced TeO3(2-) uptake generated by cultures harboring arsABC from the plasmid R773, which has been previously shown to be an oxyanion efflux transporter, was used as the standard . Uptake curves were found to be essentially identical among E . coli cultures harboring the tellurite resistance plasmids RK2Ter, pMER610, pHH1508a, and pMUR and cultures harboring tellurite-sensitive control plasmids . Cultures harboring clones of the tehAB operon from E . coli showed no change in the TeO3(2-) accumulation . Cultures harboring R478 demonstrated reduced uptake . However, a subclone containing only the tellurite resistance determinant displayed no reduced uptake . This suggests that there may be another determinant on R478 other than the primary tellurite resistance determinant that gives rise to TeO3(2-) efflux . These results demonstrate that neither reduced uptake nor increased efflux is responsible for the tellurite resistance in the resistance determinants investigated here. Can J Microbiol, 1995 Jan, 41(1), 27 - 34 Nucleotide sequence and transcriptional analysis of the celD beta-glucanase gene from Ruminococcus flavefaciens FD-1; Vercoe PE et al.; The nucleotide sequence of the celD gene, which encodes endoglucanase and xylanase activity, from Ruminococcus flavefaciens FD-1 was determined . The DNA sequence of celD contains an open reading frame of 1215 nucleotides that encodes a polypeptide of 405 amino acids with a molecular mass of 44,631 Da . The primary amino acid sequence of CelD was screened against the GenBank data base for similar polypeptide sequences and the analysis indicated that CelD has common features with endoglucanases from the family E cellulases . Both hydrophobic cluster and BESTFIT (Genetics Computer Group (University of Wisconsin) package) analyses confirmed this relationship . Pairwise alignments using BESTFIT revealed that CelD was most closely related to endE4 from Thermomonospora fusca over a 160 amino acid window . The histidine, aspartate, and glutamate residues identified as being essential for catalytic activity in family E cellulases are conserved in CelD . A Shine-Dalgarno-like sequence was present 5 base pairs (bp) upstream of the translation start site . Primer extension analysis indicated that different transcription initiation sites are used to initiate transcription of celD in Escherichia coli and R . flavefaciens . In the case of R . flavefaciens the transcription initiation site is at a T residue (nucleotide 273) 16 bp upstream from the translational start site . A region resembling a sigma 70-like-10 promoter sequence is present upstream from the transcription initiation site but there is no apparent-35 region . In contrast, transcription in E . coli is initiated at a C residue 258 bp upstream from the translational start site and a sequence resembling a omega 70-like-10 region is present 5 bp upstream of this residue.(ABSTRACT TRUNCATED AT 250 WORDS) Clin Exp Allergy, 1995 Jan, 25(1), 73 - 9 Blood inflammatory response to inhaled endotoxin in normal subjects; Michel O et al.; Previously we have reported that in asthmatics an inhalation of 20 micrograms lipopolysaccharide (LPS) produces a bronchial obstruction associated with an inflammatory blood response . The aim of the present study was to evaluate this response in normal subjects . Eight normal non-atopic subjects were challenged by inhalation of a solution containing 20 micrograms LPS (from Escherichia coli 026:B6) a week after bronchial challenge with control solution . The lung function response was evaluated by the changes in forced expiratory volume in one second (FEV1), in specific conductance and in airway resistance while the blood inflammatory response was evaluated by serial measures of total white blood cells (WBC) and polymorphonuclear neutrophils (PMN) count, luminol enhanced-chemiluminescence (luminol-CL, as a marker of the PMN degree of activation), C-reactive protein (CRP), haptoglobin, complement fraction C3, tumour necrosis factor-alpha (TNF-alpha) and adrenocorticotropic hormone (ACTH) . No response in lung function was observed for 6 h after the LPS inhalation . The count in WBC and PMN increased 300 (P < 0.01) and 360 (P < 0.01) min after the LPS challenge associated with an increase in the level of luminol-CL (P < 0.001) . This rise in luminol-CL level was significant at 120 min (P < 0.05) before any change in the PMN count . After 24 and 48 h the acute-phase protein CRP raised significantly (P < 0.01), the other proteins C3 and haptoglobin being unchanged . A slight increase in ACTH was observed 240 and 360 min (P < 0.05) after the LPS challenge while the TNF alpha detectable level was not modified.(ABSTRACT TRUNCATED AT 250 WORDS) Acta Anaesthesiol Scand, 1995 Jan, 39(1), 50 - 9 Inhibitory effects of diclofenac on the endotoxin shock response in relation to endothelin turnover in the pig; Weitzberg E et al.; During sepsis vasoactive arachidonic acid metabolites of the cyclo-oxygenase pathway and the endothelium-derived vasoconstrictor endothelin-1 (ET-1) are released . The effects of cyclo-oxygenase pathway inhibition by diclofenac on the endotoxin shock response and ET-1 turnover, were investigated in five groups of pigs . In the first group (n = 7; controls) endotoxin (15 micrograms.kg-1.h-1 i.v.) was infused for two hours . In a second endotoxin group (n = 7), the animals were pretreated with diclofenac (3 mg.kg-1 i.v.) . In a third group (n = 7), high-dose ET-1 was infused (20 pmol.kg-1.min-1 i.v.) and in a fourth group (n = 7), the ET-1 infusion was preceded by diclofenac . In a fifth group (n = 4), a low and intermediate dose of ET-1 (0.2 and 4 pmol.kg-1.min-1) was infused . A significant increase in ET-1-like immunoreactivity (LI) plasma levels was observed in both endotoxin groups, but in the diclofenac group the increase was comparatively delayed . Furthermore, this group showed a more stable haemodynamic course and in the biphasic increase of pulmonary vascular resistance seen in endotoxin controls, the initial peak was abolished by diclofenac . Exogenous ET-1 infusion indicated that not only locally released but possibly also circulating ET-1 could be a mediator of vascular responses to endotoxin . Indications of release from the lungs were seen during endotoxin infusion . Diclofenac had no effect on basal ET-1-LI plasma levels or on the disappearance rate from plasma of ET-1-LI and the haemodynamic changes seen on ET-1 infusion . The inhibition of cyclo-oxygenase pathway by diclofenac resulted in prevention of the initial pulmonary hypertension and a delayed increase in plasma ET-1-LI levels in porcine endotoxin shock and this latter effect is not due to an increased rate of disappearance from plasma but rather to a decreased release of ET-1. Mol Biol (Mosk), 1995 Jan-Feb, 29(1), 91 - 6 {Stability of phosphodiester tRNA bonds lacking minor nucleosides, in aminoacyl tRNA-synthetase complexes}; Afasozjev RD; The stability of phosphodiester bonds in tRNA(Trp) (E . coli, bovine) and tRNA(Phe) (yeast) synthesized in vitro was studied in the complexes with cognate aminoacyl-tRNA synthetases . In contrast to E . coli tRNA(Gln) where synthetase-induced cleavage has been reported, we have failed to observe this effect with tRNA(Trp) and tRNA(Phe) . Consequently, the weakness of sugar-phosphate backbone of tRNA-transcripts in the complexes is not a common feature for all tRNA-synthetase pairs . The cleavage mechanism in the case of tRNA(Gln) remains obscure. J Comp Pathol, 1995 Jan, 112(1), 1 - 10 Histopathological features in the small intestine of pigs infected with F4ac+ non-enterotoxigenic or enterotoxigenic strains of Escherichia coli; Vijtiuk N et al.; Four porcine strains of Escherichia coli were examined for their effects on the small intestine of 4-week-old weaned pigs infected orogastrically . The strains used experimentally were: strain 1467 (adhesin negative, non-toxigenic); strains 2407 and 1466 (adhesin positive, non-toxigenic), derived by genetical engineering from strain 1467 and containing a wild type plasmid and a recombinant plasmid, respectively, encoding the F4 antigen (adhesin); and strain M1823 (adhesin positive, toxigenic) . In addition, 2-week-old pigs that died from natural colibacillosis associated with two strains ("Ihan 1 and 2"; adhesin positive, toxigenic) were examined . Strain M1823 and the Ihan strains produced moderate and marked lesions, respectively . Strain 1467 did not cause mucosal damage or an inflammatory response . Strains 1466 and 2407 caused a mild to moderate leucocyte (mononuclear and polymorphonuclear) infiltration in the jejunal (but not ileal) lamina propria . However, unlike strain 1466, strain 2407 did not cause damage to the small intestinal mucosa and should be further studied as a potential oral vaccine strain for post-weaning E . coli diarrhoea. Immunol Lett, 1995 Jan, 44(1), 25 - 30 Molecular characterization of a human anti-HIV 1 monoclonal antibody revealed a CD26-related motif in CDR2; Chin LT et al.; Genes encoding the immunoglobulin variable regions of a human anti-HIV-1 IgG1 kappa monoclonal antibody were rescued from a hybridoma, derived from a sero-negative donor, using PCR cloning and expression in Escherichia coli . The ELISA binding results obtained from the expressed Fab fragment confirmed the anti-V3 loop specificity for HIV-1 (LAI) of the original antibody . In addition, an amino acid sequence derived from the second complementarity determining region (CDRH2) of the heavy chain was found to be very similar to the catalytic motif of CD26, a T-cell activation antigen . Furthermore, synthetic peptides containing both the catalytic domain of CD26 and CDRH2 of the antibody showed specific binding to an HIV peptide representing the V3 region in a dose-dependent manner . This suggests an involvement of CD26 as a possible coreceptor for HIV-1. Clin Diagn Lab Immunol, 1995 Jan, 2(1), 30 - 4 Antigenicity and immunogenicity of recombinant glutamate-rich protein of Plasmodium falciparum expressed in Escherichia coli; Theisen M et al.; A recombinant Plasmodium falciparum glutamate-rich protein (GLURP) was produced in Escherichia coli as a nearly full-length protein . In order to map immunodominant regions on GLURP, the nonrepetitive amino-terminal region (R0) as well as the central repeat region (R1) and the carboxy-terminal repeat region (R2) were also produced as separate products . All four purified gene products reacted specifically with serum samples from adults living in an area of Liberia where malaria is holoendemic . It appears that the human immune response against GLURP is primarily directed against the R2 region because 94% of the serum samples reacted with this region in an immunoassay . Antibody reactivity against the R0 region was also observed in 75% of the serum samples, while the R1 region showed only weak antibody-binding activity . When the nearly full-length GLURP molecule was adsorbed to Al(OH)3 it was found to be immunogenic in mice . In these experiments, the antibody response was almost exclusively directed against the R2 region . When anti-GLURP sera were obtained from rabbits immunized with the three regions, R0, R1, and R2, respectively, they recognized in immunoprecipitation experiments authentic GLURP from P . falciparum grown in vitro . These results demonstrate that GLURP produced in E . coli can induce a humoral immune response against GLURP derived from blood-stage parasites. Clin Diagn Lab Immunol, 1995 Jan, 2(1), 10 - 3 Intestinal immune response of volunteers ingesting a strain of enteroadherent (HEp-2 cell-adherent) Escherichia coli; Gomez HF et al.; Enteroadherent Escherichia coli (EAEC) strains identified by adherence to HEp-2 tissue culture cells have been incriminated epidemiologically as important etiologic agents of diarrheal disease in both adult travelers and children in developing countries . One strain, JM 221, with no recognized E . coli virulence characteristics other than adherence to HEp-2 cells, caused diarrhea in 5 of 16 volunteers ingesting it . We studied the secretory immunoglobulin A (sIgA) responses to EAEC JM 221 of five volunteers with diarrhea and five volunteers who remained healthy after challenge . sIgA was extracted from stools obtained prechallenge and 7 days postchallenge . Total sIgA was standardized for all specimens . Specific sIgA titers were determined by dot blotting with the following JM 221 antigens: water-extractable surface antigens, whole cells, lipopolysaccharides, and outer membrane proteins . All five subjects who became ill had fourfold or greater rises in titers against each of the four antigens . The five subjects who remained healthy following challenge did not exhibit significant rises in titers to any JM 221 antigens, but their mean titers were significantly higher than the mean prechallenge titers of the volunteers with diarrhea, suggesting that high intestinal sIgA titers may be protective . The significant increases in intestinal antibody against JM 221 in the subjects who became ill is further evidence of the enteropathogenicity of EAEC strains. Nat Struct Biol, 1995 Jan, 2(1), 69 - 76 The allosteric ligand site in the Vmax-type cooperative enzyme phosphoglycerate dehydrogenase; Schuller DJ et al.; The crystal structure of the phosphoglycerate dehydrogenase from Escherichia coli is unique among dehydrogenases . It consists of three clearly separate domains connected by flexible hinges . The tetramer has approximate 222 symmetry with the principal contacts between the subunits forming between either the nucleotide binding domains or the regulatory domains . Two slightly different subunit conformations are present which vary only in the orientations of the domains . There is a hinge-like arrangement near the active site cleft and the serine effector site is provided by the regulatory domain of each of two subunits . Interdomain flexibility may play a key role in both catalysis and allosteric inhibition. Nat Struct Biol, 1995 Jan, 2(1), 25 - 6 A ribosomal protein module in EF-G and DNA gyrase; Murzin AG; A novel common fold observed in the structures of elongation factor G, DNA gyrase B, and ribosomal protein S5 displays a rare topological feature suggesting the high probability of an evolutionary relationship. Plant Cell Physiol, 1995 Jan, 36(1), 29 - 36 Molecular cloning and characterization of S-adenosyl-L-methionine:scoulerine-9-O-methyltransferase from cultured cells of Coptis japonica; Takeshita N et al.; S-Adenosyl-L-methionine:scoulerine-9-O-methyltransferase (SMT) catalyzes the transfer of the S-methyl group of S-adenosyl-L-methionine to the 9-hydroxyl group of scoulerine during the biosynthesis of berberine . We have isolated functionally active cDNA clones (pCJSMTs) from a cDNA library prepared from cultured cells of Coptis japonica . The longest cDNA insert (pCJSMT1) had an open reading frame that encoded 351 amino acids, but the calculated molecular mass (38,364 Da) of the deduced product was slightly lower than the experimentally determined molecular mass of purified SMT . Rapid amplification of the 5' end of the cDNA indicated that the full-length cDNA of SMT consisted of 1,458 nucleotides that encoded 381 amino acids . When the full-length cDNA was expressed in E . coli, the molecular mass of the expressed SMT was greater than that of native SMT in Coptis cells . This result suggests that SMT might be produced in a pre-mature form and processed post-translationally . SMT was also found to exhibit sequence homology to other O-methyltransferases from plants and N-terminal region of the SMT polypeptide appeared to be necessary for enzymatic activity. Radiats Biol Radioecol, 1995 Jan-Feb, 35(1), 47 - 52 {Cellular effects of microwaves of thermal intensity}; Morozov II et al.; The thermal and specific effects of cm range microwaves on cell systems was investigated . It was shown that cell damage (killing and permeability disturbance) in Escherichia coli B/r, Escherichia coli Bs-1, Saccharomyces cerevisiae and ACE were induced by microwave heating more effectively than by equivalent thermal heat. FEMS Immunol Med Microbiol, 1995 Jan, 10(2), 125 - 31 Serological variety of flagellar antigen H1 in natural Escherichia coli population; Ratiner Y et al.; Variation of the Escherichia coli flagellar antigen H1 was studied among 120 human isolates belonging to more than 25 O:K serovars . Factor-specific antisera were prepared and shown to be useful in the identification of serological subtypes of H1 . The three subtypes found were defined as H1abc, H1acd and H1abe . The H1abc corresponded to the standard flagellar antigen H1 present in 84% of all strains . It was found in all O groups except O15, O17 and O83 . Eight O15 and two O17 strains studied were of subtype H1abe, while the one O83 strain studied was H1acd . Both subtypes H1abc and H1acd were found among strains within O6:K5, O6:K13 or O-non-typeable:K5 serovars. Vet Microbiol, 1995 Jan, 43(1), 41 - 52 Construction of recombinant Shiga-like toxin-IIv (SLT-IIv) and its use in monitoring the SLT-IIv antibody status of pigs; Franke S et al.; We constructed and purified recombinant B-subunits of the SLT-IIv as well as tested their usefulness in an immunoblot assay . The slt-IIvB gene amplified by PCR was ligated into the fusion vector pGEX-2T, and expressed in E . coli K 12 laboratory strains . Deletion of the signal sequence was necessary for optimal expression . High quantities of the fusion protein could be purified by affinity chromatography and subsequently used as antigen for immunoblot analysis with serum samples from diseased pigs and healthy controls . IgG antibodies against SLT-IIv were detected in the sera of 11 of 52 (21.15%) healthy pigs . By contrast, only in 1 of 28 (3.57%) serum samples of pigs with edema disease caused by SLT-IIv-producing E . coli we could demonstrate SLT-IIv-specific antibodies . During an outbreak of edema disease, sera from 10 pigs were taken at 4, 20, and 40 days after disease onset to investigate the immune response elicited by SLT-IIv . Immunoblot analysis with the recombinant SLT-IIv fusion protein revealed that the number of IgG-positive serum samples increased within this period of 40 days from one on day 4, to seven on day 20, to ten on day 40; the number of IgM-positive samples also increased from one after 4 days to eight after 20 days . Forty days after disease onset, IgM reactivity was no longer detectable . Since all animals seroconverted in the follow-up sera, the antigenicity of SLT-IIv during infection of pigs seems to differ from that of SLT-II in human hemolytic uremic syndrome where only a minority of patients are known to mount an immune response . The recombinant SLT-IIvB described here may be a possible candidate for vaccination trials. Vet Microbiol, 1995 Jan, 43(1), 13 - 9 Identification of Escherichia coli strains from cows with clinical mastitis by serotyping and DNA polymorphism patterns with REP and ERIC primers; Lipman LJ et al.; A number of Escherichia coli strains was isolated during a study of clinical mastitis on seven farms in the Netherlands . From these E . coli strains, 30 were characterised with regard to their serotype and their DNA polymorphism pattern with REP and ERIC primers . Special attention was given to recurrent E . coli mastitis in cows . The combination of serotype and DNA pattern observed, was used to study the epidemiology of clinical E . coli mastitis . The results demonstrated that the PCR reaction with the ERIC primers can be used for differentiation of E . coli strains . The DNA polymorphism patterns showed that E . coli strains isolated from cases of clinical mastitis have a great variability in genotype . More 3 than one case of clinical mastitis associated with E . coli during the same lactation period occurred infrequently . However when it took place, E . coli strains isolated from the separate episodes of inflammation, were in most instances of the same serotype and had the same DNA amplification pattern. J Mol Evol, 1995 Jan, 40(1), 86 - 93 Genetics of selection-induced mutations: I . uvrA, uvrB, uvrC, and uvrD are selection-induced specific mutator loci; Hall BG; Selection-induced mutations, sometimes called "directed," "adaptive," or "Cairnsian" mutations, are spontaneous mutations that occur as specific responses to environmental challenges, usually during periods of prolonged stress, and that occur more often when they are selectively advantageous than when they are selectively neutral . In this study I show that lesions in uvrA, uvrB, uvrC, or uvrD increase the mutation rate from trpA46 to trpA+ by 10(2)- to 10(4)-fold during tryptophan starvation, but those same lesions do not affect random mutation rates in growing cells when tryptophan is present . The increased selection-induced mutation rates remain specific to the gene that is under selection in that no increase in the mutation rate from trpA46 to trpA+ is detected during proline starvation . Evidence is presented showing that proline starvation produces a state of cellular stress which results in a burst of mutations from trpA46 to trpA+ when proline-starved cells are plated onto medium lacking tryptophan but containing proline . These results are consistent with the hypermutable state model for selection-induced mutagenesis. J Med Virol, 1995 Jan, 45(1), 99 - 105 Expression of the Epstein-Barr virus DNA polymerase in Escherichia coli for use as antigen for the diagnosis of nasopharyngeal carcinoma; Lin LS et al.; Epstein-Barr virus (EBV) encoded DNA polymerase (POL) was cloned and over-expressed in Escherichia coli . Western blot analysis confirmed the presence of antibody to this POL protein in sera from nasopharyngeal carcinoma (NPC) patients . By Western blot analysis, moderate to high concentration of IgG POL-specific antibodies were present in 43 of 48 NPC sera and only 4 of 48 healthy, seropositive controls . The POL-specific IgG antibodies appear as early as stage I of NPC, suggesting that the recombinant POL protein can be a useful diagnostic marker for early diagnosis of the disease . It was also found that human sera containing high titer of cytomegalovirus (CMV) antibodies or herpes simplex virus type 1 (HSV-1) antibodies did not cross-react with the recombinant EBV POL, despite the homology shared by DNA polymerase proteins of these viruses. Br J Pharmacol, 1995 Jan, 114(1), 8 - 12 Endotoxin inhibition of distension-stimulated gastric acid secretion in rat: mediation by NO in the central nervous system; Barrachina MD et al.; 1 . The involvement of nitric oxide in the acute inhibitory effects of low doses of endotoxin, following intracerebroventricular (i.c.v.) or intravenous (i.v.) administration, on gastric acid secretion stimulated by distension or i.v . infusion of pentagastrin has been investigated in the continuously perfused stomach of the anaesthetized rat . 2 . The i.c.v . administration of E . coli endotoxin (800 ng kg-1) abolished the acid secretory response induced by gastric distension (20 cm water intragastric pressure) within 30 min of administration . 3 . By contrast, submaximal rates of acid secretion induced by i.v . infusion of pentagastrin (8 micrograms kg-1 h-1) were not inhibited by i.c.v . administration of endotoxin (800 ng kg-1) . 4 . Prior i.c.v . administration of the NO synthase inhibitor, NG-nitro-L-arginine methyl ester (L-NAME, 800 micrograms kg-1) restored the acid secretory responses to distension in rats treated with endotoxin (i.c.v.) . 5 . Likewise, i.v . administration of endotoxin (5 micrograms kg-1) abolished the acid secretory response induced by gastric distension within 30 min of administration . Prior i.c.v . injection of L-NAME (800 micrograms kg-1) or its i.v . administration (10 mg kg-1) restored acid secretory responses in rats receiving i.v . endotoxin . 6 . The reversal by L-NAME (i.v.) of the acid inhibitory effects of endotoxin (i.v.) was prevented by L-arginine (12 mg kg-1, i.c.v . or 100 mg kg-1, i.v.), but not by its enantiomer D-arginine . 7 . The present results imply the existence of an acute response to endotoxin involving NO synthesis in the brain.(ABSTRACT TRUNCATED AT 250 WORDS) Br J Pharmacol, 1995 Jan, 114(1), 13 - 8 Significance of nitric oxide in the stimulation of intestinal fluid absorption in the rat jejunum in vivo; Schirgi-Degen A et al.; 1 . The effects of inhibiting nitric oxide (NO)-synthase on fluid transport, mucosal cyclic GMP and cyclic AMP levels and intraluminal prostaglandin E2 (PGE2)-release were studied in a model of ligated jejunal loops of anaesthetized rats in vivo . Experiments were performed under basal conditions as well as under conditions, when net fluid secretion was induced by Escherichia coli heat stable enterotoxin a (E . coli STa) or PGE2 . 2 . Intravenous infusion of the NO-synthase inhibitor N omega-nitro-L-arginine methyl ester (L-NAME, 0.25-50 mg kg-1, 45 min) dose-dependently reversed net fluid absorption to net secretion, whereas infusion of D-NAME, the inactive enantiomer of L-NAME, in corresponding doses did not influence net fluid transport . N omega-nitro-L-arginine (L-NOARG, 25 mg kg-1), another NO-synthase inhibitor, also elicited net secretion of fluid . 3 . L-NAME (25 mg kg-1)-induced net fluid secretion was reversed to net absorption by infusion of L-arginine (400 mg kg-1) or sodium nitroprusside (1 mg kg-1) and s.c . administration of indomethacin (10 mg kg-1) . Hexamethonium (1 mg kg-1, s.c.), a ganglionic blocker and granisetron (100 micrograms kg-1, s.c.), a 5-HT3-receptor antagonist, did not influence L-NAME-induced net secretion . 4 . Net fluid secretion induced by intraluminal instillation of E . coli STa (10 units ml-1) was enhanced by infusion of L-NAME (25 mg kg-1) and was inhibited by infusion of L-arginine (400 mg kg-1) and sodium nitroprusside (1 mg kg-1).(ABSTRACT TRUNCATED AT 250 WORDS) Biophys J, 1995 Jan, 68(1), 104 - 10 The F0 complex of the ATP synthase of Escherichia coli contains a proton pathway with large proton polarizability caused by collective proton fluctuation; Bartl F et al.; The F0 complex of the Escherichia coli ATP synthase embedded into cardiolipin liposomes was studied by FT-IR spectroscopy . For comparison, respective studies were performed with dried F0 liposomes and with F0 liposomes treated with N,N'-dicyclohexyl-carbodiimide (DCCD), which binds to Asp-61 of subunit c . Furthermore, the effect of H2O-->D2O exchange on the infrared spectrum was investigated . With F0 liposomes an infrared continuum is observed beginning at about 3000 cm-1 and extending toward smaller wavenumbers . In the DCCD-treated sample, this continuum is no longer observed . It vanishes also with drying of the liposomes . After H2O-->D2O exchange, this infrared continuum begins at about 2350 cm-1 and is less intense . All of these results demonstrate that a proton pathway in native F0 is present, in which the protons are shifted in a hydrogen-bonded chain with large proton polarizability due to collective proton tunneling . With the D2O-hydrated system, deuteron polarizability due to collective deuteron motion is observed, but the polarizability due to collective deuteron motion is smaller . Such pathways are very efficient, because they conduct protons or deuterons within picoseconds . These pathways lose their polarizability if the F0 complex is blocked by DCCD or if the liposomes are dried . On the basis of our results on the proton polarizability of hydrogen bonds and hydrogen-bonded systems and on the basis of structural data from the literature, the nature of the proton pathway of the F0 complex of E . coli is discussed. Bioorg Khim, 1995 Jan, 21(1), 9 - 16 {Expression vectors enabling synthesis of recombinant proteins as hybrids with metal-binding peptides}; Efimov VA et al.; Plasmid vectors providing a high level expression in Escherichia coli cells of genes for heterologous polypeptides and proteins fused to peptides containing homohistidine clusters have been constructed . The obtained vectors have been used to achieve expression of genes for streptavidin, proinsulin and calcitonin . The hybrid proteins were isolated by affinity chromatography on Ni-agarose columns. Arch Virol, 1995, 140(2), 245 - 58 A type-specific serological test to distinguish antibodies to equine herpesviruses 4 and 1; Crabb BS et al.; We describe a type-specific ELISA, which distinguishes antibody to equine herpesvirus 4 (EHV4; equine rhinopneumonitis) and EHV1 (equine abortion virus) thereby identifying horses that have been infected with either or both of these antigenically related viruses . The antigens used are parts of the EHV4 and EHV1 glycoprotein G (gG) homologues expressed in E . coli as fusion proteins {Crabb and Studdert, 1993: J Virol 67: 6332-6338) . The expressed proteins comprise corresponding regions of the gG molecules that are highly divergent and encompass strong, typespecific epitopes . Plasma samples from 97 Thoroughbred and 174 Standardbred horses were tested, all of which were unvaccinated . All horses were strongly EHV4 ELISA positive while 30% were EHV1 ELISA positive . The type-specificity of the EHV1 gG antigen was tested in cross-absorption experiments and it was found that 96% (66 of 69) of EHV1 ELISA positive horses were true EHV1 antibody positives . It was also shown that 100% (26 of 26) horses known to have been exposed to EHV1, either by infection or immunisation with EHV1, had significant levels of antibody against the EHV1 gG antigen (i.e., all horses recognised the EHV1 epitope(s) contained within this molecule) . Maintenance of EHV1 gG antibody was examined by testing sera obtained from mares four years after confirmed EHV1 abortion . Seven out of 10 of these mares remained EHV1 ELISA positive . In summary, the ELISA is highly specific and is sufficiently sensitive to detect all horses previously infected with EHV4 and most previously infected with EHV1. Arch Microbiol, 1995 Jan, 163(1), 76 - 8 Betaine modulates intracellular thiol and potassium levels in Escherichia coli in medium with high osmolarity and alkaline pH; Smirnova G et al.; Glycine betaine stimulates the growth rate of various bacteria in high osmolarity medium . In our studies, glycine betaine stimulated the growth rate of Escherichia coli K12 in minimal medium with normal osmolarity at alkaline pH (pH 8.2) . Betaine also caused a reduction in the intracellular pools of K+ and low molecular weight thiols in E . coli growing both in medium with high osmolarity and at alkaline pH . These effects of betaine were absent at pH 7.0 . In cells growing in high osmolarity medium, 10 mM sodium acetate or 10 microM N-ethylmaleimide reduced expression of the osmosensitive gene proU to the same extent as treatment with betaine; however, under these conditions, sodium acetate and N-ethylmaleimide did not stimulate the growth of E . coli . It is proposed that low molecular weight thiols and intracellular pH may participate in the response of E . coli to betaine. Genetics, 1995 Jan, 139(1), 19 - 33 A molecular investigation of genotype by environment interactions; Dean AM; The fitnesses conferred by seven lactose operons, which had been transduced into a common genetic background from natural isolates of Escherichia coli, were determined during competition for growth rate-limiting quantities of galactosyl-glycerol, a naturally occurring galactoside . The fitnesses of these same operons have been previously determined on lactose and three artificial galactosides, lactulose, methyl-galactoside and galactosyl-arabinose . Analysis suggests that although marked genotype by environment interactions occur, changes in the fitness rankings are rare . The relative activities of the beta-galactosidases and the permeases were determined on galactosyl-glycerol, lactose, lactulose and methyl-galactoside . Both enzymes display considerable kinetic variation . The beta-galactosidase alleles provide no evidence for genotype by environment interactions at the level of enzyme activity . The permease alleles display genotype by environment interactions with a few causing changes in activity rankings . The contributions to fitness made by the permeases and the beta-galactosidases were partitioned using metabolic control analysis . Most of the genotype by environment interaction at the level of fitness is generated by changes in the distribution of control among steps in the pathway, particularly at the permease where large control coefficients ensure that its kinetic variation has marked fitness effects . Indeed, changes in activity rankings at the permease account for the few changes in fitness rankings . In contrast, the control coefficients of the beta-galactosidase are sufficiently small that its kinetic variation is in, or close to, the neutral limit . The selection coefficients are larger on the artificial galactosides because the control coefficients of the permease and beta-galactosidase are larger . The flux summation theorem requires that control coefficients associated with other steps in the pathway must be reduced, implying that the selection at these steps will be less intense on the artificial galactosides . This suggests that selection intensities need not be greater in novel environments. Clin Sci (Lond), 1995 Jan, 88(1), 59 - 66 Comparison of the modulatory influence of maize and olive oils and butter on metabolic responses to endotoxin in rats; Besler HT et al.; 1 . n-3 polyunsaturated fatty acids decrease responses to cytokines and inflammatory agents . The present study examines how different intakes of n-6 and n-9 fatty acids influence the metabolic response to endotoxin in Wistar rats . 2 . Weanling male rats were, for 4 weeks, fed diets containing 50, 100 or 200 g/kg fat in the form of maize oil (rich in linoleic acid), butter (poor in linoleic acid, rich in oleic acid) or olive oil (adequate in linoleic acid, rich in oleic acid) or standard laboratory chow . All butter and olive oil diets included 10 g/kg maize oil, in total fat, to avoid essential fatty acid deficiency . 3 . Rats subsequently received 800 micrograms/kg Escherichia coli endotoxin or sterile saline subcutaneously . Twenty-four hours after injection, the rate of tissue protein synthesis was measured in liver, lung, kidney, tibialis muscle and spleen by the 'flooding dose' method . Protein and zinc concentrations were assayed in all tissues and serum albumin and caeruloplasmin measured . 4 . In animals fed chow, protein synthetic rate increased by 18%, 29% and 27% in liver, lung and kidney respectively . Tissue zinc concentrations increased by 33% in kidney, and tissue protein increased by 17%, 23% and 17% in liver, lung and kidney respectively . Serum caeruloplasmin increased by 60% and albumin concentration fell by 14% . 5 . In animals consuming the 50 g/kg maize oil diet, protein synthetic rate increased by 56%, 36% and 34% in liver, lung and kidney respectively.(ABSTRACT TRUNCATED AT 250 WORDS) Can J Vet Res, 1995 Jan, 59(1), 34 - 9 Alterations in clinical, hematological and metabolic variables in bovine neonatal endotoxemia; Gerros TC et al.; Endotoxemia is an important cause of morbidity and mortality in the neonate . Although many models are used to study the problem, none completely simulates the natural disease . To more clearly define a bovine neonatal endotoxemia model we studied the effects of dose of endotoxin on clinical, hematological and biochemical variables . Thirty-four neonatal calves were administered Escherichia coli endotoxin (LPS) at 0 (0.9% saline solution), 0.2, 2.0 or 20 micrograms/kg, by either IV bolus or infusion over 50 minutes . Variables monitored included mean arterial blood pressure (MAP), leukocyte (WBC) count, plasma glucose and lactate concentrations and clinical status . All LPS-treated calves displayed similar clinical signs within one hour . Dose-dependent differences in response to LPS among groups became evident over time . Substantial dose-dependent changes in attitude, appetite, mucous membrane character, capillary refill time, MAP, plasma glucose and lactate concentrations, and WBC count were noted in LPS-treated calves . Higher doses of LPS induced a more prolonged clinical response and significantly (p < 0.05) greater hypotension, lacticemia and hypoglycemia . While dose altered the response to endotoxin, the method of administration had no overall effect on the variables measured. Chem Res Toxicol, 1995 Jan-Feb, 8(1), 157 - 63 Miscoding by the exocyclic and related DNA adducts 3,N4-etheno-2'-deoxycytidine, 3,N4-ethano-2'-deoxycytidine, and 3-(2-hydroxyethyl)-2'-deoxyuridine; Zhang W et al.; 3,N4-Etheno-2'-deoxycytidine, 3-(hydroxyethyl)-2'-deoxyuridine, and 3,N4-ethano-2'-deoxy-cytidine are found in DNA of cells treated with either vinyl chloride or 1,3-bis(2-chloroethyl)-nitrosourea . These exocyclic and related DNA adducts were incorporated into oligodeoxynucleotides, which were then used as templates for primer extension in reactions catalyzed by the Klenow fragment of Escherichia coli DNA polymerase I . The miscoding potential of each lesion was determined quantitatively . DNA primers were readily extended on an epsilon dC-modified template; dAMP and dTMP were incorporated opposite the lesion . With high concentrations of DNA polymerase, small amounts of fully extended reaction products containing dAMP and dGMP or one-base and two-base deletions opposite ethano-dC were formed . Primer extension was blocked partially on templates containing 3-(hydroxyethyl)-dU; dAMP and smaller amounts of dTMP and dCMP were incorporated . The frequencies of nucleotide insertion opposite each of the three lesions and the frequencies of chain extension from the 3'-primer terminus, determined by kinetic analysis, were consistent with results of experiments utilizing polyacrylamide gel electrophoresis . We conclude from these studies that epsilon dC, ethano-dC, and 3-(hydroxyethyl)-U are potentially miscoding lesions; only epsilon dC facilitates translesional synthesis. Vet Surg, 1995 Jan-Feb, 24(1), 78 - 85 Hemodynamic responses of horses to anesthesia and surgery, before and after administration of a low dose of endotoxin; Wagner AE et al.; Seven horses, which were part of an investigation of the effect of endotoxin administration on vascular reactivity, were anesthetized on two separate occasions for surgical excision of 4-cm sections of palmar digital artery and vein . On the first occasion, the horses were given an infusion of 1 L 0.9% NaCl solution intravenously (i.v.) just before induction of anesthesia (control); on the second occasion, the horses received an infusion of 1 L 0.9% NaCl containing Escherichia coli endotoxin, 0.1 microgram/kg (endotoxin) . On both occasions, anesthesia was induced with xylazine, guaifenesin, and ketamine, and maintained with halothane in oxygen . Hemodynamic measurements were made with the horses under anesthesia immediately before beginning surgery (period 1), during skin incision (period 2), during dissection and excision of the vessels (period 3), during skin suturing (period 4), and after completion of surgery during bandaging (period 5) . Hemoglobin concentration and mixed venous oxygen content were greater at all periods in horses that received endotoxin . Otherwise, there were no significant differences in hemodynamic parameters between control horses and horses administered endotoxin before beginning surgery (period 1) . During surgery and bandaging, horses administered endotoxin had significantly higher heart rate (periods 3, 4, and 5), cardiac index (periods 3, 4, and 5), and oxygen delivery (periods 2, 3, 4, and 5) than did control horses, and mean arterial blood pressure (period 2) and systemic vascular resistance (periods 2, 3, 4, and 5) were less than in control horses . Compared with period 1, surgical stimulation in control horses was associated with increased mean arterial blood pressure and systemic vascular resistance (periods 2, 3, 4, and 5), but cardiac index and oxygen delivery were decreased (periods 3, 4, and 5).(ABSTRACT TRUNCATED AT 250 WORDS) Pediatr Res, 1995 Jan, 37(1), 75 - 80 Disparate in vitro inhibition of adhesion of enteropathogenic Escherichia coli RDEC-1 by mucins isolated from various regions of the intestinal tract; Mack DR et al.; Escherichia coli RDEC-1 (serotype O15:H-) is a rabbit enteropathogen in which in vivo enteroadherence is both site specific and age related . To determine whether these differences could be related to mucins, we evaluated inhibition of binding of AF/R1 piliated RDEC-1 by mucins isolated from various segments of intestine of rabbits at different ages . Mucin was purified from intestinal crude mucus by cesium chloride serial ultracentrifugation . RDEC-1 was grown to promote the expression of hydrophobic mannose-resistant AF/R1 pili . Quantitation of in vitro bacterial binding was determined using a crystal violet colorimetric assay . In postweanling rabbits, inhibition of RDEC-1 binding by purified mucin derived from ileal segments (45.1 +/- 2.6%, mean +/- SEM) and proximal colonic segments (46.0 +/- 5.5%) was less than purified mucins derived from jejunal segments (70.0 +/- 2.0%) and distal colonic segments (71.0 +/- 3.7%, p < 0.05) of the intestinal tract . In all age groups, mucins derived from jejunal segments inhibited RDEC-1 binding to a greater level than mucins derived from ileal segments . In addition, inhibition of binding by mucin derived from proximal small intestine of postweanling rabbits (70.0 +/- 2.0%) was greater than that of weanling rabbits (55.2 +/- 3.5%, p < 0.05) with suckling rabbit inhibition (62.1 +/- 3.5%) between these two levels . We conclude that mucin inhibition of RDEC-1 adhesion is both age and region related and therefore may contribute to both age-related and site localization of bacterial infections of the intestinal tract. Plant Cell, 1995 Jan, 7(1), 105 - 15 Reconstitution of Arabidopsis casein kinase II from recombinant subunits and phosphorylation of transcription factor GBF1; Klimczak LJ et al.; In contrast to the well-defined tetrameric structure of animal and yeast casein kinase II (CKII), plant CKII is found in two forms: a monomeric form and an oligomeric form whose subunit composition is not well defined . The Arabidopsis homologs of the catalytic subunit alpha (CKA1) and the regulatory subunit beta (CKB1) of CKII were expressed in Escherichia coli to examine their ability to form complexes, the effect of CKB1 on the catalytic activity, and the relationship of the recombinant enzymes to those isolated from plant material . Both subunits were found mainly in the inclusion body fraction in the bacterial expression strains, and they were solubilized and renatured with the recovery of catalytic (CKA1) and stimulatory (CKB1) activities . The combination of purified CKA1 and CKB1 proteins resulted in up to 100-fold stimulation of casein kinase activity compared with the CKA1 activity alone, showing that CKB1 has biochemical properties similar to those of the beta subunit from animals . CKA1 and CKB1 spontaneously assembled into a tetrameric complex, CKA1(2)CKB12, which had properties very similar to those of the oligomeric CKII form isolated from broccoli . However, the properties of the catalytic subunit CKA1 alone differed from those of the broccoli monomeric form of CKII-like activity . Phosphorylation of transcription factor GBF1 with the reconstituted CKA1(2)CKB1(2) enzyme resulted in stimulation of its DNA binding activity and retardation of the protein-DNA complex; these results are identical to those obtained previously with isolated nuclear CKII from broccoli. Anticancer Drug Des, 1995 Jan, 10(1), 75 - 95 A homology model of the three-dimensional structure of human O6-alkylguanine-DNA alkyltransferase based on the crystal structure of the C-terminal domain of the Ada protein from Escherichia coli; Wibley JE et al.; O6-Alkylguanine-DNA alkyltransferase (EC 2.1.1.63) repairs O6-alkylguanine lesions in DNA . A homology model of the human protein (hAT) was built, based on the crystal structure of the C-terminal domain of the Ada protein, which carries out a similar repair in Escherichia coli . Sequence alignments of known O6-alkylguanine-DNA alkyltransferases were used to aid the model building using QUANTA and CHARMm software . Despite low homology in the N-terminal half (hAT residues 1-85), a well-defined topology over this region in Ada permitted successful modelling . The C-terminal half of hAT (residues 92-207) was modelled almost entirely by residue-for-residue superposition onto the Ada structure up to residue hAT175 . The model was solvated to a residue radius of 8.0 A {corrected} and then minimized using CHARMm . This structural model was used to rationalize findings from site-directed mutagenesis experiments on hAT, to make further predictions on the relationship between structure and function for the alkyltransferase family of proteins, and to explain the specificity towards known small-molecule inhibitors of the protein. Bioelectromagnetics, 1995, 16(3), 188 - 96 Effect of millimeter waves on survival of UVC-exposed Escherichia coli; Rojavin MA et al.; Bacterial cells of the strain Escherichia coli K12 were exposed to millimeter electromagnetic waves (mm waves) with and without additional exposure to ultraviolet light lambda = 254 nm (UVC) . The mm waves were produced by a medical microwave generator emitting a 4-GHz-wide band around a 61 GHz center frequency and delivering an irradiation of 1 mW/cm2 and a standard absorption rate (SAR) of 84 W/kg to the bacteria . Exposure to the mm waves alone for up to 39 minutes did not change the survival rate of bacteria . Exposure to mm waves followed by UVC irradiation also did not alter the number of surviving E . coli cells in comparison to UVC-treated controls . When mm waves were applied after the UVC exposure, a dose-dependent increase of up to 30% in the survival of E . coli was observed compared to UVC + sham-irradiated bacteria . Because sham controls and experimental samples were maintained under the same thermal conditions, the effect is not likely to be due to heating, although the possibility of nonuniform distribution of microwave heating in different layers of irradiated bacterial suspension cannot be ruled out . The mechanism for this effect appears to involve certain DNA repair systems that act as cellular targets for mm waves. Przegl Epidemiol, 1995, 49(1-2), 73 - 5 {A problem of hospital infection: conclusions from a practice in n German hospitals}; Michowicz A et al.; The aim of this publication is to focus on a worldwide problem of hospital infections, that exists not only in Poland but also in other countries around the world . Authors tried to mention not only an epidemiologic and a financial aspect of hospital infections, but also the possible ways of fighting them . In Germany, problems of a sterilization, disinfection and hospital hygiene are of the same importance as are the diagnostics and medical treatment . About 4.5-12 percent of patients in Germany hospitals are treated because of hospital infections (like in the USA) . Authors hope that newly established Polish Society of Hospital Infection will help not only in the exchange of experiences but will also coordinate the measures aimed at the improvement of hygiene status of Polish hospitals. Chemotherapy, 1995, 41 Suppl 1, 33 - 9 Pathogenesis of traveler's diarrhea; DuPont HL; Diarrhea is the most common medical complication among persons venturing into tropical and semitropical regions of the developing world from industrialized regions . The illness is characteristically caused by one of a variety of bacterial agents, of which enterotoxigenic Escherichia coli is the most important . Intestinal electrolyte fluid movement explains the pathophysiology of most cases while in certain situations osmotic diarrhea or altered intestinal motility may lead to passage of unformed stools . In 1-2% of traveler, diarrhea lasts more than 1 month . Most of the patients will have diarrhea that is eventually self-limiting . The cause and mechanisms of diarrhea in these settings are largely unknown although a focal intestinal inflammation lesion may be found. JPEN J Parenter Enteral Nutr, 1995 Jan-Feb, 19(1), 33 - 40 Effects of chemically defined structured lipid emulsions on reticuloendothelial system function and morphology of liver and lung in a continuous low-dose endotoxin rat model; Pscheidl E et al.; BACKGROUND: This study was undertaken to determine the effect of chemically defined structured lipids on nonspecific host defense and on histologic patterns of liver and lungs compared with a physical mixture of long-chain triglycerides and medium-chain triglycerides in a continuous low-dose endotoxin rat model . METHODS: Forty male Sprague-Dawley rats, divided into four feeding groups (structured lipids, structured lipids+endotoxin, physical mixture, physical mixture+endotoxin), received total parenteral nutrition for 48 hours . During the first part of the study, 24 animals were given an injection of live Escherichia coli labeled with radioactive iron (59Fe) to investigate the function of the reticuloendothelial system . During the second part of the study, the liver and lungs of 16 animals were histologically examined using light and electron microscopy . RESULTS: Despite the similar values in the control groups, the animals receiving structured lipids+endotoxin sequestered a significantly greater percentage of bacteria in the liver and spleen (p < or = .01) and a significantly lesser percentage in the lung (p < or = .05) compared with the animals given physical mixture+endotoxin as part of their diet . Moreover, rats in the physical mixture+endotoxin group showed a microscopically evaluated higher fatty infiltration in the liver than did the structured lipids+endotoxin group . CONCLUSIONS: The results of this study indicate that chemically defined structured lipids reduce fatty infiltration of the liver compared with a physical mixture of the same compounds in an animal model of metabolic stress . They were accompanied by a better function of the reticuloendothelial system and a lesser bacterial sequestration in the lungs. Acta Biochim Pol, 1995, 42(1), 109 - 14 Synthesis, cloning and expression in Escherichia coli of the gene coding for the trypsin inhibitor from Cucurbita pepo; Rempooa B et al.; A chemically synthesized gene coding for the serine proteinase inhibitor CPTI II was cloned in E . coli and its expression was investigated in cytoplasmic and secretion systems . Under all conditions investigated the biologically active form of the inhibitor was found only in the latter system, although the yield was rather low. Scand J Clin Lab Invest Suppl, 1995, 220, 9 - 25 Molecular diagnosis and characterization of medium-chain acyl-CoA dehydrogenase deficiency; Andresen BS et al.; Medium-chain acyl-CoA dehydrogenase (MCAD) deficiency is the most common defect in mitochondrial beta-oxidation in humans . It is an autosomal recessive disorder which usually presents in infancy . The disease manifests itself in periods of metabolic stress to the beta-oxidation system and may be fatal . Four years ago we identified a prevalent disease-causing mutation (G985) which causes an amino acid change (K304E) in the mature MCAD protein . Using a Polymerase Chain Reaction (PCR) based assay for this mutation we have demonstrated: 1 . that the G985 mutation is present in 90% of the disease alleles from patients from all over the world; 2 . that the allele frequency of G985 in the general population from most European countries is very high (the carrier frequency ranges from 1/68 to 1/333); 3 . that MCAD deficiency is not, as has previously been suggested, related to Sudden Infant Death Syndrome (SIDS) . Moreover, investigation by Restriction Fragment Length Polymorphism (RFLP) analysis of several families with diagnosed MCAD deficiency revealed that the G985 mutation is only present in chromosomes of a particular RFLP haplotype, suggesting a common chromosomal background for this mutation . The other mutations in the MCAD gene are distributed to all known MCAD RFLP haplotypes . Because 80% of the patients are homozygous for the G985 mutation, DNA based diagnosis of most patients is now fast and easy . In order to make DNA based diagnosis possible for the remaining 20% of patients we have set up PCR/solid-phase based semi-automated sequencing of all 12 exons of the MCAD gene . We have so far identified the mutation in 33 of 45 non-G985 homozygous families with verified MCAD deficiency, thereby bringing the number of known mutations in the MCAD gene up to 26 . In order to investigate in detail the molecular defects of the mutant MCAD proteins we overexpressed them in COS-7 and in an E . coli based expression system with and without co-overexpression of the molecular chaperones GroES and GroEL . The expression studies revealed that the primary effect of all the identified mutations is on formation of correct enzyme structure, and does not directly affect the catalytically active regions of the enzyme . We find that our diagnostic set up, consisting of an initial testing by the G985 assay, followed by semi-automated sequencing of DNA from those patients who were indicated to be compound heterozygous, is an important improvement to the diagnosis of MCAD deficiency.(ABSTRACT TRUNCATED AT 400 WORDS) Microbiol Immunol, 1995, 39(4), 243 - 7 Analysis of the NH2-terminal 83rd amino acid of Escherichia coli GyrA in quinolone-resistance; Yonezawa M et al.; Artificial mutations of Gyrase A protein (GyrA) in Escherichia coli by site-directed mutagenesis were generated to analyze quinolone-resistant mechanisms . By genetic analysis of gyrA genes in a gyrA temperature sensitive (Ts) background, exchange of Ser at the NH2-terminal 83rd position of GyrA to Trp, Leu, Phe, Tyr, Ala, Val, and Ile caused bacterial resistance to the quinolones, while exchange to Gly, Asn, Lys, Arg and Asp did not confer resistance . These results indicate that it is the most important for the 83rd amino acid residue to be hydrophobic in expressing the phenotype of resistance to the quinolones . These findings also suggest that the hydroxyl group of Ser would not play a major role in the quinolone-gyrase interaction and Ser83 would not interact directly with other amino acid residues. Methods Enzymol, 1995, 250, 630 - 40 Glycosylphosphatidylinositol-phospholipase D: a tool for glycosylphosphatidylinositol structural analysis; Deeg MA et al.; Cleavage by the GPI-PLD provides definitive evidence of a minimal GPI structure: glucosamine-phosphatidylinositol . Unlike the case for PI-PLC, cleavage by the GPI-PLD is unaffected by acylation of the inositol ring . Thus the GPI-PLD provides an excellent simple enzymatic tool for analyzing the basic core structure of GPI anchors. J Interferon Cytokine Res, 1995 Jan, 15(1), 31 - 7 The use of Zwittergent 3-14 in the purification of recombinant human interferon-beta Ser17 (Betaseron); Russell-Harde D et al.; A new method for purifying human interferon-beta SER17 from E . coli-derived inclusion bodies has been developed . This procedure eliminates the need for strong denaturants, such as sodium dodecyl sulfate or chaotropes . The procedure makes use of a nondenaturing detergent and a brief incubation at pH 12 to solubilize interferon-beta Ser17 from inclusion bodies . The detergent used was Zwittergent 3-14 (nonionic and pH-insensitive), which is included in the class of sulfobetaines (RN+ (CH3)2(CH2)xSO3-) . Zwittergent 3-14 was used in combination with urea to produce a urea/Zwittergent 3-14 washed inclusion body preparation enriched in human interferon-beta Ser17 (Betaseron) . Solubilization of inclusion bodies was accomplished by employing a brief (1 minute) shift to pH 12 in the presence of 2.5% Zwittergent 3-14 followed by rapid adjustment to pH 8.0 . Solubilization was complete, and the solution could be rapidly adjusted to pH 8 without any observable precipitation of protein . The resultant supernatant could be successfully subjected to a number of chromatographic and analytic procedures, many of which are not compatible with strong anionic detergents, such as SDS . Betaseron was purified from Zwittergent 3-14 solubilized inclusion body lysates using both ion-exchange and size-exclusion chromatography . Purified Betaseron retained bioactivity and could be refolded by simple dialysis against a nonreducing buffer . This method represents a novel procedure for purifying Betaseron from inclusion bodies using a nondenaturing detergent and ion-exchange chromatography. Planta, 1995, 196(3), 477 - 84 The maize brittle 1 gene encodes amyloplast membrane polypeptides; Sullivan TD et al.; A chimeric protein, formed of 56 amino acids from the carboxy terminus of the maize (Zea mays L.) wild-type Brittle1 (Bt1) protein fused to the glutathione-S-transferase gene, was synthesized in Escherichia coli, and used to raise antibodies . Following affinity purification, the antibodies recognized a set of 38- to 42-kDa proteins in endosperm from wild-type Bt1 plants, as well as from brittle2, shrunken2 and sugary1 plants, but not in mutant bt1 endosperm . Bt1 proteins were not detected with the preimmune antibodies . A low level of Bt1-specific proteins was detected at 10 d after pollination (DAP) and increased to a plateau at 16 DAP . At the same time, the ratio of slow- to fast-migrating forms of the protein decreased . During endosperm fractionation by differential centrifugation and membrane sedimentation in sucrose gradients, the Bt1 proteins co-purified with the carotenoid-containing plastid membranes . They were localized to amyloplasts by electron-microscopic immunocytochemistry; most of the signal was detected at the plastid periphery . These results are consistent with predictions made from the deduced amino-acid sequence and previous in-vitro experiments that the bt1 locus encodes amyloplast membrane proteins. Rapid Commun Mass Spectrom, 1995, 9(8), 693 - 6 252Cf-plasma desorption mass spectrometry analysis of lipids A obtained by an elimination reaction under mild conditions; Lebbar S et al.; Lipids A are the hydrophobic domains of bacterial endotoxic lipopolysaccharides . Since they are responsible for most of the biological activities (both pathogenic and beneficial) of endotoxins, the characterization of their structure is crucial to the understanding of their mode of action . However, the inadequacy of existing methods for preparing certain lipids A has prompted us to devise a new, mild procedure which gives intact products . Use was made of the special features of 252Cf-plasma desorption mass spectrometry for forming molecular ions from these species and giving qualitative and quantitative information from the primary mass spectrum. Biochimie, 1995, 77(3), 194 - 203 A structure-based multiple sequence alignment of all class I aminoacyl-tRNA synthetases; Landes C et al.; The superimposable dinucleotide fold domains of MetRS, GlnRS and TyrRS define structurally equivalent amino acids which have been used to constrain the sequence alignments of the 10 class I aminoacyl-tRNA synthetases (aaRS) . The conservation of those residues which have been shown to be critical in some aaRS enables to predict their location and function in the other synthetases, particularly: i) a conserved negatively-charged residue which binds the alpha-amino group of the amino acid substrate; ii) conserved residues within the inserted domain bridging the two halves of the dinucleotide-binding fold; and iii) conserved residues in the second half of the fold which bind the amino acid and ATP substrate . The alignments also indicate that the class I synthetases may be partitioned into two subgroups: a) MetRS, IleRS, LeuRS, ValRS, CysRS and ArgRS; b) GlnRS, GluRS, TyrRS and TrpRS. Arch Virol, 1995, 140(7), 1321 - 8 The Newcastle disease virus V protein binds zinc; Steward M et al.; The V protein of Newcastle disease virus (NDV) is produced by the insertion of a single nontemplated G residue at a specific point during transcription of the phosphoprotein (P) gene, accessing a new reading frame upon translation . The V protein, in common with its counterpart in other paramyxoviruses contains a highly cysteine rich motif near the carboxyl terminus, suggestive of a zinc-binding domain . By constructing E . coli overexpression plasmids for the NDV P and V proteins, and monitoring the binding of 65ZnCl2 to proteins electroblotted onto nitrocellulose membranes, we have demonstrated that the V protein strongly binds zinc. Vet Res Commun, 1995, 19(2), 149 - 57 Pentoxifylline pretreatment fails to block the acute-phase response to Escherichia coli endotoxin in dwarf goats; Van Duin CT et al.; The purpose of this study was to assess whether pentoxifylline, a drug that can inhibit the production and action of pro-inflammatory cytokines, had beneficial effects on the acute-phase response to E . coli endotoxin in the dwarf goat . First, the effects of 0.5 mg/kg per min pentoxifylline given intravenously over 15 min were examined in five goats . One week later, the clinical changes caused by i.v . injection of E . coli endotoxin (LPS: 0.1 microgram/kg) were determined . This endotoxin induced fever, tachycardia, inhibition of rumen motility, and decreases in plasma zinc and iron concentrations . Three weeks later, the effect of E . coli LPS were again determined immediately after pentoxifylline infusion in the same group of animals . It was concluded that a pharmacological dose of pentoxifylline has no protective effects on the acute-phase response reactions induced by a pyrogenic dose of E . coli LPS. Environ Mol Mutagen, 1995, 26(1), 9 - 15 Relative sensitivity of the endogenous hprt gene and lacI transgene in ENU-treated Big Blue B6C3F1 mice; Skopek TR et al.; Three-week-old Big Blue (BB) B6C3F1 mice were given a single i.p . injection of ENU . Three weeks later, splenic T cells were isolated from each animal by ficoll gradient centrifugation and divided into two samples . One sample was cultured to measure hprt- mutation and the other was used to extract DNA for lacI- analysis . T cells from BB mice exposed to 0, 4.5, 13.5, and 40 mg ENU/kg (9 or 10 animals per group) displayed dose-related increases in the frequency of both hprt- and lacI- mutations . Within each treatment group, the ENU-induced mutation frequency (average observed mutation frequency minus average control frequency) was remarkably similar at the two loci . This suggests that treatments that increase mutation frequency at the endogenous hprt gene also produce similar incremental increases at the BB lacI transgene . However, because of the ten-fold higher spontaneous mutation rate at lacI, the fold-increase over background produced by ENU at this locus was significantly less than the fold-increase produced at hprt . For example, the 4.5 mg ENU/kg treatment produced a 5.2-fold increase above background at hprt (P = 0.001), whereas only a 1.5-fold increase was produced at lacI (P = 0.140) . Consequently, mutagenic insults that produce up to a fivefold increase in mutation frequency at an endogenous locus may be difficult to detect at the lacI transgene . Finally, the ENU-induced response at hprt in BB mice was identical to that in generic B6C3F1 mice, suggesting that there are no inherent differences between transgenic and normal mice in their response to this mutagenic agent. Environ Mol Mutagen, 1995, 26(1), 16 - 25 Effect of isopropyl-beta-D-thiogalactopyranosid induction of the lac operon on the specificity of spontaneous and doxorubicin-induced mutations in Escherichia coli; Veigl ML et al.; Previous studies of doxorubicin-induced mutations employing F' lacl/lacO as an endogenous gene target have focused on properties of large deletions with 3' endpoints residing in the lacO region of the target gene . This study considers the influence of Lac repressor binding on the distribution of these deletions . Results of the DNA sequence level analysis of spontaneous and doxorubicin-induced i-d and lacO mutations in Escherichia coli uvrB- are reported for mutants isolated under conditions where Lac repression is relieved by isopropyl-beta-D-thiogalactopyranosid (IPTG; an inducer that prevents repressor binding to lacO) . The location of deletions isolated from doxorubicin-treated cultures in the presence and absence of IPTG suggests that doxorubicin preferentially focuses deletion endpoints adjacent to its binding sites in lacO and that the distribution of these deletion endpoints is not modulated by Lac repressor binding . In contrast, spontaneous deletion endpoints are preferentially clustered in the loop away from the palindromic sequences under conditions of repression . However, when the Lac repressor/lacO binding complex is dissociated by IPTG, the spontaneous 3'-deletion endpoints distribute proportionally between the putative stem and loop of the lacO palindrome . The single most striking effect of IPTG induction of the Lac operon was elimination of a "hot spot" for T:A-->C:G transitions at position +6 in lacO . This base substitution "hot spot," which accounted for 17.6% of total doxorubicin-induced mutants and 16.4% of spontaneous mutants in repressed bacterial cultures, accounted for approximately 1% of total mutations in similar experiments carried out in the presence of IPTG . A large number of mutations at the +6 position are induced only by doxorubicin in the absence of IPTG, however, suggesting that both doxorubicin-induced and spontaneous mutation at this transition "hot spot" are mediated by Lac repressor binding to lacO. Environ Mol Mutagen, 1995, 26(1), 1 - 8 Increased frequency of mutations at A:T base pairs in the bone marrow of B6C3F1 lacI transgenic mice exposed to 1,3-butadiene; Recio L et al.; We have examined the spectra of mutations in a collection of 74 lacI mutants isolated from the bone marrow of B6C3F1 lacI transgenic mice exposed to 1,250 ppm 1,3-butadiene (BD) . Of the 49 independent mutations analyzed in the present study, 30 of 49 (61%) were point mutations at G:C base pairs, and 10 of 49 (20%) were point mutations at A:T base pairs . The remaining mutations consisted of small deletions and insertions and a single tandem change . An analysis of variance (ANOVA) on the frequency of specific mutations observed in each animal in this mutagenicity experiment {present study; also Sisk SC et al . (1994): Carcinogenesis 15:471-477} indicated that the frequency of point mutations at A:T base pairs is significantly greater (P < 0.05) in BD-exposed mice than in the air controls . In addition, there was a decrease (P < 0.05; Fisher's exact test) in the frequency of G:C-->A:T transitions at 5'-CpG-3' sites in BD-exposed mice (27%) relative to air controls (51%) . These data indicate that subchronic exposures to BD induces an increased frequency of in vivo mutation at A:T base pairs in the bone marrow of B6C3F1 lacI transgenic mice. Virchows Arch, 1995, 426(5), 487 - 91 Morphology of cardiac muscle in septic shock . Observations with a porcine septic shock model; Hauptmann S et al.; The morphology of cardiac muscle was investigated in a porcine model of septic shock, created by intermitted application of Escherichia coli-endotoxin . The earliest lesions, found after 18 h of septic shock, were endothelial cell swelling, marked leucostasis and slight ischaemic alterations of the muscle fibres . At the end point of the experiments, after 48 h, some fibrin thrombi were found associated with more pronounced ischaemic alterations of cardiac muscle cells and some necrotic fibres . Comparing these findings with the severe endothelial and muscle fibre lesions found in skeletal muscle, the endothelial cells of the heart microvasculature, are clearly more resistant to the attack of the endotoxins and mediators liberated in septic shock. Microbios, 1995, 82(332), 157 - 70 Quantitative effects of redox-cycling chemicals on the oxidant-sensitive enzyme dihydroxy-acid dehydratase; Babu BN et al.; The {4Fe-4S} cluster-containing enzyme dihydroxy-acid dehydratase (DHAD) is susceptible to inactivation by dioxygen and active oxygen species including superoxide with an inactivation rate constant of 10(6) m-1 sec-1 . Based on this property, DHAD was used to quantify and investigate the biological oxidant stress activity of various redox-cycling chemicals . Exponentially growing cultures of Escherichia coli were used as a sensitive source of the DHAD enzyme . The effects on DHAD of compounds with and without established redox activity, under aerobic and anaerobic (control) conditions were measured . Paraquat, juglone, nitrofurantoin, the nitrofuran related compound NF-963 which is 6,7-dihydro-3-(5-nitro-2-furyl-5H-imidazo-{2,1-b} thiazolium chloride), plumbagin, benzoquinone, duroquinone, hydralazine, and naphthalene inhibited DHAD activity and the concentrations required for 50% inhibition ranged from 3.5 microM for paraquat to 950 microM for naphthalene . Eleven other agents tested (including 4,4-dipyridyl which is a non-redox-cycling compound similar to paraquat and an extract and two compounds of plant origin) did not inhibit DHAD . The DHAD technique described is a useful means of detecting and comparing the oxidant-stress toxicity and mechanism of action of chemicals by a biological means on a quantitative scale relatable to paraquat. Hematol Pathol, 1995, 9(1), 37 - 47 Neutrophil functions in essential thrombocythemia; Carulli G et al.; Chronic myeloproliferative diseases, such as chronic myeloid leukemia and polycythemia vera, are associated with neutrophil dysfunction . Very little data is available on essential thrombocythemia (ET) . In the current study we evaluated 21 patients with ET . All patients were studied at least 16 weeks after any cytostatic therapy and 10 days after any other therapy . Neutrophil functions were investigated as follows: flow cytometric evaluation of whole blood phagocytosis of opsonized FITC-conjugated E . coli; whole blood chemiluminescence after stimulation with opsonized zymosan and evaluation by an automated, computer-assisted luminometer (LB 950, Berthold); and chemiluminescence and superoxide anion generation by purified neutrophils after f-MLP and PMA stimulation . Chemiluminescence and superoxide anion generation after f-MLP stimulation were found to be significantly lower than in normal subjects, whereas values within the normal ranges were registered after PMA stimulation . Phagocytosis-associated chemiluminescence was found to be impaired both by using zymosan opsonized with autologous plasma and zymosan opsonized with normal plasma, despite a normal phagocytic activity . These data show the presence in ET of a complex neutrophil dysfunction that may be related to an impaired signal transduction during both the phagocytic process and f-MLP stimulation. Adv Biophys, 1995, 31, 49 - 65 Molecular mechanisms of Holliday junction processing in Escherichia coli; Shinagawa H et al.; Recent genetic and biochemical studies revealed the mechanisms of late stage of homologous recombination in E . coli . A central intermediate of recombination called "Holliday structure", in which two homologous duplex DNA molecules are linked by a single-stranded crossover, is formed by the functions of RecA and several other proteins . The products of the ruvA and ruvB genes, which constitute an SOS regulated operon, form a functional complex that promotes migration of Holliday junctions by catalyzing strand exchange reaction, thus enlarging the heteroduplex region . RuvA is a DNA-binding protein specific for these junctions, and RuvB is a motor molecule for branch migration providing energy by hydrolyzing ATP . The product of the ruvC gene, which is not regulated by the SOS system, resolves Holiday junctions by introducing nicks at or near the crossover junction in strands with the same polarity at the same sites . The recombination reaction is completed by sealing the nicks with DNA ligase, resulting in spliced or patched recombinants . The product of the recG gene provides an alternative route for resolving Holliday junctions . RecG has been proposed to promote branch migration in the opposite direction to that promoted by RecA protein . The atomic structure of RuvC protein revealed by crystallographic study, when combined with mutational analysis of RuvC, provides mechanistic insights into the interactions of RuvC with Holliday junction. Adv Biophys, 1995, 31, 23 - 48 Analysis of the DNA binding site of Escherichia coli RecA protein; Morimatsu K et al.; To investigate the DNA binding site of RecA protein, we constructed 15 recA mutants having alterations in the regions homologous to the other ssDNA binding proteins . The in vivo analyses showed that the mutational change at Arg243, Lys248, Tyr264, or simultaneously at Lys6 and Lys19, or Lys6 and Lys23 caused severe defects in the recA functions, while other mutational changes did not . Purified RecA-K6A-K23A (Lys6 and Lys23 changed to Ala and Ala, respectively) protein was indistinguishable from the wild-type RecA protein in its binding to DNA . However, the RecA-R243A (Arg243 changed to Ala) and RecA-Y264A (Tyr264 changed to Ala) proteins were defective in binding to both ss- and ds-DNA . In self-oligomerization property, RecA-R243A was proficient but RecA-Y264A was deficient, suggesting that the RecA-R243A protein had a defect in DNA binding site and the RecA-Y264A protein was defective in its interaction with the adjacent RecA molecule . The region of residues 243-257 including the Arg243 is highly homologous to the DNA binding motif in the ssDNA binding proteins, while the eukaryotic RecA homologues have a similar structure at the amino-terminal side proximal to the nucleotide binding core . The region of residues 243-257 would be a part of the DNA binding site . The other parts of this site would be the Tyr103 and the region of residues 178-183, which were cross-linked to ssDNA . These three regions lie in a line in the crystal structure. Adv Biophys, 1995, 31, 209 - 22 IS1-encoded proteins, InsA and the InsA-B'-InsB transframe protein (transposase): functions deduced from their DNA-binding ability; Sekino N et al.; Insertion sequence IS1 encodes a transframe protein, InsA-B'-InsB, which is produced from two out-of-phase reading frames, insA and B'-insB, by translational frameshifting at a run of adenines . Unless the frameshifting event occurs, the InsA protein is produced from IS1 . We found that cells harboring a plasmid carrying an IS1 mutant with a single adenine insertion in the run of adenines contained miniplasmids . Cloning and DNA sequencing analyses of the miniplasmids revealed that they had a deletion extending from an inverted repeat (IR) at the left end of IS1 . This indicates that they were generated by IS1-mediated deletion due to efficient production of the InsA-B'-InsB transframe protein that is IS1 transposase . Both the InsA protein and transposase were partially purified as a fusion protein with collagen-LacZ by LacZ-specific affinity column chromatography . The InsA* and the collagenolyzed InsA* were found to bind specifically to a 24-bp region within each of the IRs at the ends of IS1 . The transposase Tnp* and the collagenolyzed Tnp* were found to bind to the sequence with or without IR, but preferentially to that with IR . The nonspecific DNA-binding ability of transposase may be involved in recognition of the target DNA, an important process of transposition of IS1 . Both InsA and transposase have the IR-specific DNA binding ability and a common polypeptide segment containing the alpha-helix-turn-alpha-helix motif, supporting the previous indication that InsA competes with transposase to bind to IRs and thus becomes a transposition inhibitor . Based on the observations described in this article, we speculate that transposase of IS1 consists of at least two domains, the N-terminal half, which almost entirely overlaps InsA, and the C-terminal half, which almost entirely overlaps B'-InsB . The frameshifting event adds the latter domain to the former to give the transposase activity recognizing IRs and the target sequence to initiate the transposition reaction. Adv Biophys, 1995, 31, 197 - 208 A novel assay for illegitimate recombination in Escherichia coli: stimulation of lambda bio transducing phage formation by ultra-violet light and its independence from RecA function; Ikeda H et al.; We developed a novel assay system for illegitimate recombination, in which the frequency of the formation of lambda Spi- phages formed during prophage induction was measured with an E . coli P2 lysogen as the indicator bacteria . Since almost all of the lambda Spi- phages thus detected contain attR, they have essentially the same structures as lambda bio transducing phages, indicating that this assay system enables us to detect specialized transducing phages that produce heterogenote transductants, thus ignoring the occurrences of docL and docR particles which carry only one cohesive end . The following results on the formation of specialized transducing phages have been obtained by this assay system to date . (1) Irradiation with UV light greatly enhanced the formation of lambda Spi- phages . (2) Treatments with other DNA-damaging agents also enhanced the formation of lambda Spi- phages . (3) Illegitimate recombination during prophage induction does not require the RecA function, indicating that enhancement of lambda Spi- phage formation is not controlled by the SOS regulatory system . (4) Preliminary results suggested that DNA gyrase is involved in the formation of lambda Spi- phage during pro-phage induction . Since the above results were consistent with most of the previous observations on the illegitimate recombination in other systems, the Spi- assay system can provide important clues to the mechanism of illegitimate recombination. Adv Biophys, 1995, 31, 181 - 93 Mating variation by DNA inversions of shufflon in plasmid R64; Komano T et al.; Gene organization of the 54-kb transfer region of IncI1 plasmid R64 was deduced from the DNA sequence . Forty-eight ORFs were found in this region . A unique DNA rearrangement designated shufflon is located at the downstream region of an operon responsible for synthesis of thin pilus . The shufflon of R64 consists of four DNA segments, designated as A, B, C, and D, which are flanked and separated by seven 19-bp repeat sequences . Site-specific recombination mediated by the product of the rci gene between any two inverted repeats results in a complex DNA rearrangement . An analysis of open reading frames revealed that the shufflon is a biological switch to select one of seven C-terminal segments of the pilV genes . The products of pilV genes were shown to be components of thin pilus which was required for liquid mating . Seven R64 derivatives where the pilV genes were fixed in the seven C-terminal segments were constructed and their transfer frequencies in liquid mating were measured using various bacterial strains as recipients . Transfer frequencies of R64 in liquid mating strongly depended on the combination of C-terminal segments of the pilV genes in donor cells and bacterial strains of recipient cells, suggesting that the shufflon determines the recipient specificity in liquid mating of plasmid R64. Life Sci, 1995, 57(6), 599 - 608 Pyrogenic stimulation of vascular resistance in conscious sheep; Parkes DG et al.; Increased arterial blood pressure following a pyrogenic reaction has been reported in previous studies, however the mechanism of this hypertension has not been examined in detail . The present study investigated the effects of both intravenous (IV) and intracerebroventricular (ICV) injection of lipopolysaccharide (LPS) from E . coli on body temperature (Tb), mean arterial pressure (MAP), heart rate (HR), cardiac output (CO), calculated total peripheral resistance (CTPR), stroke volume (SV) and plasma levels of adrenocorticotropin (ACTH) and arginine vasopressin (AVP) in conscious, chronically instrumented sheep . IV injection of LPS (1 microgram) increased Tb in a biphasic manner from 38.7 +/- 0.1 to 39.5 +/- 0.2 degrees C after 50 min and to 39.9 +/- 0.2 degrees C after 130 min, and MAP increased biphasically from 64 +/- 1 to 70 +/- 4 mmHg after 40 min and to 78 +/- 3 mmHg after 130 min . CO initially decreased from 4.4 +/- 0.1 to 3.5 +/- 0.1 after 40 min followed by a secondary rise to 4.8 +/- 0.1 l/min after 100 min . This occurred together with a large, biphasic increase in CTPR from 14.5 +/- 1.0 to 22.0 +/- 2.0 mmHg/l/min at 40 min, and to 18.1 +/- 0.1 mmHg/l/min at 120 min . HR increased from 68 +/- 4 to 97 +/- 4 b/min and SV decreased from 65 +/- 2 to 41 +/- 4 ml/beat during the first phase of activation . Plasma ACTH increased from 22 +/- 9 to 1043 +/- 175 pg/ml after 80 min, and plasma AVP increased from 0.7 +/- 0.2 to 12 +/- 4.0 pg/ml after 60 min . ICV injection of LPS produced a long-lasting increase in Tb and MAP, but had no effect on HR or plasma AVP . Plasma ACTH increased from 30 +/- 12 to 427 +/- 110 pg/ml . These changes suggest that intravenous pyrogenic infection produces a potent vasoconstrictor action in sheep to increase blood pressure, possibly mediated by the actions of AVP within the CNS, or other pyrogenically released vasoconstrictor factors . Furthermore, the duration of activation of the cardiovascular system following peripheral and central LPS administration is different, which together with the contrasting effects on ACTH and AVP, indicate the involvement of several hypertensive mechanisms. Nippon Saikingaku Zasshi, 1995, 50(2), 551 - 5 {Adherence to HEp-2 cells of enterotoxemic Escherichia coli O-group 139 from pigs with edema disease}; Nakazawa M et al.; One hundred and one strains of enterotoxemic Escherichia coli (ETEEC) O-group 139 isolated from swine with edema disease were investigated for their adherence to HEp-2 cells in the presence of D-mannose . All strains adhered in large numbers to the cells (21 to 60 bacteria/cell) . No correlation was found between the presence of F107 fimbria on the organisms and the adherence to the cells . Adhesion-inhibition tests showed that anti-K12 serum inhibited the adhesion ETEEC O-group 139 (an inhibition rate of 63 to 65%), but anti-F107 or anti-O139 sera did not . These results indicate that the capsular K12 antigen may be one of the pathogenic factors of ETEEC O-group 139. Urol Res, 1995, 23(1), 33 - 8 Cytokine and lymphocyte activation during experimental acute pyelonephritis; Roberts JA et al.; We studied the cellular and humoral events which follow experimental acute pyelonephritis from P-fimbriated Escherichia coli to gain insight into the relationships among cells and specifically cytokines to determine how early events in untreated infection lead to renal damage . Cynomolgus (Macaca fascicularis) monkeys were studied after they were subjected to unilateral ureteral bacterial inoculation . We evaluated the blood for leukocytosis and studied lymphocyte subsets using flow cytometry and monoclonal antibodies to the subsets and serum, complement, cytokines and antibody titers . Interleukin-1, 2 and 6 and tumor necrosis factor (TNF) were assayed by enzyme-linked immunoadsorbent assay (ELISA), using monoclonal and polyclonal antibodies . Leukocytosis was marked and there were significant elevations in serum cytokines, interleukin-1 alpha, 2 and 6 with only small changes in the level of TNF . Interleukin-2 levels were sustained and may have upregulated the homing receptor for virgin lymphocytes . The studies illustrated the unique relationship between cytokines and lymphocytes and the response to bacterial infection, showing that the inflammatory response is regulated not only by cytokine activity but also by lymphocyte activation. DNA Seq, 1995, 5(3), 195 - 7 The gene (aroK) encoding shikimate kinase I from Escherichia coli; Griffin HG et al.; Shikimate kinase I (SKI), encoded by the aroK gene, converts shikimate to shikimate 3-phosphate, an intermediate in the biosynthesis of the aromatic amino acids . This paper provides evidence that the E . coli aroK reading frame is in a different place to that reported previously and presents a predicted SKI sequence of 173 residues and a molecular mass of 19.5 kDa . The translational start point of aroK is some 84 bp downstream of the site proposed earlier and the reading frame ends 169 bp beyond the stop site reported in the previous sequence . The SKI sequence reported here more closely resembles those from other organisms and the predicted molecular mass approximates more closely that determined by experiment. DNA Seq, 1995, 5(3), 185 - 9 Sequence of a transposon identified as Tn1000 (gamma delta); Broom JE et al.; We report the complete sequence of a transposon found in a cosmid clone of a human DNA sequence . The transposon is identified as the Escherichia coli transposon Tn1000 (also known as gamma delta) on the basis of the identity of the restriction map of the new sequence with that previously recorded for Tn1000 and homology between parts of the new sequence and that of published fragments of Tn1000 sequence . The transposon, which comprises 5,981 nucleotides including two 35 bp inverted terminal repeat sequences (ITRs), contains three open reading frames . The sequence of the resolvase coding region (tnpR) is identical to that published by others . A second reading frame can be identified as the tnpA gene, coding for the transposase, on the grounds of its strong homology with the corresponding gene from transposon Tn3 . The third reading frame has the potential to code for a protein of unknown function containing 698 amino acids. Arch Virol, 1995, 140(6), 1061 - 74 Expression of the nucleoprotein of rabies virus in Escherichia coli and mapping of antigenic sites; Goto H et al.; Investigations were performed to delineate the antigenic sites I and IV of rabies virus nucleoprotein (N), the former of which is well conserved among the rabies and rabies-related viruses . The N cDNA of the RC-HL strain was inserted into an expression vector pET3a, with which the E . coli BL21(DE3) was transformed . Upon induction with isopropyl-1-thio-beta-D-galactoside, the transformants produced a protein with a size (56 k-Da) almost identical to that of the authentic N protein . The protein also reacted with a panel of our N protein-specific monoclonal antibodies (N-MAbs) including the antibodies against the antigenic sites I and IV . By using the cDNA, various deletion mutants were generated and expressed in E . coli to examine the reactivity of mutant proteins with N-MAbs by Western blot analysis . Deletion of the C-terminal 67 amino acid residues did not abolish their reactivity with any of the N-MAbs specific for the sites I and IV . When 91 residues or more were deleted from the C-terminus, however, the protein lost the reactivity, indicating that the antigenic sites I and IV are mapped to a small region which is comprised of at most 24 amino acid residues from positions 360 to 383 . Comparison of the 24-amino acid sequence with the corresponding region of N protein of several other Lyssavirus strains suggests that the antigenic site I is mapped to positions 360 to 369. Patol Fiziol Eksp Ter, 1995 Jan-Mar, (1), 31 - 4 {Corneal wound healing in rabbits as affected by substances changing the cyclic nucleotide level}; Chesnokova NB et al.; A rabbit corneal deepthelization model was used to study in vivo the effects of agents changing the level of cyclic nucleotides on the time of its corneal epithelial wound healing . Cholera toxin (CT) and thermostable E.coli toxin (ST toxin) were employed as agents elevating the level of cAMP and cHMP . The studies showed that a healing process in rabbits could be divided into 4 periods, depending on the epithelization rate . CT instilled in doses of 0.25-2.00 micrograms per year reduced the healing of an epithelial defect by 10 hours, increased the epithelization rate 2-fold at hours 4 to 20, but failed to affect in the remaining periods . It is suggested that by elevating the intracellular cAMP level in this period, CT accelerates migration of epithelial cells, while ST toxin increasing the level of cHGP fails to affect the time of corneal wound epithelization . It is concluded that it is promising to search for new drugs for accelerating the healing of corneal wounds among the agents elevating the level of intracellular cAMP. Nucl Med Commun, 1995 Jan, 16(1), 38 - 46 Influence of endogenous biotin on the biodistribution of labelled biotin derivatives in mice; Rusckowski M et al.; Previously, this laboratory reported that in mice pre-targeted with unlabelled streptavidin, the biodistribution of 111In administered on one biotin derivative (EB1) was superior to that of another derivative (DB2) . In addition, a Scatchard analysis showed that the affinity constant of 111In-EB1 is lower by seven orders of magnitude from that of 111In-DB2 . Therefore, this paper considers the role that endogenous biotin may play in these observations . Both 111In-labelled EB1 and DB2 were bound to streptavidin and incubated at 37 degrees C in mouse blood with increasing concentrations of d-biotin . As determined by Sephadex G-50 chromatography, only an 8-fold molar excess of d-biotin relative to labelled streptavidin was required to displace 90% of label in the case of EB1, whereas even a 20-fold molar excess provided no detectable displacement of DB2 . That this displacement was also occurring in vivo was established in a mouse model bearing an infected thigh: increasing the serum biotin level (by intraperitoneal administration of d-biotin) had no effect on the biodistribution of 111In when administered on DB2; however, the target to non-target ratio decreased in the case of EB1 . We have also observed that the biodistribution is no longer favourable when EB1 is administered radiolabelled with 99Tcm . When 111In was substituted with 99Tcm on EB1, chromatography of blood samples showed that similar displacement was occurring; however, in this case, the displaced label bound to serum proteins.(ABSTRACT TRUNCATED AT 250 WORDS) Methods Cell Biol, 1995, 46, 139 - 51 Use of the yeast two-hybrid system for identifying the cascade of protein interactions resulting in apoptotic cell death; Bemis LT et al.; Use of the yeast two-hybrid system allows rapid identification of interacting protein or proteins for a specific target protein . The technique is readily applied and allows immediate isolation of a cDNA encoding the interacting protein . One consideration might be to outline criteria for continued study of the interactors once they are identified . Our criterion for further study of an interactor is its presence in the mammary gland at a developmental time when the target protein is also present . Further characterization of interactors may involve immunoprecipitation, enzyme assays, or other techniques applicable to the specific protein. Can J Microbiol, 1995, 41 Suppl 1, 200 - 6 The function of ackA and pta genes is necessary for poly(3-hydroxybutyrate-co-3-hydroxyvalerate) synthesis in recombinant pha+ Escherichia coli; Rhie HG et al.; In Escherichia coli carrying the poly(3-hydroxyalkanoate) (PHA) biosynthesis pathway on a plasmid (pha+), the function of the ackA (acetate kinase) and pta (phosphotransacetylase) genes is necessary for efficient incorporation of 3-hydroxyvalerate (3-HV) into the copolymer, poly(3-hydroxybutyrate-co-3-hydroxyvalerate) (P(3HB-co-3HV)) . Recombinant pha+ E . coli fadR atoC(Con) strains possessing mutations in ackA, pta, or both ackA and pta exhibited substantially reduced levels of 3-HV formation . Conversely, the same strains carrying the ackA gene on a multicopy plasmid exhibited an increase in 3-HV formation concomitant with a large increase in acetate kinase activity . However, if the strain possessing the multicopy ackA+ plasmid was mutant at the pta locus, it lost the ability to incorporate significant amounts of 3-HV into P(3HB-co-3HV) . In addition to the ackA pta pathway, there is an inducible activity that can also mediate the incorporation of 3-HV into P(3HB-co-3HV) . This pathway is repressed by glucose and is not normally operative in P(3HB-co-3HV) production in recombinant pha+ E . coli strains that are grown using glucose as the major carbon source . It appears likely that this activity is due to an inducible acetyl-CoA synthetase that converts propionate to propionyl-CoA. Arch Insect Biochem Physiol, 1995, 29(2), 159 - 73 Genetic mechanisms involved in cellular recovery from oxidative stress; Eisenstark A et al.; Sophisticated biochemical networks allow organisms such as bacteria and insects to switch from very rapid growth and development in ideal environments to dormancy during severely unfavorable conditions . These switches may be accompanied by abrupt changes in oxidation/reduction involving reactive oxygen species (ROS) . ROS have the potential of damaging nucleic acids, proteins, and membranes . In Escherichia coli, certain genetically regulated circuits (regulons) turn on synthesis of anti-oxidant enzymes to protect against distinct ROS excesses (superoxide, hydrogen peroxide, organic or lipid peroxides, etc.) . As examples, the soxRS regulon controls synthesis of Mn-superoxide dismutase, oxyR controls catalase HPI, rpoS positively regulates HPII, and fur regulates several oxidative reactions that involve iron uptake . Our studies have focused on the regulatory role of rpoS, known to be a sigma factor (sigma 38) that combines with RNA polymerase and is a regulator of those gene products needed to protect cells during dormancy . Since insect cells, during both active growth and dormancy, endure severe environments, analogous protective gene products may be induced . Examples are presented of insect anti-oxidant metabolism, including those involved in the aging process . In addition, we searched several DNA and protein sequence data banks to compare resemblances between anti-oxidant gene products of bacteria and insects. Mol Gen Mikrobiol Virusol, 1995 Jan-Mar, (1), 9 - 14 {Isolation of a group of hybrid proteins consisting of tumor necrosis factor alpha and thymosin alpha 1}; Shmelev VA et al.; New plasmid DNA coding for the synthesis of three hybrid proteins differing by the position of alpha 1-thymosin at N- and C-terminals of tumor necrosis factor were optimized and constructed . The instability of plasmids at culturing of E . coli strains stored in solid nutrient media was demonstrated . Newly obtained transformants and preserved cells were cultured, this providing a high level of synthesis of hybrid proteins . The effects of culturing temperature and protein structure on protein solubility were shown . Hybrid proteins were purified by chromatography on hydrophobic anion-exchange carriers and blue agarose in the presence of 7 M urea . After dialysis the proteins displayed different cytotoxicity in L-929 cells and were fit for immunobiological studies. Biochimie, 1995, 77(1-2), 92 - 8 The nucleoside deaminases for cytidine and adenosine: structure, transition state stabilization, mechanism, and evolution; Carter CW Jr; Enzymatic deamination of cytidine and adenosine bases in RNA have recently been shown to be mechanisms for changing the coding specificity of messenger and transfer RNAs . The structures of the enzymes that carry out deamination of the corresponding nucleosides have been analyzed by X-ray crystallography . They are quite different from one another in most respects, including quaternary and tertiary structure, but they have similar chemical groups in their active sites . Both enzymes envelope their nucleoside substrates completely, perhaps accounting for the fact that they are inactive on RNA substrates . Much has been learned about catalytic mechanisms from the structures of the enzymes and their complexes with transition state analog inhibitors . Catalysis proceeds with the activation by zinc of a bound water molecule, presumably to hydroxide ion, which attacks the appropriate carbon to generate a tetrahedral intermediate . The detailed stereochemistry of the two resulting chiral centers is diastereoisomeric . Details of the ensuing proton transfer steps necessary to generate and release the products are also apparently different in the two enzymes . Thus, the active site similarities are probably the result of convergent evolution. Biochimie, 1995, 77(1-2), 62 - 5 tRNA-m1G methyltransferase interactions: touching bases with structure; Holmes WM et al.; m1G methyltransferase of Escherichia coli is being examined with regard to how specific tRNA substrates are recognized . This enzyme appears to require the entire tRNA structure of optimal activity . Recognition may require specific base contacts as well as phosphate backbone structures embodied in the tRNA structure. Biochimie, 1995, 77(1-2), 125 - 34 Site-selected introduction of modified purine and pyrimidine ribonucleosides into RNA by automated phosphoramidite chemistry; Agris PF et al.; The study of modified nucleoside contributions to RNA chemistry, structure and function has been thwarted by the lack of a site-selected method of incorporation which is both versatile and adaptable to present synthetic technologies . A reproducible and versatile site-selected incorporation of nine differently modified nucleosides into hepta- and octadecamer RNAs has been achieved with automated phosphoramidite chemistry . The 5'-O-(4,4'-dimethoxytrityl-2'-O-tert-butyldimethylsilyl-ribonucleoside- 3'-O-(2-cyanoethyl-N,N-diisopropyl)phosphoramidite syntheses of m5C, D, psi, riboT, s2U, mnm5U, m1G and m2A were designed for compatibility with the commercially available major and 2'OH methylated ribonucleoside phosphoramidites . The synthesis of the m5C phosphoramidite was uniquely designed, and the first syntheses and incorporation of the two modified purine ribonucleosides are reported in detail along with that of psi, s2U, and mnm5U . Cleavage of RNA product from the synthesis support column, deprotection of the RNA, its purification by HPLC and nucleoside composition analysis are described . Modified nucleoside-containing tRNA domains were synthesized and purified in mumol quantities required for biophysical, as well as biochemical, studies . The anticodon domain of yeast tRNA(Phe) was synthesized with modified nucleosides introduced at the native positions: Cm32, Gm34, m1G37 (precursor to Y), psi 39 and m5C40 . The T loop and stem was synthesized with riboT54 and the D loop and stem with D16 and D17 . The E coli tRNA(Glu2) anti-codon codon domain was synthesized with mnm5U at wobble position 34, but an attempt at incorporating s2U at the same position failed . The unprotected thio group was labile to the oxidation step of the cyclical process . Chemically synthesized anticodon and T domains have been used in assays of tRNA structure and function (Guenther et al (1994) Biochimie 76, 1143-1151). Biochimie, 1995, 77(1-2), 104 - 8 The ability of bovine mitochondrial transfer RNAMet to decode AUG and AUA codons; Takemoto C et al.; The ability of bovine mitochondrial tRNA(Met) with the anticodon f5CAU (where f5C is 5-formylcytidine) to decode AUG and AUA codons was examined in a codon-dependent ribosomal binding assay . The AUG codon stimulated the binding of Met-tRNA(Met) to mitochondrial ribosomes in the presence of EF-Tu/TSmt . In contrast, the AUA codon did not promote the binding to mitochondrial Met-tRNA to the ribosome . To investigate the translation of the AUG and AUA codons more fully, an in vitro translation system from bovine liver mitochondria was developed . The activity of this system was greatly enhanced by the addition of 1 mM spermine and reached about half the activity observed with a comparable translational system from E coli . Two types of mRNA containing either AUG or AUA codons were synthesized using T7 RNA polymerase to transcribe their chemically synthesized genes . In the E coli system, the AUG-containing mRNA was translated as Met and the AUA-containing mRNA was translated as Ile . The AUG-containing mRNA but not the AUA-containing mRNA was translated as Met by the mitochondrial translational system . The process by which the AUA codon is translated as Met in the mitochondrial system remains to be clarified. Annu Rev Pharmacol Toxicol, 1995, 35, 145 - 64 Transgenic animal models for detection of in vivo mutations; Mirsalis JC et al.; Transgenic rodent models for measuring mutations provide a tool for assessing tissue-specific mutations following in vivo treatment . These systems are based on the insertion into the rodent genome Escherichia coli lacI (lac repressor) or lacZ (beta-galactosidase) genes that serve as targets for mutations . Following in vivo treatment of animals, genomic DNA is isolated from tissues of interest, and the target gene is screened for mutations using either lambda-phage packaging or isolation of the target gene with magnetic affinity capture . In this paper we review the various experimental methods used in the conduct of transgenic mutation assays and discuss critical factors that affect the interpretations of results of these assays. Ophthalmic Res, 1995, 27(1), 23 - 31 Angiogenic activity of vitreous extract obtained from rabbit eyes with endotoxin-induced uveitis; Naveh N et al.; The effect of rabbit vitreous with endotoxin-induced uveitis was investigated for its role in the modulation of rat corneal neovascularization (NV) . Elvax pellets implanted into at cornea contained either uveitic vitreous, intact vitreous or Elvax alone . The latter two kinds of pellets did not elicit NV . Uveitic vitreous pellets caused, in 100% of eyes, a persistent NV with maximal growth on the 10th day post implantation . Prostaglandin E2 and leukotriene B4 levels in the uveitic rabbit vitreous at 36 h postendotoxin injection were 7- and 5-fold higher than baseline, respectively . Histopathologically, the neovascularized cornea showed a significant inflammatory reaction attributable to the uveitic vitreous extract. Exp Brain Res, 1995, 105(1), 59 - 66 Transplantation of embryonic retinal donor cells labelled with BrdU or carrying a genetic marker to adult retina; Seiler MJ et al.; After transplantation of embryonic retinal cells to injured adult retina, it is often difficult to distinguish donor from host cells . To overcome this problem, two methods were applied: labelling donor cells with the nuclear marker bromodeoxyuridine (BrdU) and use of transgenic donor tissue . BrdU was injected into timed-pregnant rats on 2 or 3 consecutive days . The donor embryos were taken 1-4 days later for transplantation . The BrdU-labelled donor tissue was examined in transplants sampled up to 1 year after grafting . Labelled donor cells were specifically identified in the transplants and in the interface with the adjacent host retina . The varying intensities of cell labelling indicated differences in the initial uptake of BrdU in the S-phase, or the dilution of the label by cell divisions after BrdU injection . The best labelled cells were presumably the ones that stopped dividing shortly after injection of BrdU . As controls, the normal development of BrdU-labelled retinas from the offspring of females that had been BrdU-injected at E16 and E17 and not used for transplantation was studied . Near the time of birth, clones of labelled cells were radially distributed . In the mature retina, labelled cells were seen in all retinal layers . Embryonic retina derived from transgenic (NSE-lacZ) mice was transplanted to 'nude', immunodeficient rats (xenografts) . These transgenic mice contain the Escherichia coli beta-galactosidase gene, coupled to the promoter for neuron-specific enolase (NSE) . Thus, all retinal donor cells that contain NSE could be identified by histochemistry or immunohistochemistry . The donor cells expressing the transgene could be detected several months after transplantation. Eur Surg Res, 1995, 27(5), 292 - 300 Effects of N-acetyl-L-cysteine on regional blood flow during endotoxic shock; Zhang H et al.; We previously reported that N-acetyl-L-cysteine (NAC), an oxygen free-radical scavenger, can increase the oxygen extraction capabilities during endotoxic shock when blood flow is progressively reduced . In the present study, we investigated whether the protective effects of NAC are related to an improvement in regional blood flow following endotoxemia . Fourteen anesthetized, saline-infused and ventilated dogs were divided into two groups: 7 dogs received NAC (150 mg/kg, followed by a 20 mg/kg.h infusion), and the other 7 dogs served as a control time-matching group . Thirty minutes later all the dogs received Escherichia coli endotoxin (2 mg/kg) i.v . A saline infusion was started 30 min after endotoxin challenge to restore pulmonary artery occlusion pressure to baseline and maintain it constant . Regional blood flow was measured by ultrasonic volume flowmeter . In the control group, arterial pressure, left ventricular stroke work index and systemic vascular resistance remained lower than baseline . Mesenteric, renal and femoral arterial blood flow increased but only femoral blood flow returned to baseline levels . In the NAC group, cardiac index and left ventricular stroke work index remained higher and systemic and pulmonary vascular resistance were lower than in the control group . Blood flow in mesenteric, renal and especially femoral arteries was higher than in the control group . Fractional blood flow increased only in the femoral artery . PaO2 and PvO2 had similar courses in the two groups . A higher venous admixture was associated with a higher cardiac index and a lower pulmonary vascular resistance in the NAC group . Oxygen delivery and oxygen-uptake were higher in the NAC-treated than in the control animals throughout the study.(ABSTRACT TRUNCATED AT 250 WORDS) Eur J Appl Physiol Occup Physiol, 1995, 71(2-3), 281 - 4 Increased phagocytic capacity of the blood, but decreased phagocytic activity per individual circulating neutrophil after an ultradistance run; Gabriel H et al.; The effect of a long strenuous endurance exercise on the phagocytic function of neutrophils was examined . 9 athletes {7 males, 2 females, age: 36-68 years, body mass: 64 (SD 10) kg, height: 175 (SD 10) cm} completed a competetive 100 km run in 8:07 (median value; range: 7:29-9:50 hours) . In a whole blood assay the phagocytosis of opsonized E . coli, the receptor density of the Fc gamma receptor 3 (CD16) and the complement receptor 3 (CD11b, direct immunofluorescence) of neutrophils were measured on a per cell basis by flow cytometry before and up to 3 hours after the race . The phagocytic rate (percentage of neutrophils incorporating bacteria) was unchanged after exercise, whereas the phagocytic activity (number of incorporated bacteria per cell) was significantly reduced by -34 (SD 8) % (Wilcoxon test, P < 0.001) . The total phagocytic capacity of the blood increased 2-3fold post exercise . The surface antigen expressions of CD11b and CD16 were unaffected by the ultradistance run . The results indicate either a reduced phagocytic function of neutrophils on a single cell basis or the mobilization of neutrophils of the marginal pool with a lower phagocytic activity . However, after a long endurance exercise the phagocytotic capacity of the blood was enhanced due to increased cell concentrations. Dev Genet, 1995, 17(2), 117 - 28 Widespread expression of the eve1 gene in zebrafish embryos affects the anterior-posterior axis pattern; Barro O et al.; The zygotic expression of the eve1 gene is restricted to the ventral and lateral cells of the marginal zone . At later stages, the mRNAs are localized in the most posterior part of the extending tail tip . An eve1 clone (pcZf14), containing a poly-A tail, has been isolated . In order to address eve1 gene function, pcZf14 transcript injections into zebrafish embryos have been performed . The injection into uncleaved eggs of a synthetic eve1 mRNA (12 pg), which encodes a protein of approximately 28 kd, produces embryos with anterior-posterior (A-P) axis defects and the formation of additional axial structures . The first category of 24 h phenotypes (87%) mainly displays a gradual decrease in anterior structures . This is comparable to previous phenotypes observed following Xhox3 messenger injection either in Xenopus or in zebrafish that have been classified according to the index of axis deficiency (zf-IAD) . These phenotypes result in anomalies of the development of the neural keel, from microphthalmia to acephaly . The second category (13%) corresponds to the phenotypes described above together with truncal or caudal supernumerary structures . Additional truncal structures are the most prominent of these duplicated phenotypes, displaying a "zipper" shape of axial structures including neural keels and notochords . Caudal duplication presents no evident axis supernumerary structures . The observation of these phenotypes suggests an important role for the eve1 gene in mesodermal cell specification and in the development of the posterior region, and more particularly of the most posterior tail tip where endogenous eve1 messengers are found. Proc Int Conf Intell Syst Mol Biol, 1995, 3, 367 - 75 Identification of human gene structure using linear discriminant functions and dynamic programming; Solovyev VV et al.; Development of advanced technique to identify gene structure is one of the main challenges of the Human Genome Project . Discriminant analysis was applied to the construction of recognition functions for various components of gene structure . Linear discriminant functions for splice sites, 5'-coding, internal exon, and 3'-coding region recognition have been developed . A gene structure prediction system FGENE has been developed based on the exon recognition functions . We compute a graph of mutual compatibility of different exons and present a gene structure models as paths of this directed acyclic graph . For an optimal model selection we apply a variant of dynamic programming algorithm to search for the path in the graph with the maximal value of the corresponding discriminant functions . Prediction by FGENE for 185 complete human gene sequences has 81% exact exon recognition accuracy and 91% accuracy at the level of individual exon nucleotides with the correlation coefficient (C) equals 0.90 . Testing FGENE on 35 genes not used in the development of discriminant functions shows 71% accuracy of exact exon prediction and 89% at the nucleotide level (C = 0.86) . FGENE compares very favorably with the other programs currently used to predict protein-coding regions . Analysis of uncharacterized human sequences based on our methods for splice site (HSPL, RNASPL), internal exons (HEXON), all type of exons (FEXH) and human (FGENEH) and bacterial (CDSB) gene structure prediction and recognition of human and bacterial sequences (HBR) (to test a library for E . coli contamination) is available through the University of Houston, Weizmann Institute of Science network server and a WWW page of the Human Genome Center at Baylor College of Medicine. Proc Int Conf Intell Syst Mol Biol, 1995, 3, 309 - 13 Subclass approach for mutational spectrum analysis; Rogozin IB et al.; Analysis and comparison of mutational spectra represents a burning question in molecular biology . We report an algorithm based upon the SEM subclass approach (SEM--stochastique, estimations, maximizations) . Any real mutational spectrum is regarded as a mixture of standard binomial distributions . The separation procedure is run by rounds . Each iteration includes simulation, maximization and estimation . The algorithm has been checked on random spectra with the preset parameters and on real mutational spectra . As has been shown, any real mutational spectrum can be represented as a mixture of two and more binomial distributions, of which one contains hotspots of mutation. Proc Int Conf Intell Syst Mol Biol, 1995, 3, 292 - 9 Investigations of Escherichia coli promoter sequences with artificial neural networks: new signals discovered upstream of the transcriptional startpoint; Pedersen AG et al.; In this paper we present a novel method for using the learning ability of a neural network as a measure of information in local regions of input data . Using the method to analyze Escherichia coli promoters, we discover all previously described signals, and furthermore find new signals that are regularly spaced along the promoter region . The spacing of all signals correspond to the helical periodicity of DNA, meaning that the signals are all present on the same face of the DNA helix in the promoter region . This is consistent with a model where the RNA polymerase contacts the promoter on one side of the DNA, and suggests that the regions important for promoter recognition may include more positions on the DNA than usually assumed . We furthermore analyze the E . coli promoters by calculating the Kullback Leibler distance, and by constructing sequence logos. Pr