Escherichia coli

Mol Med, 1996 Jan, 2(1), 134 - 42
Response of Mycobacterium tuberculosis to reactive oxygen and nitrogen intermediates; Garbe TR et al.; BACKGROUND: Mycobacterium tuberculosis is a significant human pathogen capable of replicating in mononuclear phagocytic cells . Exposure to reactive oxygen and nitrogen intermediates is likely to represent an important aspect of the life cycle of this organism . The response of M . tuberculosis to these agents may be of significance for its survival in the host . MATERIALS AND METHODS: Patterns of de novo proteins synthesized in M . tuberculosis H37Rv exposed to compounds that generate reactive oxygen and nitrogen intermediates were studied by metabolic labeling and two-dimensional electrophoresis . RESULTS: Menadione, a redox cycling compound which increases intracellular superoxide levels, caused enhanced synthesis of seven polypeptides, six of which appeared to be heat shock proteins . Chemical release of nitric oxide induced eight polypeptides of which only one could be identified as a heat shock protein . Nitric oxide also exhibited a mild inhibitory action on general protein synthesis in the concentration range tested . Hydrogen peroxide did not cause differential gene expression and exerted a generalized inhibition in a dose-dependent manner . Cumene hydroperoxide caused mostly inhibition but induction of two heat shock proteins was detectable . CONCLUSIONS: The presented findings indicate major differences between M . tuberculosis and the paradigms of oxidative stress response in enteric bacteria, and are consistent with the multiple lesions found in oxyR of this organism . The effect of hydrogen peroxide, which in Escherichia coli induces eight polypeptides known to be controlled by the central regulator oxyR, appears to be absent in M . tuberculosis . Superoxide and nitric oxide responses, which in E . coli overlap and are controlled by the same regulatory system soxRS, represent discrete and independent phenomena in M . tuberculosis.

Arch Virol, 1996, 141(9), 1689 - 701
The M(r) 43K major capsid protein of rice ragged stunt oryzavirus is a post-translationally processed product of a M(r) 67,348 polypeptide encoded by genome segment 8; Upadhyaya NM et al.; The nucleotide sequence of DNA complementary to rice ragged stunt oryzavirus (RRSV) genome segment 8 (S8) of an isolate from Thailand was determined . RRSVS8 is 1914 bp in size and contains a single large open reading frame (ORF) spanning nucleotides 23 to 1810 which is capable of encoding a protein of M(r) 67,348 . The N-terminal amino acid sequence of a approximately 43K virion polypeptide matched to that inferred for an internal region of the S8 coding sequence . These data suggest that the 43K protein is encoded by S8 and is derived by a proteolytic cleavage . Predicted polypeptide sizes from this possible cleavage of S8 protein are 26K and 42K . Polyclonal antibodies raised against a maltose binding protein (MBP)-S8 fusion polypeptide (expressed in Escherichia coli) recognised four RRSV particle associated polypeptides of M(r) 67K, 46K, 43K and 26K and all except the 26K polypeptide were also highly immunoreactive to polyclonal antibodies raised against purified RRSV particles . Cleavage of the MBP-S8 fusion polypeptide with protease Factor X produced the expected 40K MBP and two polypeptides of apparent M(r) 46K and 26K . Antibodies to purified RRSV particles reacted strongly with the intact fusion protein and the 46K cleavage product but weakly to the 26K product . Furthermore, in vitro transcription and translation of the S8 coding region revealed a post-translational self cleavage of the 67K polypeptide to 46K and 26K products . These data indicate that S8 encodes a structural polypeptide, the majority of which is auto-catalytically cleaved to 26K and 46K proteins . The data also suggest that the 26K protein is the self cleaving protease and that the 46K product is further processed or undergoes stable conformational changes to a approximately 43K major capsid protein.

Bioelectromagnetics, 1996, 17(4), 312 - 21
Resonance effect of millimeter waves in the power range from 10(-19) to 3 x 10(-3) W/cm2 on Escherichia coli cells at different concentrations; Belyaev IY et al.; The effect of millimeter waves (MMWs) on the genome conformational state (GCS) of E . coli AB1157 cells was studied by the method of anomalous viscosity time dependencies (AVTD) in the frequency range of 51.64-51.85 GHz . The 51.755 GHz resonance frequency of the cell reaction to MMWs did not depend on power density (PD) in the range from 10(-19) to 3 x 10(-3) W/cm2 . The half-width of the resonant reaction of cells showed a sigmoid dependence on PD, changing from 3 MHz to 100 MHz . The PD dependence of the half-width had the same shape for different concentrations of exposed cells (4 x 10(7) and 4 x 10(8) cells/ml), whereas the magnitude of the 51.755 GHz resonance effect differed significantly and depended on the PD of MMW exposure . Sharp narrowing of the 51.755 GHz resonance in the PD range from 10(-4) to 10(-7) W/cm2 was followed by an emergence of new resonance frequencies . The PD dependence of the MMW effect at one of these resonance frequencies (51.674 GHz) differed markedly from the corresponding dependence at the 51.755 GHz resonance, the power window occurring in the range from 10(-16) to 10(-8) W/cm2 . The results obtained were explained in the framework of a model of electron-conformational interactions . The frequency-time parameters of this model appeared to be in good agreement with experimental data.

Hum Mutat, 1996, 8(3), 236 - 46
PKU mutation (D143G) associated with an apparent high residual enzyme activity: expression of a kinetic variant form of phenylalanine hydroxylase in three different systems; Knappskog PM et al.; We have used three complementary in vitro systems to express the human phenylalanine hydroxylase (PAH) gene at high levels . Recombinant PAH was expressed in Escherichia coli (as a fusion protein), in human kidney cells and in a cell-free in vitro transcription-translation system . These systems were used to characterize a novel kinetic variant form (D143G) of the enzyme . The recombinant D143G mutant enzyme had the same physicochemical properties as the wild-type PAH and was stable when expressed in eukaryotic cells . Enzyme activity studies of the D143G mutant enzyme, produced in the three expression systems, revealed a kinetic variant form with reduced affinity for L-Phe (about 2.4-fold increase in the S0.5 value) as well as reduced affinity for tetrahydrobiopterin (BH4) (about 2-fold increase in the apparent Km) . At standard assay conditions (1 mM L-Phe, t5 microM BH4) the residual activity of the mutant enzyme was high and variable (52%, 33%, and 102%) when analysed in the three different systems . The high residual activities of the mutant enzyme obtained at these conditions were not in agreement with the classical PKU phenotype found in a patient compound heterozygous for the termination mutation G272X and the novel D143G mutation . However, when the D143G mutant enzyme was assayed at lower concentrations of L-Phe (100-300 microM) and BH4 (10 microM) the residual activities were compatible with severely reduced hydroxylation of L-Phe and the classical PKU phenotype.

Kidney Blood Press Res, 1996, 19(3-4), 205 - 8
Typical and atypical hemolytic uremic syndrome; Proesmans W; The hemolytic uremic syndrome is the most frequent cause of acute renal failure in childhood . In the vast majority of patients, the syndrome of acute hemolysis, thrombopenia and renal dysfunction is preceded by an episode of diarrhea with or without bloody stools . This colitis is caused by different strains of Escherichia coli which produce shiga like toxins . These toxins are responsible for both hemolysis and renal disease . There are good reasons for distinguishing patients with D (+) HUS and those without prodromal diarrhea {D (-) HUS}, especially since the outcome in the latter group is less predictable and on average fairly unfavorable . D (+) HUS has also been labeled 'typical HUS' and D (-) HUS as 'atypical HUS' . This has led to some oversimplification, in that atypical has become synonymous with poor outcome . Our experience comprises 20 D (-) HUS patients, of whom 14 did extremely well . Scrutinizing the data of our patients lead to the conclusion that, within the D (-) group, some have a 'typical course' and display complete cure whereas those with an 'atypical course' either die or have severe sequelae.

Biochem Cell Biol, 1996, 74(3), 363 - 72
The yeast nucleoporin Nsp1 binds nuclear localization sequences in vitro; Barth W et al.; Facilitated transport of proteins into the nucleus requires nuclear localization sequences (NLSs) be present in the protein destined for the nucleus . The specific binding of NLSs by components of the nuclear transport apparatus is essential for these targeting reactions . We now report that the yeast nucleoporin Nsp1 binds specifically nuclear localization sequences in vitro . This nucleoporin recognizes several NLSs that are functional for nuclear targeting in vivo, including the NLS of SV40 T-antigen and of the yeast transcription factor Ga14 . Nsp1 is organized into three domains, and we have located NLS binding sites to the N-terminal portion and the middle repetitive region of the protein . For the interaction between the NLS of SV40 T-antigen and Nsp1, we obtained association constants of 1.2 x 10(7) M-1 and 5 x 10(7) M-1 . An association constant of 5 x 10(7) M-1 was determined for NLS binding to the repetitive domain of Nsp1 . We analyzed binding of Nsp1 and its domains to a mutant version of the NLS derived from SV40 T-antigen, which poorly functions for nuclear targeting in vivo . The affinity for the mutant signal was about two orders of magnitude lower than for the wild-type NLS.

Virus Genes, 1996, 12(3), 265 - 74
Hamster polyomavirus-encoded proteins: gene cloning, heterologous expression and immunoreactivity; Ulrich R et al.; The hamster polyomavirus (HaPV) is associated with spontaneously appearing skin epithelioma of the Syrian hamster Z3 strain . Virus particles prepared from the skin epithelioma cause lymphoma and leukemia when injected into newborn hamsters from a distinct Syrian hamster colony (HaP); in contrast to the skin epithelioma the hemopoietic tumors are virus free but accumulate viral DNA . To study the humoral immune response of HaPV-infected Z3 hamsters we produced recombinant HaPV proteins in Escherichia coli as beta-galactosidase-, TrpE- and dihydrofolate reductase-fusion proteins or as non-fused proteins . Recombinant plasmids carried segments of all putative early and late HaPV proteins . The recombinant proteins were detected in stained SDS polyacrylamide gels and in Western blots using monoclonal anti-TrpE and anti-beta-galactosidase antibodies and sera of HaPV-infected hamsters . Sera from HaPV-infected Z3 hamsters and crude lysates of all clones were applied to Western blots to characterize the humoral immune response in the animals . HaPV-specific antibodies were found to be directed against early protein segments translated from the first common exon and from the second unique exon of LT and MT, resp., as well as against the late proteins VP1 and VP2/3 . The almost complete VP2 was recognized by all sera whereas VP1 was detected only by a half of the sera . Our data suggest the presence of at least 2 immunodominant regions in VP2, one in the C-terminal VP1 and at least 4 in early proteins.

Proc Int Conf Intell Syst Mol Biol, 1996, 4, 176 - 81
Genome-scale DNA sequence recognition by hybridization to short oligomers; Milosavljevic A et al.; Recently developed hybridization technology (Drmanac et al . 1994) enables economical large-scale detection of short oligomers within DNA fragments . The newly developed recognition method (Milosavljevic 1995b) enables comparison of lists of oligomers detected within DNA fragments against known DNA sequences . We here describe an experiment involving a set of 4,513 distinct genomic E.coli clones of average length 2kb, each hybridized with 636 randomly selected short oligomer probes . High hybridization signal with a particular probe was used as an indication of the presence of a complementary oligomer in the particular clone . For each clone, a list of oligomers with highest hybridization signals was compiled . The database consisting of 4,513 oligomer lists was then searched using known E.coli sequences as queries in an attempt to identify the clones that match the query sequence . Out of a total of 11 clones that were recognized at highest significance level by our method, 8 were single-pass sequenced from both ends . The single-pass sequenced ends were then compared against the query sequences . The sequence comparisons confirmed 7 out of the total of 8 examined recognitions . This experiment represents the first successful example of genome-scale sequence recognition based on hybridization data.

Proc Int Conf Intell Syst Mol Biol, 1996, 4, 165 - 75
Applications of GeneMark in multispecies environments; McIninch JD et al.; This paper is supposed to bridge the gap between practical experience in using GeneMark for a rapidly widening repertoire of genomes, and the available publications that determine and compare the gene prediction accuracy of the GeneMark method for different genomes . Here we focus on the genome-specific variability of prediction error rates and their sources . DNA sequence inhomogeneity is present both in training and control sets of coding and non-coding regions . Coding region inhomogeneity, caused by differences in sequence composition between "native" and horizontally transferred genes or between genes expressed at different levels, contributes to the false negative error rate . Inhomogeneity of non-coding region may frequently be caused by the presence of unnoticed genes and contributes to the false positive error rate . We have documented such unnoticed genes in GenBank sequences for several species Some of protein products of these genes have been characterized by similarity search methods . For others, which we call "pioneer genes", no significant similarity has been found at a protein sequence level although the confidence of GeneMark prediction is high . For instance, to date a majority of those pioneer gene predictions made for E . coli now show strong similarity to more recently characterized proteins that have been added to protein sequence database . Another practical question is related to genomic sequence inhomogeneity at interspecies level: if GeneMark has not been trained for a particular species, is it possible to apply models derived for phylogenetically close genomes? The answer is, yes . The results of cross-species gene prediction experiments show that cross-species prediction can often be reasonably accurate.

Proc Int Conf Intell Syst Mol Biol, 1996, 4, 116 - 24
HinCyc: a knowledge base of the complete genome and metabolic pathways of H . influenzae; Karp PD et al.; We present a methodology for predicting the metabolic pathways of an organism from its genomic sequence by reference to a knowledge base of known metabolic pathways . We applied these techniques to the genome of H . influenzae by reference to the EcoCyc knowledge base to predict which of 81 metabolic pathways of E . coli are found in H . influenzae . The resulting prediction is a complex hypothesis that is presented in computer form as HinCyc: an electronic encyclopedia of the genes and metabolic pathways of H . influenzae . HinCyc connects the predicted genes, enzymes, enzyme-catalyzed reactions, and biochemical pathways in a WWW-accessible knowledge base to allow scientists to explore this complex hypothesis.

Proc Int Conf Intell Syst Mol Biol, 1996, 4, 109 - 15
A probabilistic classification system for predicting the cellular localization sites of proteins; Horton P et al.; We have defined a simple model of classification which combines human provided expert knowledge with probabilistic reasoning . We have developed software to implement this model and have applied it to the problem of classifying proteins into their various cellular localization sites based on their amino acid sequences . Since our system requires no hand tuning to learn training data, we can now evaluate the prediction accuracy of protein localization sites by a more objective cross-validation method than earlier studies using production rule type expert systems . 336 E . coli proteins were classified into 8 classes with an accuracy of 81% while 1484 yeast proteins were classified into 10 classes with an accuracy of 55% . Additionally we report empirical results using three different strategies for handling continuously valued variables in our probabilistic reasoning system.

Chin J Biotechnol, 1996, 12(1), 17 - 24
High level expression of oryzacystatin in Escherichia coli; Zhou Z et al.; A rice cDNA library of immature seeds has already been constructed, from which colonies carrying oryzacystatin cDNA were isolated . The coding region of the oryzacystatin (a thiol proteinase inhibitor) gene was amplified by the Polymerase Chain Reaction (PCR) and inserted downstream of lambdaPRPL promoter of E . coli thermal-inducible expression vector pBV220 . An oryzacystatin expression plasmid pBVC9 was therefore obtained . Shifting the culture temperature of E . coli DH5 alpha (pBVC9) from 30 degrees C to 42 degrees C led to a high level expression of oryzacystatin . The result of SDS-PAGE showed a distinct band of 12.0 kDa which accounts for at least 10% of the total soluble proteins . The inhibitory activity of the total soluble proteins of E . coli DH5 alpha (pBVC9) toward papain was confirmed by using that of E . coli DH5 alpha (pBV220) as a control.

Cell Motil Cytoskeleton, 1996, 34(4), 313 - 23
Site-directed mutagenesis enabled preparation of a functional fluorescent analog of profilin: biochemical characterization and localization in living cells; Tarachandani A et al.; The preparation of fluorescent profilin analogs for binding and spectroscopic studies, in vitro and in vivo, has been hampered by the poor chemical reactivity of this protein in its native form . We have addressed this problem by labeling a mutant, chemically reactive form of profilin . Site-directed mutagenesis was first used to replace a serine residue in a non-essential domain with a reactive cysteine residue . The mutant protein was expressed in Escherichia coli and reacted with tetramethylrhodamine iodoacetamide . In vitro assays indicated that the fluorescent profilin maintained its ability to bind actin, polyproline, and PIP2, to inhibit actin polymerization, and to stimulate actin nucleotide exchange . Fluorescence spectroscopy showed that neither the excitation nor the emission of the analog was sensitive to the interaction with actin or polyproline . However, binding of PIP2 caused a 75% quenching of the fluorescent signal, suggesting a dramatic change in the immediate environment of the probe . When the fluorescent profilin was microinjected into living NRK cells, it became localized at cell-cell junctions and discrete sites near the anterior end, where it colocalized with aggregates of unpolymerized actin . Different engineered forms of profilin with fluorophores located at defined sites should greatly facilitate the study of its interactions with various ligands and cellular structures.

Microbiol Immunol, 1996, 40(1), 71 - 5
Characterization by Western blotting of mouse intestinal glycoproteins bound by Escherichia coli heat-labile enterotoxin type I; Shida K et al.; Escherichia coli heat-labile enterotoxin type I (LT-I)-binding galactoproteins, which were not recognized by cholera toxin, were detected in intestinal epithelial cells of BALB/c mouse by Western blotting . Inhibitory studies using lectins and modifications of sugar chain suggest that LT-I recognizes certain mucin-type sugar chains containing the terminal Galbeta1-3GalNAc sugar sequence in the galactoproteins . The terminal sugar sequence is identical to that of GM1 ganglioside, the well-documented functional receptor for LT-I.

Microbiol Immunol, 1996, 40(1), 33 - 8
Crystallization of synthetic Escherichia coli-type lipid A; Kato N et al.; Synthetic Escherichia coli-type lipid A formed hexagonal plate crystals when it was precipitated by the addition of 2 volumes of 95% ethanol containing 375 mM MgCl2 and incubated in 70% ethanol containing 250 mM MgCl2 at 4 C for 10 days . Analyses of crystals by electron diffraction and synchrotron X-ray diffraction showed that crystals consist of hexagonal lattices with the lattice constant (a side of the lozenge as a unit cell on the basal plane) of 4.62 angstrom and the longitudinal axis (perpendicular to the basal plane) of 49.3 +/- 1.3 angstrom . Results suggest that the previous finding that various kinds of R-form lipopolysaccharides crystallized but free lipid A isolated by acid hydrolysis from Re lipopolysaccharide did not crystallize under the same experimental conditions (Kato et al, J . Bacteriol., 172:1516-1528, 1990) is due to structural changes of lipid A occurring during the procedure of isolation of free lipid A.

HPB Surg, 1996, 9(2), 61 - 9
Intestinal endotoxins as co-factors of liver injury in obstructive jaundice; Mentes BB et al.; The concept of endotoxin-mediated rather than direct liver injury in biliary obstruction was investigated using the experimental rat model of bile duct ligation (BDL) and small bowel bacterial overgrowth (SBBO) . Small identical doses of intravenous endotoxin (bacterial LPS) caused a significantly more severe liver injury in rats with BDL, compared with sham-operated rats, suggesting the possible contribution of LPS in this type of liver damage . BDL was then combined with surgically created jejunal self-filling blind loops, which resulted in SBBO . Plasma LPS level increased significantly, and once again a more severe liver injury, determined by liver histology and serum gamma-glutamyl transpeptidase levels, was observed compared with the control group of rats with BDL+self-emptying blind loops . The data presented suggest that small amounts of exogenous LPS and/or the ordinarily innocous amounts of LPS constantly absorbed from the intestinal tract may be critical in the hepatic damage caused by obstruction of the biliary tract.

Adv Food Nutr Res, 1996, 40, 95 - 106
Molecular biology in nutrition research: modeling of folate metabolism; Lin BF et al.; Model CHO cells obtained by transfecting CHO mutants with the E . coli and human folylpolyglutamate synthetase genes have proven useful for assessing the role of folylpolyglutamates in one carbon metabolism and for delineating how folate intracellular stores are regulated . Cells expressing enzymes in specific subcellular compartments, expressing enzymes with different substrate specificity's, and expressing enzyme activity at different levels, all in a common background, in this case the CHO cell, has allowed the development of kinetic models for assessing the role of folypolyglutamate synthetase in folate retention and in the cytotoxicity of antifolates.

EXS, 1996, 77, 57 - 78
Involvement of molecular chaperones in intracellular protein breakdown; Sherman MY et al.; In all cells and organelles, there exist multiple molecular chaperones, which not only can facilitate the proper folding, transport and assembly of multimeric structures, but also appear to function in intracellular protein degradation . Recent findings in E . coli indicate that the major chaperones of the Hsp70 (DnaK) and Hsp60 (GroEL) families and their cofactors (DnaJ, GrpE or GroEL and Trigger Factor) associate with certain short-lived proteins (e.g . mutant polypeptides or regulatory proteins) and promote their degradation by the ATP-dependent proteases, La (lon or ClpP) . Moreover, ATPases of ClpA/B family not only function in ATP-dependent proteolysis in association with the Clp protease, but by themselves can facilitate or act as chaperones in protein assembly . In eukaryotes, Hsp70 and their cofactors, the DnaJ homologs, are essential for the ubiquitination of certain abnormal and regulatory proteins and in the breakdown of certain polyubiquitinated polypeptides by 26S proteasome . It is likely that the chaperones function in proteolysis either as elements that faciliate the recognition of unfolded proteins or that the chaperones partially unfold substrates to make them more susceptible to proteases or ubiquitinating enzymes.

Nephron, 1996, 73(4), 587 - 96
Escherichia coli-macrophage interactions modulate mesangial cell proliferation and matrix synthesis; Sharma S et al.; Patients with chronic renal interstitital diseases often develop glomerular lesions (focal segmental glomerular sclerosis) . Because mesangial expansion (enhanced mesangial cell (MC) growth and matrix accumulation) has been demonstrated to precede the development of focal segmental glomerulosclerosis, we studied the effect of the interaction between bacteria such as Escherichia coli and macrophages on MC proliferation and matrix synthesis . We determined the effect of control media (CM), E . coli supernatant (ESP), serum-free macrophage supernatant (MSP), and E . coli-treated macrophage supernatants (HB101-MSP, H10-MSP) on the proliferation of MCs and synthesis of laminin (a component of mesangial matrix) . ESP did not alter MC growth, whereas E . coli MSP increased the mean MC number by 5- to 6-fold when compared to cells treated with CM . Both HB101-MSP and H10-MSP stimulated greater (p < 0.05) incorporation of {3H}thymidine when compared with MSP (HB101-MSP 3.1 +/- 0.4, H10-MSP 2.7 +/- 0.3 vs . MSP 1.6 +/- 0.2 x 10(6) cpm/micrograms protein) . When MC proliferation was judged by incorporation of bromodeoxyuridine, both HB101-MSP- and H10-MSP-treated cells showed a greater (p < 0.01) number of proliferating cells compared with cells treated with either MSP or CM . MC treated with H10-MSP grew in a specific pattern and showed a tendency to form hillocks (foci of cell proliferation and matrix aggregation) . Both HB101-MSP and H10-MSP enhanced (p < 0.01) synthesis of laminin compared with CM . HB101-MSP-induced enhanced laminin synthesis was attenuated when MCs were treated with anti-transforming growth factor (TGF)-beta antibodies . HB101-MSP also increased mRNA expression of TGF-beta by MCs . These results indicate that E . coli-macrophage interaction has the potential to cause mesangial expansion.

Arch Virol, 1996, 141(8), 1493 - 507
Epitope mapping of capsid proteins VP2 and VP3 of infectious bursal disease virus; Yamaguchi T et al.; Twenty hybridoma cell lines producing monoclonal antibodies (MAbs) against serotype 1 infectious bursal disease virus (IBDV) of GBF-1 and the attenuated GBF-1E strains were produced . The MAbs recognized major structural proteins VP2 and VP3 . MAb recognition sites were mapped using recombinant Escherichia coli clones which expressed N-terminal and (or) C-terminal truncated virus antigens, and competitive-binding assays . At least 3 conformation-dependent serotype 1 specific virus neutralizing antigenic sites and a linear antigenic site were defined on VP2 and VP3, respectively . Two of the conformational virus neutralizing antigenic sites were localized in the central area of VP2 consisting of 156 amino acid residues, and the linear epitope was localized in C-terminal 105 amino acid residues of VP3 . Another conformational virus neutralizing antigenic site recognized with the virus neutralizing MAb GK-5 was not defined because GK-5 did not react with virus antigen expressed in recombinant E . coli . The conformational antigenic site was supposed to be composed of tertiary or quaternary protein structure, which may not be constructed in recombinant E . coli.

Surg Today, 1996, 26(8), 610 - 7
Induction mechanism of small intestinal lesions caused by intravenous injection of endotoxin in rats; Shindo M et al.; The pathogenesis of intestinal damage caused by bolus intravenous injection of endotoxin (ETX; 3 mg/kg) was investigated . Administration of ETX to rats induced reddish discoloration suggestive of bleeding, increased hemoglobin amounts, and leakage of plasma protein in the intestine . However, light microscopic examination of the intestine demonstrated blood congestion of the microvessels . Plasma accumulation was partially inhibited by combined pretreatment with a histamine H1 antagonist and a serotonin (5-HT) antagonist . Neither a 5-lipoxygenase inhibitor, a soybean trypsin inhibitor, nor atropine was observed to inhibit plasma accumulation . Both the intestinal leakage of plasma and the accumulation of hemoglobin were completely inhibited by indomethacin, a selective thromboxane A synthetase inhibitor (OKY 1581), and a stable PGI2 analogue (beraprost) . Intravital microscopic observation of the microvessels of the small intestinal villi demonstrated microthrombus formation within several minutes after the injection of ETX, and pretreatment with OKY 1581 attenuated the formation of microthrombus . Platelet counts decreased significantly 10 min after ETX administration, and the decrease was not inhibited by pretreatment with either OKY 1581 or beraprost . Prothrombin time (PT) and activated partial thromboplastin time (APTT) were not prolonged . These observations thus suggest that microcirculatory disturbances by platelet thrombus, which are mediated by thromboxane A2 at least in part, play an important role in ETX-induced intestinal damage.

Free Radic Biol Med, 1996, 21(3), 261 - 73
Cupric ion/ascorbate/hydrogen peroxide-induced DNA damage: DNA-bound copper ion primarily induces base modifications; Drouin R et al.; The kinetics of frank DNA strand breaks and DNA base modifications produced by Cu(II)/ascorbate/H2O2 were simultaneously determined in purified human genomic DNA in vitro . Modified bases were determined by cleavage with Escherichia coli enzymes Nth protein (modified pyrimidines) and Fpg protein (modified purines) . Single-stranded lesion frequency before (frank strand breaks) and after (modified bases) Nth or Fpg protein digestion was quantified by neutral glyoxal gel electrophoresis . Dialysis of EDTA-treated genomic DNA purified by standard proteinase K digestion/phenol extraction was necessary to remove low molecular weight species, probably transition metal ions and metal ion chelators, which supported frank strand breaks in the presence of ascorbate + H2O2 without supplemental copper ions . We then established a kinetic model of the DNA-damaging reactions caused by Cu(II) + ascorbate + H2O2 . The principal new assumption in our model was that DNA base modifications were caused exclusively by DNA-bound Cu(I) and frank strand breaks by non-DNA-bound Cu(I) . The model was simulated by computer using published rate constants . The computer simulation quantitatively predicted: (1) the rate of H2O2 degradation, which was measured using an H2O2-sensitive electrode, (2) the linearity of accumulation of DNA strand breaks and modified bases over the reaction period, (3) the rate of modified base accumulation, and (4) the dependence of modified base and frank strand production on initial Cu(II) concentration . The simulation significantly overestimated the rate of frank strand break accumulation, suggesting either that the ultimate oxidizing species that attacks the sugar-phosphate backbone is a less-reactive species than the hydroxyl radical used in the model and/or an unidentified hydroxyl radical-scavenging species was present in the reactions . Our experimental data are consistent with a model of copper ion-DNA interaction in which DNA-bound Cu(I) primarily mediates DNA base modifications and nonbound Cu(I) primarily mediates frank strand break production.

Eur Urol, 1996, 30(1), 96 - 102
Human papillomavirus in tissue of bladder and bladder carcinoma specimens . A preliminary study; Ludwig M et al.; OBJECTIVE: To evaluate the significance of HPV type 6b, 11, 16 and 18 together with type-specific antibodies in the serum of bladder carcinoma . METHODS: The prevalence of HPV type 6b, 11, 16 and 18 in bladder tumor, normal bladder and urethra together with type-specific antibodies in serum was investigated in 23 patients with bladder cancer and 9 patients with chronic cystitis . HPV DNA analysis was done by polymerase chain reaction (PCR) . Open reading frames of HPV were expressed in Escherichia coli as beta-galactosidase fusion proteins . RESULTS: HPV 6b was demonstrated in the tumor tissue of 6 patients (19%), and in the nonmalignant specimens of 6 further patients (19%) . HPV 16/18 was only found in the urethral swabs of 2 patients (6%) . Anti-HPV antibodies were positive in 7 patients (22%) . There was no association between the demonstration of HPV 6b and the occurrence of bladder tumor in this study . CONCLUSION: Though, in this study, HPV was not associated with bladder cancer, further investigation is necessary to elucidate the role of HPV 6b in bladder tissue possibly by a semiquantitative PCR in tissue samples and of anti-HPV antibodies in serum.

RNA, 1996 Jan, 2(1), 88 - 101
Methyltransferase-specific domains within VP-39, a bifunctional protein that participates in the modification of both mRNA ends; Shi X et al.; VP39 is a bifunctional vaccinia virus protein that acts as both a cap- dependent 2'-O-Methyltransferase and a poly(A) polymerase processivity factor . An analysis of C-terminal truncation mutants of a GST-VP39 fusion protein indicated the presence of a protease-sensitive C-terminal "tail" 36-43 amino acids in length that is non-essential for VP39 function . Fourteen new VP39 pointmutants, containing either single or multiple-clustered amino acid substitutions, were expressed in Escherichia coli . Of the eight that retained either one or both of the activities of VP39, seven were specifically methyltransferase-defective . None was specifically defective in adenylyltransferase stimulation . The nature of the methyltransferase defects in 10 of the methyltransferase-specific defectives, identified both herein and in a previous study (Schnierle BS, Gershon PD, Moss B, 1994, J biol Chem 269:20700-20706), was investigated using two novel substrate-binding assays . Three of the mutants (and possible a fourth), whose lesions were juxtaposed and centrally located within VP39, exhibited anomalous S-adenosyl-(L)-methionine (AdoMet) binding behavior, identifying residues important for AdoMet binding and possible also for catalysis . A surface plasmon resonance-based assay measured the interaction of VP39 with uncapped and 5'-cap 0-terminated oligo(A) . A cap 0- dependent association-rate enhancement was observed for wild-type VP39 and 4 of the 10 mutant proteins . Two others were identified as defective in cap binding, and a third as partially defective . The lesions within the latter three mutants were closely apposed, and located toward the N-terminus of VP39 . We have thus identified regions of VP39 important for interaction with its two substrates for cap-dependent methyltransferase activity: AdoMet and cap 0.

RNA, 1996 Jan, 2(1), 24 - 37
Ribosomal protein L4 from Escherichia coli utilizes nonidentical determinants for its structural and regulatory functions; Li X et al.; Escherichia coli ribosomal protein L4 has two functions: it is a structural component of the 50S ribosomal sub-unit and it is a repressor of both transcription and translation of its own transcription unit, the 11-gene S10 operon . Genetic and biochemical studies have suggested that L4 can interact with 23S rRNA as well as with both RNA interactions . However, no significant similarities between its two RNA targets can be found at the primary or secondary structure level . To test if identical determinants of L4 are involved in both ribosome assembly and autogenous control, we have isolated L4 mutants defective in either of these functions and asked if a mutant protein divested of one function is also deficient in the other . Several mutations eliminated autogenous control, but still allowed assembly of the mutant L4 protein into functional ribosomes . Conversely, several mutant L4 proteins that could not be detected in 50S subunits nevertheless could regulate expression of the S10 operon . These results indicate that the L4 determinants required for autogenous regulation and ribosome incorporation are not congruent.

Biotechnol Prog, 1996 Jan-Feb, 12(1), 51 - 6
Extractive cultivation of recombinant Escherichia coli using aqueous two-phase systems for production and separation of intracellular heat shock proteins; Umakoshi H et al.; The extractive cultivation of recombinant Escherichia coli cells to produce, release, and separate heat shock proteins (HSPs; GroEL and GroES) using poly(ethylene glycol) (PEG)/dextran (Dex) aqueous two-phase systems was developed . The growth rate of E . coli OW10/pND5 cells in the PEG/Dex two-phase media was almost the same value as that in the control media . The addition of 0.1 M potassium phosphate salts (KPi) increased the productivity of HSPs with keeping the growth rate of E . coli cells relatively high . The partition coefficients of HSPs were improved to greater values when phosphate salts were added at a concentration of more than 0.1 M . As a result, PEG/Dex systems supplemented with 0.1 M KPi were found to be the optimized two-phase systems for the extractive cultivation of E . coli cells . In the systems, the HSPs were selectively partitioned to the top phase while cells occupied the bottom phase and the interface between the two phases . This integrated process was extended to a semicontinuous operating mode, where the top phase containing the HSPs was recovered following intermittent heating and ultrasonic irradiation . The bottom phase containing cells and cell debris was recycled together with new top phase solution to repeat production and recovery of HSPs.

Indian J Gastroenterol, 1996 Jan, 15(1), 26 - 7
Neonatal cholestasis syndrome due to galactosemia; Kumar M et al.; We report here a patient with neonatal cholestasis syndrome due to galactosemia, a rare entity . The child died of Escherichia coli septicemia.

J Protein Chem, 1996 Jan, 15(1), 103 - 13
Characterization of gamma-crystallin from the eye lens of bullfrog: complexity of gamma-crystallin multigene family as revealed by sequence comparison among different amphibian species; Lu SF et al.; gamma-Crystallin is the major and most abundant lens protein present in the eye lens of lower vertebrates such as amphibian and piscine species . To facilitate structural characterization of gamma-crystallins isolated from the lens of the bullfrog (Rana catesbeiana), a cDNA mixture was synthesized from the poly(A)+mRNA isolated from fresh eye lenses . cDNA encoding gamma-crystallin was then amplified using polymerase chain reaction (PCR) based on two primers designed according to the relatively conserved N- and C-terminal sequences of known gamma-crystallins from teleostean fishes . PCR-amplified product corresponding to gamma-crystallin isoforms was obtained, which was then subcloned in pUC18 vector and transformed into Escherichia coli strain JM109 . Plasmids containing amplified gamma-crystallin cDNAs were purified and prepared for nucleotide sequencing by the dideoxynucleotide chain-termination method . Sequencing several clones containing DNA inserts of about 0.54 kb revealed the presence of two isoforms with an open reading frame of 534 base pairs, covering two gamma-crystallins each with a deduced protein sequence of 177 amino acids including the translation-initiating methionine . These gamma-crystallins of pI 6.364 and 6.366 contain a low-methionine content of 2.81%, in contrast to 11-16% obtained for those gamma-crystallins with high-methionine content from most teleostean lenses . Pairwise sequence comparison of bullfrog gamma-crystallins with those published sequences of gamma-crystallins from carp, shark, Xenopus and another Rana frog, bovine, and human lenses indicates that there is only 46-63% sequence similarity among these species, revealing that amphibians possess a very complex and heterogeneous group of gamma-crystallins even from closely related species of Rana frogs . The sequence analysis and comparison of various isoforms of the frog gamma-crystallin family provide a firm basis for identifying these lens proteins as members of a multigene family more complex than that reported for mammalian gamma-crystallins.

J Protein Chem, 1996 Jan, 15(1), 59 - 61
Do "antisense proteins" exist?
Chou KC, Zhang CT, Elrod DW.
A DNA double helix consists of two complementary strands antiparallel with each other . One of them is the sense chain, while the other is an antisense chain which does not directly involve the protein-encoding process . The reason that an antisense chain cannot encode for a protein is generally attributed to the lack of certain preconditions such as a promotor and some necessary sequence segments . Suppose it were provided with all these preconditions, could an antisense chain encode for an "antisense protein"? To answer this question, an analysis has been performed based on the existing database . Nine proteins have been found that have a 100% sequence match with the hypothetical antisense proteins derived from the known Escherichia coli antisense chains.

J Protein Chem, 1996 Jan, 15(1), 45 - 58
A fluorescence study of Tn10-encoded tet repressor; Wasylewski Z et al.; Steady-state fluorescence quenching and time-resolved measurements have been performed to resolve the fluorescence contributions of the two tryptophan residues, W43 and W75, in the subunit of the homodimer of the Tet repressor from Escherichia coli . The W43 residue is localized within the helix-turn-helix structural domain, which is responsible for sequence-specific binding of the Tet repressor to the tet operator . The W75 residue is in the protein matrix near the tetracycline-binding site . The assignment of the two residues has been confirmed by use of single-tryptophan mutants carrying either W43 or W75 . The FQRS (fluorescence-quenching-resolved-spectra) method has been used to decompose the total emission spectrum of the wild-type protein into spectral components . The resolved spectra have maxima of fluorescence at 349 and 324 nm for the W43 and W75 residues, respectively . The maxima of the resolved spectra are in excellent agreement with those found using single-tryptophan-containing mutants . The fluorescence decay properties of the wild type as well as of both mutants of Tet repressor have been characterized by carrying out a multitemperature study . The decays of the wild-type Tet repressor and W43-containing mutant can be described as being of double-exponential type . The W75 mutant decay can be described by a Gaussian continuous distribution centered at 5.0 nsec with a bandwidth equal to 1.34 nsec . The quenching experiments have shown the presence of two classes of W43 emission . One of the components, exposed to solvent, has a maximum of fluorescence emission at 355 nm, with the second one at about 334 nm . The red-emitting component can be characterized by bimolecular-quenching rate constant, kq equal to 2.6 x 10(9), 2.8 x 10(9), and 2.0 x 10(9) M-1 sec-1 for acrylamide, iodide, and succinimide, respectively . The bluer component is unquenchable by any of the quenchers used . The W75 residue of the Tet repressor has quenching rate constant equal to 0.85 x 10(9) and 0.28 x 10(9) M-1 sec-1 for acrylamide and succinimide, respectively . These values indicate that the W75 is not deeply buried within the protein matrix . Our results indicate that the Tet repressor can exist in its ground state in two distinct conformational states which differ in the microenvironment of the W43 residue.

J Protein Chem, 1996 Jan, 15(1), 27 - 34
Properties of Cys21-mutated muscle acylphosphatases; Modesti A et al.; Cys21 is an invariant residue in muscle acylphosphatases, but is absent in the erythrocyte isozymes . To assess the importance of this residue in the muscle isozymes for catalytic, structural, and stability properties, two gene mutants have been prepared by oligonucleotide-directed mutagenesis and expressed in Escherichia coli cells; in these mutants, the codon for Cys21 was replaced by those for Ser and Ala, respectively . The two mutant enzymes, purified by immunoaffinity chromatography, showed kinetic and structural properties similar to those of the wild-type recombinant enzyme; however, the specific activity of the two mutants, especially that of the C21A mutant, was lower . The urea and thermal stabilities of the mutant enzymes were reduced with respect to those of the wild-type form, contrary to the susceptibility to inactivation by mercuric ions . The reported data support the possibility that Cys21 is involved in the stabilization of the enzyme active-site conformation.

Acta Neuropathol (Berl), 1996, 91(3), 254 - 62
Neuronal and vascular pathology produced by verocytotoxin 2 in the rabbit central nervous system; Mizuguchi M et al.; To study the pathogenesis of the central nervous system (CNS) involvement associated with verocytotoxin-producing Escherichia coli infection, we developed an animal model by administering verocytotoxin 2 to rabbits either intravenously or intrathecally . After an interval of 2-9 days, the rabbits became paralyzed in a dose-dependent manner and in the absence of renal impairment . The minimal intravenous and intrathecal doses that produced these neurological signs were 250 and 0.4 ng/kg, respectively . After intravenous administration, most of the toxin was cleared from the serum within 24 h, with concomitant transition of a small amount into the cerebrospinal fluid . Pathological examination revealed that neurons in various CNS regions showed atrophy, cytoplasmic hyperchromasia and nuclear pyknosis as early as 6 h after administration . The distribution of affected neurons was constant and irrespective of the route of administration . Abnormalities of the blood vessels, such as the thickening of arterioles walls, were noted from 2 days after administration . The vascular lesions became more prominent after the intrathecal injection, which caused thrombosis and multiple infarction . Selective deposition of the toxin on the vessel walls was demonstrated immunohistochemically . Thus, the pathological manifestations of verocytotoxin 2 neurotoxicity consisted essentially of two types of lesions, early neuronal and late vascular, both of which might have developed under the influence of the toxin that had entered the CNS by crossing or circumventing the blood-brain barrier.

Ann Clin Lab Sci, 1996 Jan-Feb, 26(1), 60 - 3
The role of tumor necrosis factor in endotoxic shock; Heidemann SM et al.; Tumor necrosis factor (TNF) has been implicated in hemodynamic changes of endotoxic shock . The temporal relationship of hypotension and TNF release in endotoxemia was studied . Carotid arteries of five intubated rats were cannulated and Escherichia coli 0127:B8 lipopolysaccharide (LPS) was infused over 10 seconds . Arterial blood pressure (ABP), heart rate, and plasma TNF concentrations were measured at 0, 5, 15, 30, and 60 mins . Five to 15 mins after LPS, there was a marked decline in ABP (146 +/- 23 vs 57 +/- 5 mm Hg, p < 0.005), without a significant rise in TNF . The heart rate did not change . From 15 to 60 mins, there was a rise in TNF concentrations (523 +/- 333 vs 5783 +/- 629 pg/ml, p < 0.005) while the same degree of hypotension persisted . It is concluded that early hypotension after acute endotoxemia is not dependent on TNF alone . However, TNF may play a role in sustaining hypotension after endotoxemia.

J Membr Biol, 1996 Jan, 149(2), 113 - 21
Permeability increase induced by Escherichia coli hemolysin A in human macrophages is due to the formation of ionic pores: a patch clamp characterization; Menestrina G et al.; Escherichia coli hemolysin is known to cause hemolysis of red blood cells by forming hydrophilic pores in their cell membrane . Hemolysin-induced pores have been directly visualized in model systems such as planar lipid membranes and unilamellar vesicles . However this hemolysin, like all the members of a related family of toxins called Repeat Toxins, is a potent leukotoxin . To investigate whether the formation of channels is involved also in its leukotoxic activity, we used patch-clamped human macrophages as targets . Indeed, when exposed to the hemolysin, these cells developed additional pores into their membrane . Such exogenous pores had properties very different from the endogenous channels already present in the cell membrane (primarily K+ channels), but very similar to the pores formed by the toxin in purely lipidic model membranes . Observed properties were: large single channel conductance, cation over anion selectivity but weak discrimination among different cations, quasilinear current-voltage characteristic and the existence of a flickering pre-open state of small conductance . The selectivity properties of the toxin channels appearing in phospholipid vesicles were also investigated, using a specially adapted polarization/depolarization assay, and were found to be completely consistent with that of the current fluctuations observed in excised macrophage patches.

Biochimie, 1996, 78(3), 209 - 12
Autogenous control of the suhB gene expression of Escherichia coli; Inada T et al.; The suhB gene encodes a 29-kDa protein that possesses inositol monophosphate activity and its mutations suppress several temperature-sensitive mutations in Escherichia coli . We found that the suhB gene is autoregulated; suhB- mutant strains oversynthesized the suhB mRNA and suhB-lacZ gene/operon fusion products . Since the half-life of the suhB transcript in the suhB mutant was much greater than the half-life in the wildtype strain, the suhB protein is implicated in the control of gene expression by modulating mRNA turnover.

Lung, 1996, 174(3), 181 - 94
Hyperpermeability of pulmonary endothelial monolayer: protective role of phosphodiesterase isoenzymes 3 and 4; Suttorp N et al.; The regulation of endothelial permeability is poorly understood . An increase in endothelial permeability in the pulmonary microvasculature, however, is critical in noncardiogenic pulmonary edema and other diffuse inflammatory reactions . In the present study thrombin and Escherichia coli hemolysin (HlyA), a membrane-perturbing bacterial exotoxin, were used to alter hydraulic permeability of porcine pulmonary artery and human endothelial cell monolayers . We also investigated the pharmacological approach of adenylyl cyclase activation/phosphodiesterase (PDE) inhibition to block endothelial hyperpermeability . Thrombin (1-5 units/ml) and HlyA (0.5-3 hemolytic units/ml) dose and time dependently (> 15 min) increased endothelial permeability . Forskolin, cholera toxin, and prostaglandin E1, which all stimulate adenylyl cyclase activity, abrogated this effect . One mM dibutyryl cAMP, a cell membrane-permeable cAMP analogue, was similarly active . Endothelial hyperpermeability was also reduced dose dependently by inhibitors of different PDE isoenzymes (motapizone, rolipram, and zardaverine, which block PDE3 and/or PDE4) . The effectiveness of PDE inhibitors was increased in the presence of adenylyl cyclase activators . Analysis of cyclic nucleotide hydrolyzing PDE activity in lysates of human umbilical vein endothelial cells showed high activities of PDE isoenzymes 2, 3, and 4 . Consistent with the functional data PDE3 and PDE4 were the major cAMP hydrolysis enzymes in intact endothelial cells . We conclude that the hyperpermeability of pulmonary endothelial monolayers, evoked by thrombin or HlyA, can be blocked by the simultaneous activation of adenylyl cyclase and inhibition of PDEs, especially of PDE3 and PDE4 . The demonstration of PDE isoenzymes 2-4 in human endothelial cells will help optimize this therapeutic approach.

Hum Mutat, 1996, 7(3), 228 - 38
PKU mutation G46S is associated with increased aggregation and degradation of the phenylalanine hydroxylase enzyme; Eiken HG et al.; The G46S mutation in the phenylalanine hydroxylase (PAH) gene was identified by fluorescence-based single-strand conformation polymorphism (F-SSCP) analysis on phenylketonuria (PKU) haplotype 5.9 alleles . DNA sequencing of PAH exon 2 revealed a G-to-A transition in cDNA position 136 . G46S mutations were present on 17 of 236 Norwegian PKU alleles (7.2%) and on 8 of 176 Swedish PKU alleles (4.5%) . Analysis of all 13 exons with the flanking regions further detected a 1316-35c > t polymorphism (PAH intron 12), associated with both G46S and haplotype 5.9 . Three patients were homozygous for the G46S mutation, two were untreated and had mild and severe mental retardation, respectively . The G46S mutation was introduced in the PAH cDNA by site-directed mutagenesis and expressed in three different systems (the pMAL/Escherichia coli system, the pcDNA3/human embryonic kidney (A293) cells, and the pcDNA3/TnT coupled in vitro transcription-translation system) . The mutant recombinant E . coli fusion protein was recovered in high yield and with a specific activity of the purified tetrameric form, which was higher than the wild-type activity . After transient expression in A293 cells, the amount of the G46S protein was only about 3% of the wild type at equal PAH mRNA levels . The fusion protein cleaved by restriction protease factor Xa, as well as the enzyme produced by in vitro transcription-translation, revealed an abnormal susceptibility to form catalytically inactive high-molecular-mass aggregates of the enzyme . This aggregation, followed by an increased cellular degradation of the G46S mutant enzyme, is compatible with the clinical/metabolic phenotype of the affected homozygous and compound heterozygous patients.

Mol Microbiol, 1996 Jan, 19(2), 389 - 96
Transcriptional activation of the dnaA gene encoding the initiator for oriC replication by IciA protein, an inhibitor of in vitro oriC replication in Escherichia coli; Lee YS et al.; Transcription of the Escherichia coli dnaA gene, encoding DnaA protein required for initiation of chromosomal DNA replication at oriC in E . coli, starts from two promoters, 1P and 2P . Gel-shift and DNase I-protection assays revealed that IciA protein, an inhibitor of initiation of in vitro E . coli chromosomal DNA replication at oriC, bound to two sites in the dnaA promoter region . One site is located upstream of promoter 1P, and the second is located downstream of promoter 2P . Whereas IciA protein did not affect transcription from the promoter 2P, transcription from the promoter 1P was specifically enhanced by IciA protein in vivo and in vitro . DnaA protein bound to the DnaA box between the two promoters 1P and 2P, acts as an transcriptional repressor . Under this condition, IciA protein counteracted the repressive effect of DnaA protein on the promoter 1P . These findings suggest that IciA protein may regulate the initiation of chromosomal DNA replication at oriC by controlling expression of the dnaA gene, as well as by inhibiting the initiation of chromosomal DNA replication at oriC.

Mol Microbiol, 1996 Jan, 19(2), 307 - 17
Ambidextrous transcriptional activation by SoxS: requirement for the C-terminal domain of the RNA polymerase alpha subunit in a subset of Escherichia coli superoxide-inducible genes; Jair KW et al.; Purified MalE-SoxS fusion protein specifically stimulated in vitro transcription of the Escherichia coli zwf, fpr, fumC, micF, nfo, and sodA genes, indicating that activation of the superoxide regulon requires only SoxS . As in vivo, a 21 bp sequence adjacent to the zwf promoter was able to activate transcription of an heterologous promoter in vitro . Activation of zwf and fpr transcription required the C-terminal domain (CTD) of the RNA polymerase alpha subunit, while stimulation of fumC, micF, nfo, and sodA transcription was independent of CTD truncation . Thus, like the catabolite gene activator protein (CAP), SoxS is an 'ambidextrous' activator, activation only requiring the alpha CTD in a subset of regulated promoters . Indeed, the -35 hexamers of the zwf and fpr promoters lie downstream of the respective MalE-SoxS binding sites, while the binding sites of fumC, micF, nfo, and sodA overlap their -35 promoter hexamers.

Mol Microbiol, 1996 Jan, 19(2), 241 - 8
Differential mRNA stability of the cspA gene in the cold-shock response of Escherichia coli; Goldenberg D et al.; Exposure of bacterial cells to temperature changes induces the synthesis of a set proteins . We investigated the control of expression of the cspA gene, coding for the major cold-shock protein of Escherichia coli . This protein was shown to be transiently induced upon shift to low temperature . We demonstrated that the cspA mRNA is extremely unstable at 37 degrees C with a half-life of approx . 10 s . Upon shift to 15 degrees C cspA mRNA becomes highly stable . This mRNA stability is transient and is lost once the cells are adapted to the low temperature . Transcription fusions of lacZ containing part or most of the cspA gene do not show the rapid degradation at high temperature . Our results suggest that mRNA stability plays a major role in the control of the cspA gene . The expression of cspA is also regulated, to a smaller extent, by the relative increase in transcription after transfer to low temperature . A model by which cspA mRNA is regulated in response to temperature shift is discussed.

Mol Microbiol, 1996 Jan, 19(2), 231 - 40
Post-transcriptional regulation of CspA expression in Escherichia coli; Brandi A et al.; The Escherichia coli cspA gene, encoding the major cold-shock protein CspA, was deprived of its natural promoter and placed in an expression vector under the control of the inducible lambda PL promoter . After induction of transcription by thermal inactivation of the lambda ts repressor, abundant expression of the product (CspA) was obtained if the cells were subsequently incubated at 10 degrees C, but poor expression was obtained if the cells were incubated at 37 degrees C or 30 degrees C . The reason for this differential temperature-dependent expression was investigated and it was found that: (i) the CspA content of the cells decreased more rapidly at 37 degrees C compared to 10 degrees C, regardless of whether transcription was turned off by addition of rifampicin; (ii) both the chemical and functional half-lives of the cspA transcript were substantially longer at 10 degrees C compared to 37 degrees C; (iii) S30 extracts as well as 70S ribosomes prepared from cold-shocked cells translated CspA mRNA (but not phage MS2 RNA) more efficiently than equivalent extracts or ribosomes obtained from control cells grown at 37 degrees C; and (iv) purified CspA stimulated CspA mRNA translation . Overall, these results indicate that a selective modification of the cold-shocked translational apparatus favouring translation of CspA mRNA, and an increased stability of this mRNA at low temperature, may play an important role in the induction of cspA expression during cold shock.

Mol Microbiol, 1996 Jan, 19(2), 213 - 9
The effect of the stringent response on mutation rates in Escherichia coli K-12; Wright BE; The reversion rates of two isogenic Escherichia coli K-12 auxotrophs differing only in relA have been determined in the absence or presence of serine hydroxamate, which provokes the stringent response . Reversion rates of leuB- and argH- were significantly higher in the relA+ than in the relA- strain, and the reversion rates in both strains were enhanced by serine hydroxamate . A positive correlation was established between reversion rates and the synthesis of guanosine-5'-diphosphate-3'-diphosphate in the absence and presence of serine hydroxamate . It is proposed that mutation rates are dependent upon rates of transcription and upon the genes which regulate the level of the signal nucleotide, guanosine tetraphosphate.

Mol Microbiol, 1996 Jan, 19(2), 197 - 204
The Escherichia coli enzoskeleton; Norris V et al.; The nature of the structure of the bacterial cell is becoming clearer . The envelope contains periseptal annuli, a discontinuous periplasm and adhesion sites, whilst the cytoplasmic membrane is probably organized into distinct proteolipid domains by the coupled transcription-translation-insertion (transertion) of membrane proteins . The structure of the nucleoid is determined by proteins which self-associate and by attachment to membrane, which is achieved in part by transertion . Metabolic pathways form multi-enzyme complexes which channel substrates and which connect membranes and nucleic acids to create the extensive, cross-linked, intracellular structure we term the 'enzoskeleton' . This enzoskeleton includes eukaryotic-like cytoskeletal structures and elements such as the MukB and FtsZ proteins . We propose that the enzoskeleton is regulated by calcium and by protein phosphorylation during adaptation to different environments and during the cell cycle.

Biosci Biotechnol Biochem, 1996 Jan, 60(1), 117 - 9
Protecting effect of a green tea percolate and its main constituents against gamma ray-induced scission of DNA; Yoshioka H et al.; Gamma ray-induced scission of puC18 plasmid DNA prepared from E . coli was examined in the presence of a green tea percolate and its main constituents, L-ascorbic acid (used as the sodium salt) and (-)-epigallocatechin gallate . Each of these showed a protecting effect against DNA scission . The relationship between the protecting effect against DNA scission and the scavenging effect of the hydroxyl radical was examined, and is discussed from the viewpoint of interaction with DNA.

Trends Microbiol, 1996 Jan, 4(1), 5 - 9
Epigenetic phase variation of the pap operon in Escherichia coli; van der Woude M et al.; Expression of the pyelonephritis-associated pilus (pap) operon in Escherichia coli is regulated by a complex epigenetic phase-variation mechanism involving the formation of differential DNA-methylation patterns . This review discusses how DNA-methylation patterns are formed by protein-DNA interactions and how methylation patterns, in turn, control pap gene expression.

Mol Microbiol, 1996 Jan, 19(1), 171 - 86
Light-induced carotenogenesis in Myxococcus xanthus: light-dependent membrane sequestration of ECF sigma factor CarQ by anti-sigma factor CarR; Gorham HC et al.; Light-induced carotenogenesis in Myxococcus xanthus is under the control of the carQRS operon . CarQ, a proposed extracytoplasmic (ECF) RNA polymerase sigma factor, is required for expression of the operon and the carC gene that encodes phytoene dehydrogenase . CarR, an inner membrane protein in Escherichia coli, is essential for carQRS promoter inactivation in the dark . CarS is required for the light-dependent expression of the promoter of the carB gene cluster that encodes the rest of the structural genes for carotenogenesis . Regulation of carQRS is dependent on the stoichiometry of CarQ and CarR . Increasing the copy number of carQ over carR led to constitutive carotenogenesis, as did loss of translational coupling between carQ and carR . The severity of the constitutive phenotype depended on the distance between the uncoupled genes . When expressed in M . xanthus, a CarR:beta-galactosidase fusion protein disappeared in the light . We propose that anti-sigma factor CarR sequesters CarQ to the membrane in the dark, but, in the light, loss of CarR leads to release of the sigma factor.

Mol Microbiol, 1996 Jan, 19(1), 125 - 37
FNR-DNA interactions at natural and semi-synthetic promoters; Green J et al.; Two rapid and convenient methods have been developed for the amplification and purification of FNR, the anaerobic transcription regulator of Escherichia coli . The overproduced proteins resemble wild-type FNR in their basic properties: oligomeric state, iron contents (up to 2.7 atoms per monomer), DNA-binding affinities and ability to activate transcription . However, unlike previous preparations, FNR could be isolated in a form containing up to 0.25 atoms of acid-labile sulphur per monomer . Incorporation of iron increased the Mr of FNR from 28,000 to 40,000 . Under anaerobic conditions, reconstituted FNR exhibited absorption maxima at 315 nm and 420 nm, which were replaced by a broad absorbance from 380 to 440 nm under aerobic conditions . These observations indicate that FNR contains one redox-sensitive {3Fe 4S} or {4Fe 4S} centre per monomer . Footprints of FNR-dependent promoters (ansB, fdn, fnr, narG, pflP6, pflP7 and nirB) showed protection at all of the predicted FNR sites except the pflP7 (-57.5), ansB (-74.5) and nirB (-89.5) sites . An unpredicted second binding site was detected at -57.5 in the narG promoter . Hypersensitive sites within regions of FNR protection indicated that FNR bends DNA in a similar way to CRP . Promoters containing binding sites for FNR (FF), CRP (CC) or hybrid sites (CF or FC) were footprinted with FNR and two derivatives (FNR-610 and FNR-573) which activate the CCmelR promoter in vivo . FNR preferentially protected the FNR site (FF) whereas FNR-610 preferred CC and FNR-573 interacted with equal affinity at all sites.

Mol Microbiol, 1996 Jan, 19(1), 101 - 12
The local repressor AcrR plays a modulating role in the regulation of acrAB genes of Escherichia coli by global stress signals; Ma D et al.; Genes acrAB encode a multidrug efflux pump in Escherichia coli . We have previously reported that transcription of acrAB is increased under general stress conditions (i.e . 4% ethanol, 0.5 M NaCl, and the stationary phase in Luria-Bertani medium) . In this study, lacZ transcriptional fusions and an in vitro gel mobility shift assay have been utilized to study the mechanisms governing the regulation of acrAB . We found that a closely linked gene, acrR, encoded a repressor of acrAB . Nevertheless, the general stress conditions increased transcription of acrAB in the absence of functional AcrR, and such conditions surprisingly increased the transcription of acrR even more strongly than that of acrAB . These results suggest that the general-stress-induced transcription of acrAB is primarily mediated by global regulatory pathway(s), and that one major role of AcrR is to function as a specific secondary modulator to fine tune the level of acrAB transcription and to prevent the unwanted overexpression of acrAB . To our knowledge, this represents a novel mechanism of regulating gene expression in E . coli . Evidence also suggests that the up-regulation of acrAB expression under general stress conditions is not likely to be mediated by the known global regulators, such as MarA or SoxS, although elevated levels of these proteins were shown to increase the transcription of acrAB.

Mol Microbiol, 1996 Jan, 19(1), 79 - 89
Expression of the groESL operon is cell-cycle controlled in Caulobacter crescentus; Avedissian M et al.; The Caulobacter crescentus groESL operon was cloned, sequenced and found to be homologous to previously described groES and groEL genes and proteins . The size of the groESL-specific transcript (2.3 kb) suggested that groES and groEL of C . crescentus are organized in a bicistronic operon . Heat-shock induction of groESL mRNA is not transient, high levels of the transcript can be observed after 2 h at 40 degrees C . Primer extension experiments showed that transcription initiated at two sites . Only the start site closer to the groES coding region was highly induced during heat shock . The promoter corresponding to the heat-shock-inducible transcript has -10 and -35 regions very similar to Escherichia coli sigma 32 promoters . At normal temperatures, transcription of the groESL operon is cell-cycle controlled and both transcripts increase co-ordinately in pre-divisional cells . Transcription fusions with a lacZ reporter gene and deletions within the promoter region of the groESL operon have shown that no sequences upstream of the heat-shock promoter are necessary for temporal control . An 11 bp inverted repeat, located between the heat-shock promoter and the translation start site of groES and very similar to inverted repeats found in front of several heat-shock genes of other bacteria, may play a role in cell-cycle control of C . crescentus groESL expression.

Mol Microbiol, 1996 Jan, 19(1), 65 - 78
Amino-terminal maturation of the Bordetella pertussis filamentous haemagglutinin; Jacob-Dubuisson F et al.; The 220 kDa filamentous haemagglutinin (FHA) is a major adhesin of Bordetella pertussis and is produced from a large precursor designated FhaB . Although partly surface associated, it is also very efficiently secreted into the extracellular milieu . Its secretion depends on the outer membrane accessory protein FhaC . An 80 kDa N-terminal derivative of FHA, named Fha44, can also be very efficiently secreted in a FhaC-dependent manner, indicating that all necessary secretion signals are localized in the N-terminal region of FhaB . A comparison of predicted and apparent sizes of FHA derivatives, in addition to immunoblot analyses of cell-associated and secreted FHA polypeptides, indicated that FhaB undergoes N-terminal maturation by the cleavage of an 8-9 kDa segment . However, phenotypic analyses of translational lacZ and phoA fusions showed that this segment does not function as a typical signal peptide . Co-expression of the Fha44-encoding gene with fhaC also did not allow for secretion of Fha44 in Escherichia coli . High levels of secretion could, however, be observed when the OmpA signal peptide was fused to the N-terminal end of Fha44 . Regardless of the OmpA signal peptide-Fha44 fusion point, the E . coli-secreted Fha44 had the same M(r) as that secreted by B . pertussis, indicating that the N-terminal proteolytic maturation does not require a B . pertussis-specific factor . Similar to FHA, the B . pertussis-secreted Fha44 contains an as yet uncharacterized modification at its N-terminus . This modification did not occur in E . coli and is therefore not required for secretion . The N-terminus of Fha44 secreted by E . coli was determined and found to correspond to the 72nd residue after the first in-frame methionine of FhaB . The N-terminal modification was also found not to be required for haemagglutination or interaction with sulphated glycoconjugates.

Mol Microbiol, 1996 Jan, 19(1), 19 - 25
Expression of the tolQRA genes of Escherichia coli K-12 is controlled by the RcsC sensor protein involved in capsule synthesis; Clavel T et al.; The tolQRABpal cluster of Escherichia coli K-12 encodes proteins involved in the maintenance of cell-envelope integrity . In addition, tol/pal mutations result in a mucoid colony phenotype at low temperature . The synthesis of capsular polysaccharides by the cps genes is controlled by the positive regulator RcsA and the two-component RcsC/RcsB system . It was shown that the mucoid phenotype of the tol/pal mutants was due to an rcsCB-dependent activation of the cps genes . Furthermore, we have identified a mutation in the rcsC gene that decreased the activity of a tolA-lac operon fusion independently of RcsA and partially independently of RcsB activators . The corresponding rcsC338 mutation resulted in a Glu to Lys substitution at residue 338 of RcsC . This mutation induced mucoidy even at high temperature . We propose that RcsC modulates the phosphorylated forms of RcsB and an uncharacterized regulatory protein involved in the control of the tolQRA genes in an opposite manner . Moreover, our findings strengthen the previous suggestion that RcsC senses some alterations in the cell surface such as those induced by tol, pal or rfa mutations, and activates capsule synthesis to protect the cell against deleterious agents.

Zhonghua Yi Xue Za Zhi (Taipei), 1996 Jan, 57(1), 7 - 15
Use of recombinant Epstein-Barr virus early antigen for detection of antibody in patients with nasopharyngeal carcinoma; Liu MT et al.; BACKGROUND: Nasopharyngeal carcinoma (NPC) is one of the most common cancers in southern China and Taiwan . Serological studies revealed the close-relationship between NPC and Epstein-Barr virus (EBV) . Elevated serum and saliva levels of anti-EBV antibodies are detected in patients with NPC . Therefore, Development Center for Biotechnology prepared the EBV-early antigen (EA-D) by recombinant DNA technique for screening the serum and throat washing samples from patients with head and neck cancers . METHODS: The BMRF1 gene for EBV early antigen (EA-D) was placed into the plasmid pDB18, then transformed into an Escherichia coli strain containing the lambda cI857 temperature-sensitive repressor . Heat treatment of the transformant, at exponential growth phase, inactivated the cI protein and induced an over-expression of the EA-D protein . Next, the EA-D was purified by chromatography and characterized as a protein of molecular weight 47 kDa, by sodium dodecyl sulfate-polyacry lamide gel electrophoresis (SDS-PAGE) and Western blot analysis using monoclonal anti-EA antibody and sera from patients with nasopharyngeal carcinoma (NPC) . Enzyme-linked immunosorbent assay (ELISA) with the purified EA-D antigen was used to screen 129 serum and throat washing (TW) samples from patients with head and neck tumors, 24 from patients with a nonmalignant disease and 44 from normal donors . RESULTS: Experimental results indicated significantly higher positive rates of EA-D IgA (69%) and EA-D IgG (91%) in NPC sera than in the sera of patients with other head and neck tumors and normal controls . TW samples from patients with NPC also showed a higher positive rate (34%) than the other groups (7-20%) . CONCLUSIONS: Results in this study demonstrate that the bacterially expressed EA-D antigen could be recognized by sera from patients with NPC and monoclonal anti-EA antibody . Thus, it has potential use in ELISA for screening EBV-related diseases such as NPC.

J Ind Microbiol, 1996 Jan, 16(1), 57 - 61
Inactivation of Brazilian wild type and enterotoxigenic Escherichia coli by chlorine; Penna TC et al.; The kinetic inactivation parameters of four wild strains and two enterotoxigenic strains of Escherichia coli exposed to commercial calcium hypochlorite were determined . The four wild strains (1A, 3C, 4D and 8H) were isolated from lettuce bought in Sao Paulo (Brazil), and the two enterotoxigenic strains (TR69 and TR101) were originally isolated from human patients . Decimal reduction time 'D', for 10 mg L-1 available chlorine at pH 6.8, varied between 71.4 s for the wild strain 4D and 31.3 s for the toxigenic strain . The 'D' values obtained for wild strain 1A exposed to 5.0 mg L-1 available chlorine at pH 6.8 varied between 111.1 s and 41.7 s . The 'D' values obtained for E . coli strain TR69 exposed to 10 mg L-1 available chlorine varied from 15.2 s at pH 5.4 up to 83.3 s at pH 8.2 . The use of the most resistant wild strain of E . coli as a biological standard assures maximal effectiveness in controlling water contamination by chlorination.

Planta, 1996, 199(4), 557 - 64
Subcellular location of the tetrapyrrole synthesis enzyme porphobilinogen deaminase in higher plants: an immunological investigation; Witty M et al.; A recombinant plasmid, pArab8, harbouring the cDNA encoding the mature form of the tetrapyrrole synthesis enzyme porphobilinogen deaminase (EC 4.3.1.8; also known as hydroxymethylbilane synthase) from Arabidopsis thaliana (L.) Heynh . has been constructed, and used to transform Escherichia coli . The porphobilinogen deaminase protein from Arabidopsis was overexpressed in this strain, and purified to homogeneity (3000-fold) with a yield of 20% . Antibodies were raised against the purified plant enzyme, and used in Western blot analysis, immunoprecipitation of enzyme activity and immuno-gold electron microscopy . The results indicate that the enzyme is confined to plastids in both leaves and roots . The implications of this finding for plant tetrapyrrole synthesis are discussed.

Planta, 1996, 199(4), 515 - 9
Disruption of an overlapping E-box/ABRE motif abolished high transcription of the napA storage-protein promoter in transgenic Brassica napus seeds; Stalberg K et al.; The storage protein napin is one of the major protein components of Brassica napus L . (oilseed rape) seeds . To investigate the transcriptional regulation of the napin promoter, different constructs of the napin gene napA promoter were fused to the Escherichia coli uidA gene and transformed into B . napus . A-152-bp promoter construct directed a strong expression of the marker gene in mature seeds . The 5' deletion of an additional 8 completely abolished this activity . This deletion disrupted sequence motifs that are similar to an E-box, (CA decreases NNTG) and an ABRE (CGCCA decreases CGTGTCC) element (identify is indicated by bold face) . Further, internal deletion of a segment corresponding to -133 to -121 caused an eightfold reduction in the activity of the -152 construct . This region contains an element, CAAACAC, conserved in many storage-protein gene promoters . These results imply that the E-box/ABRE-like sequence is a major motif of the napA promoter and suggest that the CAAACAC sequence is important for high activity of the napA promoter . Similar results have been obtained by analysing some of the constructs in transgenic tobacco, suggesting that many of the cis-elements in the napA promoter are conserved, at least in dicotyledonous species.

Biochimie, 1996, 78(2), 117 - 22
IbpA and IbpB, the new heat-shock proteins, bind to endogenous Escherichia coli proteins aggregated intracellularly by heat shock; Laskowska E et al.; IbpA/B, 16 kDa heat-shock proteins were recently described as recognizing heterologous protein inclusion bodies in Escherichia coli cells; the corresponding genes formed an operon regulated by the rpoH gene product, sigma 32 protein (Burland et al (1993) Genomics 16, 551; Allen et al (1992) J Bacteriol 174, 6938; Chuang et al (1993) Gene 134, 1; Chuang and Blattner (1993) J Bacteriol 175, 5242) . We have found that IbpA/Bs also recognize endogenous bacterial proteins aggregated intracellularly by heat shock . IbpA/B proteins were isolated and purified from the aggregates (the S fraction), identified by amino acid microsequencing and used as immunogen for anti-IbpA/B serum preparation . Western blotting with the serum showed that in cells growing at 30 degrees C IbpA/B were located in the bacterial outer membrane and appeared in the S fraction after heat shock . Then the cellular level of the IbpA/B proteins increased about 20-fold as estimated by densitometry of the Western blots . In the E coli rpoH strain the level of IbpA/B was higher than in wild type before the heat shock and rose to still higher levels after it . This result pointed to a regulation of ibpA/B operon by another factor, besides that of sigma 32.

Biochimie, 1996, 78(2), 85 - 9
Conformation of plasmid DNA from Escherichia coli deficient in the repair systems protecting DNA from 8-oxyguanine lesions; Wojcik A et al.; 8-Oxyguanine (8ohG) is a major oxidation product of guanine and a biomarker of oxidative stress in mammal . We have attempted to estimate the level of 8ohG residues in plasmid DNA (pGW2123 and pBR322) grown in various bacterial strains (fpg, mutY, or mutT, plus mutT fpg and mutT mutY double mutants) differing in the system protecting cells against the mutagenic effects of 8ohG in DNA . The method was based on digestion of plasmid DNA with Fpg, and agarose gel electrophoresis . Fpg converts pDNA from covalently closed circular to the open circular (ccc-->oc) form of pDNA when there is at least one 8ohG, or apurinic site, per ccc pDNA molecule . It was found that: i) the content of 8ohG in pDNA grown in any of the tested bacteria is below one 8ohG per 10(4) base pairs; ii) a substantial part of pGW2123 is isolated from the bacteria in the oc form; iii) the ratio of oc/ccc in pGW2123 depends on the bacterial host and is the lowest when the plasmid was harvested from mutY- deficient cells; iv) pBR322, unlike pGW2123, is isolated predominantly in the ccc form; and v) of the pBR322 grown in the tested bacteria apparently the most resistant to Fpg digestion was pBR322 grown in the mutY strain . It is proposed that this reflects the compact structure of pDNAs when they are grown in bacteria deficient in mutY gene product.

Annu Rev Biochem, 1996, 65, 169 - 214
Mechanisms of helicase-catalyzed DNA unwinding; Lohman TM et al.; DNA helicases are essential motor proteins that function to unwind duplex DNA to yield the transient single-stranded DNA intermediates required for replication, recombination, and repair . These enzymes unwind duplex DNA and translocate along DNA in reactions that are coupled to the binding and hydrolysis of 5'-nucleoside triphosphates (NTP) . Although these enzymes are essential for DNA metabolism, the molecular details of their mechanisms are only beginning to emerge . This review discusses mechanistic aspects of helicase-catalyzed DNA unwinding and translocation with a focus on energetic (thermodynamic), kinetic, and structural studies of the few DNA helicases for which such information is available . Recent studies of DNA and NTP binding and DNA unwinding by the Escherichia coli (E . coli) Rep helicase suggest that the Rep helicase dimer unwinds DNA by an active, rolling mechanism . In fact, DNA helicases appear to be generally oligomeric (usually dimers or hexamers), which provides the helicase with multiple DNA binding sites . The apparent mechanistic similarities and differences among these DNA helicases are discussed.

Annu Rev Biochem, 1996, 65, 101 - 33
Mismatch repair in replication fidelity, genetic recombination, and cancer biology; Modrich P et al.; Mismatch repair stabilizes the cellular genome by correcting DNA replication errors and by blocking recombination events between divergent DNA sequences . The reaction responsible for strand-specific correction of mispaired bases has been highly conserved during evolution, and homologs of bacterial MutS and MutL, which play key roles in mismatch recognition and initiation of repair, have been identified in yeast and mammalian cells . Inactivation of genes encoding these activities results in a large increase in spontaneous mutability, and in the case of mice and men, predisposition to tumor development.

Acta Microbiol Immunol Hung, 1996, 43(1), 55 - 65
Detection of potential cytokine gene(s) in an Alu-PCR library from the human chromosome 4; Balogh I et al.; Jumping and linking libraries have been constructed in order to have more information of chromosome 4 . Two types of jumping libraries (Not I and Xma III jumping) and one linking library were prepared . The jumping clones represent DNA regions between two rare-cutting enzymes sites (such is Not I or Xma III) whereas linking clones represent only the area which surrounds the rare-cutting site . These clones were used as target DNA in Alu-PCR . The Alu-PCR fragments were cloned into pGEM-T vector and transformed into Escherichia coli JM 109 competent cells . The colonies were screened for inserts by restriction analysis . More than 50% of the colonies were shown to contain inserts . Inserts from each library were screened by PCR . One of the jumping libraries appeared to have inserts containing CpG islands and AT-rich sequences.

Curr Biol, 1996 Jan 1, 6(1), 70 - 5
Two GTPases, Cdc42 and Rac, bind directly to a protein implicated in the immunodeficiency disorder Wiskott-Aldrich syndrome; Aspenstrom P et al.; BACKGROUND: Members of the Rho family of small GTPases play an essential role in controlling the motile behaviour of animal cells . Specifically, Cdc42 and Rac have been shown to induce the formation of filopodia and lamellipodia, respectively, at the cell periphery of Swiss 3T3 fibroblasts . In addition, both GTPases are required for progression through G1 phase of the cell cycle, possibly by regulating the activity of the Jun N-terminal kinase (JNK) signalling pathway . In order to examine more closely the mechanisms underlying the diverse functions of Rho GTPases in mammalian cells, we searched for downstream targets of these proteins . RESULTS: A yeast two-hybrid screen for proteins interacting with the human Cdc42 GTPase identified WASP, a protein implicated in the immunodeficiency disorder Wiskott-Aldrich syndrome (WAS) . Recombinant WASP, expressed in Escherichia coli, also bound to Cdc42 and weakly to Rac, but not at all to Rho . The Cdc42/Rac-binding domain was identified in a region between amino acids 201-321 of WASP, and binding was dependent on Cdc42 and Rac being in the GTP-bound conformation . Furthermore, WASP did not catalyze GTPase activation or nucleotide exchange activity on Cdc42 . CONCLUSIONS: Positional cloning has implicated WASP in causing WAS, and the protein is defective in patients suffering from the disease . WASP is expressed exclusively in cells of hematopoietic lineage, and lymphocytes from WAS patients have a distorted cell-surface and exhibit reduced proliferative capacity . WASP has recently been found to bind to the Src-homology 3 (SH3) domain of the adapter protein Nck . This observation, and the results presented here, suggest that WAS is the result of defects in signal transduction pathways regulated by Cdc42/Rac and Nck.

Cell Motil Cytoskeleton, 1996, 33(4), 252 - 62
Microinjection of intact MAP-4 and fragments induces changes of the cytoskeleton in PtK2 cells; Yoshida T et al.; The molecular cloning and sequencing of microtubule-associated protein (MAP)-4 identified microtubule-binding repeats near the C-terminus and a projection domain near the N-terminus . Although it is well known that MAP-4 stimulates the assembly of and stabilizes microtubules (MT) in vitro, the function of MAP-4 in vivo is still unclear . In this study, we examined the function of MAP-4 in the cytoskeleton both in vitro and in vivo . Intact MAP-4 was prepared from bovine adrenal cortex, and the truncated fragments of the N- and the C-terminal halves (named NR and PA4 fragments, respectively) were expressed in Escherichia coli and isolated . In vitro studies demonstrated that in a solution containing a physiological concentration of NaCl, intact MAP-4 and the PA4 fragment were bound to MT, but not to F-actin . The NR fragment was not bound to MT or to F-actin . We also examined the MT changes in PtK2 cells after they had been microinjected with intact MAP-4 and the truncated fragments of PA4 and NR . The injection of intact MAP-4 or PA4 into the cells induced an increase in the number of cytoplasmic MT, as well as MT bundling . The NR fragment did not affect the MT array . Injected MAP-4 and PA4 were associated with the increased MT . In addition, injection with MAP-4 and PA4 stabilized MT in relation to treatment with the MT-disrupting drug, nocodazole . These results indicated that intact MAP-4 and the PA4 fragment promoted MT assembly and stabilized MT, by binding to MT, in vivo as well as in vitro . Further, the injection of the PA4 fragment induced an increase in stress fibers . However, these proteins did not show any association with the stress fibers . Our results suggest that there is an indirect effect of MAP-4 on stress fibers rather than a direct interaction between MAP-4 and stress fibers.

Annu Rev Biophys Biomol Struct, 1996, 25, 259 - 86
Electron paramagnetic resonance and nuclear magnetic resonance studies of class I ribonucleotide reductase; Graslund A et al.; Ribonucleotide reductase catalyses the reduction of ribonucleotides to the corresponding deoxyribonucleotides needed for DNA synthesis . This review describes recent studies on the iron/tyrosyl free radical site in the R2 protein of iron-containing (class I) ribonucleotide reductases . The active enzyme is composed of two homodimeric proteins, R1 and R2 . Active protein R2 contains a diiron-oxygen site and a neighboring free radical on a tyrosyl residue per polypeptide chain . The properties of the different redox states of the diiron center in protein R2 are discussed, as well as the formation of the iron/radical site and its possible involvement in long range electron transfer from the substrate binding site in protein R1 . The EPR properties of oxidized neutral tyrosyl free radicals are described, and also of tryptophan free radicals found in studies of a mutant of the R2 protein, which lacks the tyrosyl radical site . NMR studies on protein R2 include observations of paramagnetically shifted resonances . Structural NMR studies have been performed on its highly mobile C-terminal domain as well as the corresponding oligopeptide which interacts with protein R1.

Annu Rev Biophys Biomol Struct, 1996, 25, 163 - 95
Use of 19F NMR to probe protein structure and conformational changes; Danielson MA et al.; 19F NMR has proven to be a powerful technique in the study of protein structure and dynamics because the 19F nucleus is easily incorporated at specific labeling sites, where it provides a relatively nonperturbing yet sensitive probe with no background signals . Recent applications of 19F NMR in mapping out structural and functional features of proteins, including the galactose-binding protein, the transmembrane aspartate receptor, the CheY protein, dihydrofolate reductase, elongation factor-Tu, and D-lactose dehydrogenase, illustrate the utility of 19F NMR in the analysis of protein conformational states even in molecules too large or unstable for full NMR structure determination . These studies rely on the fact that the chemical shift of 19F is extremely sensitive to changes in the local conformational environment, including van der Waals packing interactions and local electrostatic fields . Additional information is provided by solvent-induced isotope shifts or line broadening of the 19F resonance by aqueous and membrane-bound paramagnetic probes, which may reveal the proximity of a 19F label to bulk solvent or a biological membrane . Finally, the effect of exchanging conformations on the 19F resonance can directly determine the kinetic parameters of the conformational transition.

Adv Exp Med Biol, 1996, 379, 95 - 104
Cloning and expression of the gene encoding a novel proteinase from Tritirachium album limber; Samal B et al.; We have isolated the cDNA and the genomic clones encoding a novel serine proteinase, named proteinase T, from the fungus Tritirachium album Limber . The coding region of the gene is interrupted by two introns . The amino acid sequence of proteinase T as deduced from the nucleotide sequence is about 56% identical to that of proteinase K . Four cysteines are present in the mature proteinase, probably in the form of disulfide bonds . We have also purified the native proteinase from Tritirachium album Limber grown in the presence of 2% skim milk . Proteinase T is extremely stable at 50 degrees C . The thermal stability is not affected in the presence of 1% SDS either at pH 8.0 or 10.0 . We have expressed the cDNA of proteinase T in Escherichia coli . The authenticity of the proteinase has been characterized by Western blotting and amino terminal analysis of the recombinant product . High level expression of proteinase T in E . coli as well as the refolding process to generate active proteinase will be discussed in detail.

Life Sci, 1996, 59(11), 945 - 51
U-19451A: a selective inducible nitric oxide synthase inhibitor; Stratman NC et al.; Drugs with high selectivity for iNOS inhibition may be useful for treatment of neurodegenerative disorders, chronic inflammatory diseases, and septic shock . Therefore, U-19451A (2-benzyl-2-thio-pseudourea hydrochloride), a potential NOS inhibitor, has been investigated for its selectivity for iNOS using tissues, primary cerebellar granule cell cultures and glial cell cultures . Lungs isolated from rats treated with intravenous injection of E coli lipopolysaccharide and glial cell cultures treated with the same bacterial toxin plus gamma-interferon were used for iNOS activity . Rat cerebellum and primary cerebellar granule cell cultures were utilized for neuronal NOS (nNOS) activity . S-methylthiourea (SMT) and L-nitroarginine methyl ester (L-NAME), selective iNOS and nNOS inhibitors, respectively, were chosen as standards . Both U-19451A and SMT were 4-times more selective for iNOS as compared to nNOS in tissues . U-19451A was more selective than SMT for iNOS inhibition using cultures . L-NAME was 16-31 times more selective for inhibiting nNOS activity . Based on the selectivity of U-19451A for iNOS inhibition, this drug would be expected to be effective in the treatment of diseases with inflammatory pathology without producing side effects associated with nNOS inhibition.

Free Radic Biol Med, 1996, 21(1), 43 - 52
Nitric oxide regulation of superoxide-dependent lung injury: oxidant-protective actions of endogenously produced and exogenously administered nitric oxide; Gutierrez HH et al.; The influence of endogenous cell .NO production and .NO derived from exogenous sources on oxidant injury to cultured fetal rat lung alveolar epithelium and an animal model of pulmonary oxidant injury was examined . Confluent fetal rat alveolar epithelial cell monolayers were stimulated to produce .NO after treatment with a combination of cytokines (IL-1 beta, TNF-alpha, IFN-gamma), LPS and zymosan-activated serum (CZ) . Cell injury, assessed by 14C-adenine release, was significantly increased compared to basal and CZ-induced cells after inhibition of .NO synthesis by L-NMMA . Cell monolayer macromolecule barrier function was determined by the rate of diffusion of 125I-albumin from the apical to basolateral side of monolayers . Following exposure to CZ and/or O2.- generated by xanthine oxidase + lumazine (XO), endogenous cell .NO production and exogenously administered .NO (from .NO donors S-nitrosyl-glutathione and S-nitroso-N-acetylpenicillamine) significantly inhibited the increased monolayer permeability induced by exposure to reactive oxygen species . Furthermore, inhalation of 5-10 ppm of .NO significantly reduced the toxicity of > 95% oxygen to adult rats . We conclude that when cultured pulmonary epithelial cells and lung tissue in vivo are subjected to inflammatory mediators or acute oxidative stress, .NO can play a protective role by inhibiting O2.(-)-dependent toxicity.

Free Radic Biol Med, 1996, 21(1), 7 - 14
Genotoxicity of photoilluminated riboflavin in the presence of Cu(II); Jazzar MM et al.; Riboflavin is known to generate superoxide anion (O2.-) and other reactive oxygen species in the presence of Cu(II) and light as well as cause fragmentation of DNA and protein in vitro . In the present study we examined the genotoxic effects of photoilluminated riboflavin in the presence of Cu(II) . Using the phage inactivation assay, a significant decline in plaque-forming unit (PFU) is seen . Results of Ames testing have suggested that probably a frameshift mutation is caused by a riboflavin-Cu(II)-mediated reaction . Using neocuproine as a Cu(I) sequestering reagent, Cu(I) has been shown to be an essential intermediate generated in the reaction between Cu(II), photoilluminated riboflavin, and DNA . Results obtained with various scavengers of active oxygen species strongly suggest that the species predominantly responsible for DNA damage is oxygen (O2) in the singlet or triplet state, together with H2O2, hydroxyl radical, and hydroxyl ion, to a lesser extent . In the case of riboflavin, a ternary complex of DNA-drug-Cu(II) is presumably formed . A redox reaction, involving riboflavin and Cu(II) in the complex, may then occur with the formation of a DNA-oxidized riboflavin-Cu(I) complex . This probably acts as a catalyst for the oxidation of Cu (I) to Cu(II), during which molecular oxygen is reduced to generate a variety of active oxygen species . The probable mechanism for the generation of these reactive oxygen species has also been proposed.

Acta Biochim Pol, 1996, 43(1), 265 - 79
Effect of reversed orientation and length of An.Tn DNA bending sequences in the -35 and spacer domains of a consensus-like Escherichia coli promoter on its strength in vivo and gross structure of the open complex in vitro; Lozinski T et al.; In continuation of an earlier study (Lozinski et al., 1991 Nucleic Acids Res . 19, 2947-2953) a series of consensus-like E . coli promoters with bending An.Tn sequences of different length (n = 3-8) and orientation in the -35 and spacer domains was constructed, cloned into the plasmid pDS3 and their strength in vivo measured in relation to an internal transcriptional standard . Gel mobilities of free DNA restriction fragments carrying these promoters and of open transcriptional complexes with cognate RNA polymerase were determined by polyacrylamide gel electrophoresis and the gross structure of the complexes interpreted in terms of the theoretically predicted superstructure of DNA restriction fragments . The results obtained together with those reported earlier show that bending of the DNA helix axis immediately upstream of the -35 domain generally lowers the promoter strength in vivo and brings about shortening of the mean square end-to-end distance between free DNA ends in the open complex in vitro . T4(-34 ...-37) and T5(-34 ...-38) tracts located in the nontemplate DNA strand had the largest and comparable effect on the promoter strength, while the A5.T5(-37 .. . -41) sequence in either orientation (A5 tract in the template or nontemplate strand) exerted a much smaller effect . Promoters with the spacer bent by about 40 degrees but in different directions, by two A(n) (n = 5 or 6) tracts aligned in phase with the B-DNA repeat and located either in the template or nontemplate strands, had somewhat lower strength in vivo but the gross geometry of the respective open complexes was the same as that of a control promoter with straight spacer . Implications of these findings are discussed in connection with the existing model of E . coli transcriptional open complex.

Acta Biochim Pol, 1996, 43(1), 115 - 24
Nucleotide probes of DNA polymerases; Wright GE; The modified nucleotides, N2-(p-n-butylphenyl)dGTP and 2-(p-n-butylanilino) dATP and related compounds have been developed as inhibitor-probes of B family DNA polymerases . Synthetic approaches to these compounds are summarized . The nucleotides are potent, non-substrate inhibitors of DNA polymerase a . In contrast, they inhibit other members of the family with less potency but act as substrates for these enzymes . Modelling of the inhibitor: enzyme binding mechanism has been done based on the known structure of E . coli DNA polymerase I, and site-directed mutagenesis experiments to evaluate this mechanism are proposed.

Acta Biochim Pol, 1996, 43(1), 53 - 64
Synthetic and biological applications of tricyclic analogues of guanosine; Golankiewicz B; Tricyclic nucleosides incorporating the 9-oxo-imidazo {1,2-a}purine (1,N2-ethenoguanine) system, natural prototypes of which occur in tRNA(Phe) as nucleosides of the wyosine series, were used for synthetic, structural and biological purposes . 1,N2-(Prop-1-ene-1,2-diyl)guanosine derivatives used as intermediates allowed to enforce on guanosine the substitution at the N-3 position and at the N2 exo-amino group, not possible to be performed directly . Wyosine and 2'-deoxywyosine together with 4,5'-anhydro-4-desmethylwyosine and its congeners were used as, respectively, 100% anti and 100% syn conformation standards in a new graphical method for the syn-anti conformational analysis of nucleosides by 1D 1H NOE difference spectroscopy . Substitution at the appended third ring allowed to modify the biological and physical properties of antiviral agents acyclovir and ganciclovir, e.g . to develop their fluorescent analogues.

Clin Exp Allergy, 1996 Jan, 26(1), 88 - 95
An allergenic polypeptide representing a variable region of hsp 70 cloned from a cDNA library of Cladosporium herbarum; Zhang L et al.; BACKGROUND: Extracts of Cladosporium herbarum, a major source of fungal aeroallergens, exhibit a complex profile of IgE-binding proteins . Yields of conventionally purified allergens from this mold have been insufficient to permit further molecular analyses . OBJECTIVE: To enhance and simplify the purification of allergens from C . herbarum, we have sought to use recombinant DNA techniques to clone, identify and bacterially express immunoselected C . herbarum allergens . METHODS: We constructed a cDNA library in lambda ZAP II using mRNA isolated from C . herbarum . From this library, phage clones encoding a new allergen were immunoselected using pooled human atopic IgE . The cloned cDNA was excised from the phage vector as a recombinant pBluescript II SK-phagemid and sequenced . Expression of the recombinant allergen was carried out in E . coli XL1-blue transformants of the phagemid . Bacterial lysates from cells induced to express the cloned allergen were immunoblotted and probed with individual human atopic IgEs . RESULTS: The cDNA clone encodes a 278 amino acid polypeptide homologous to the C-terminal portion of 70 kDa heat shock protein (hsp 70) . The polypeptide possesses features common to other hsps 70, i.e . a similar hydropathic profile and a variable C-terminal region with conserved sequence at the very C-terminus . Binding of the recombinant peptide to IgE from 38% of atopic sera or plasma from individuals allergic to C . herbarum was demonstrated . CONCLUSION: These results indicate that amino acid substitutions are relatively conserved even in the variable C-terminal regions of hsp 70 species . Thus, this study should draw attention to the possibility of induction of anaphylactic responses in a sensitized individual when hsp 70 from any pathogenic species is administered for vaccination.

Anal Biochem, 1996 Jan 1, 233(1), 76 - 86
Excision of oxidative cytosine modifications from gamma-irradiated DNA by Escherichia coli endonuclease III and human whole-cell extracts; Wagner JR et al.; The possible release from gamma-irradiated DNA of eight oxidatively modified cytosine bases by Escherichia coli endonuclease III was examined by trimethylsilylation and gas chromatography/electron impact/mass spectrometry . The results indicated that endonuclease III induced the release of 5-hydroxyhydantoin (1), 5-hydroxyuracil (2), cis-uracil 5,6-glycol (3), 5-hydroxycytosine (4), trans-uracil 5,6-glycol (5), and trans-1-carbamoyl-2-oxo-4,5-dihydroxyimidazolidine (8) . The release of these products increased with the initial amount of damage in DNA, i.e., the dose of gamma-radiation (0-100 Gy), giving 4.6 +/- 1.0 fmol of 1, 5.8 +/- 0.3 fmol of 2, 4.9 +/- 0.5 fmol of 3, 11.2 +/- 1.2 fmol of 4, 10.7 +/- 2.1 fmol of 5, and 1.5 +/- 0.5 fmol of 8, per microgram DNA per 10 Gy . In addition, we estimated that the relative rates of excision were 5 approximately equal to 3 > (1.2-fold) 1 > (1.5-fold) 4 > (3.3-fold) 2 on the basis of their initial yields in DNA and initial rates of release as a function of incubation time . The excision of 5-hydroxyuracil (2) and 5-hydroxycytosine (4) lesions was studied in greater detail by enzymatic digestion and HPLC coupled to electrochemical (EC) detection which determines the amounts of these products in DNA . The results showed that the excision of 4 was more efficient than that of 2 (2.7-fold) with greater than 50% of the lesions remaining in DNA after treatment . Finally, we examined the excision of products 2 and 4 from irradiated DNA (50 Gy) by whole human cell extracts . The release of product 2 into the hydrosylate was 5.2 +/- 1.4 fmol per microgram of DNA as measured by fluorobenzylation coupled to gas chromatography/electron capture negative-ion chemical ionization/mass spectrometry . In identical samples, the amount of product 2 was reduced by 45.0 +/- 2.6% (225 from 500 fmol per microgram of DNA) and that of product 4 by 7.0 +/- 3.1% (42 from 600 fmol per microgram of DNA) as measured by HPLC/EC analysis.

Mol Gen Mikrobiol Virusol, 1996 Jan-Mar, (1), 28 - 32
{Effect of the length of the loop segment of local mRNA secondary structure in the region of lacZ gene translation initiation on its expression}; Nikolenko GN et al.; A number of p50FNL plasmids with a polycistronic operon IX-VIII-IFN-LacZ have been constructed . These plasmids provide the synthesis of polycistronic mRNA translational terminator of gene IFN situated in the coding region of lacZ gene at 41 nucleotide downstreams from its initiation codon . The coding region of IFN gene has been altered by insertions, substitutions, and deletions which provided the formation in the region of local secondary structures differing only by the loop size . The effect of this characteristic of mRNA secondary structure on lacZ translation efficiency has been assessed by measuring the levels of beta-Gal synthesis in E . coli harboring the corresponding plasmids . The level of lacZ expression increased with increase of the loop size . These data confirm that the rate of formation of mRNA secondary structure in the region of translation initiation has a crucial effect on the translation efficacy.

Dev Biol Stand, 1996, 86, 41 - 7
USDA: progress toward in vitro tests and other trends; Goodman SA; The Animal and Plant Health Inspection Service of the United States Department of Agriculture has demonstrated a commitment toward replacement, reduction and refinement of animal use in the development and control of biological products . This presentation describes some specific approaches with which APHIS has reduced the number of animals used in testing by replacing host or laboratory animal potency tests with validated in vitro tests, reduced the number of animals required for tests by allowing sequential use of animals for tests of immunologically distinct entities, and replaced host or laboratory animal challenge studies with serological tests . It also describes APHIS' plans to reduce pain and suffering of animals by allowing euthanasia when death from causes unrelated to the test is expected . Finally, it reports on refinements in extraneous agent testing, which began when host animal tests were replaced with an in vitro test method and continued when the in vitro test was replaced with a more sensitive status of these approaches is discussed in the context of APHIS' current regulatory framework.

Rev Latinoam Microbiol, 1996 Jan-Mar, 38(1), 31 - 7
Correspondence between radioactive and functional methods in the quality control of DNA restriction and modifying enzymes; Trujillo LE et al.; We evaluated the use of two radiolabeled lambda DNA/Hpa II substrates to detect 5'-->3', 3'-->5' single and double stranded DNA dependent exonuclease and phosphatase activities found as contaminants in restriction and modifying enzyme preparations . Looking for the meaning of the radioactive assays results in a real cloning experience, we performed a cloning simulation assay using the same conditions established for the radioactive assay (enzyme units and pmols of DNA ends) . As a result, we found that for degradation percentages of the radioactive DNA substrate per enzyme unit below 0.5, the false positives in the cloning stimulation assay were less than 5% . This conditions could ensure a good performance of the enzyme preparations for cloning experiments . Finally, we described the use of the radiolabeled {gamma 33P} ATP lambda Hpa II DNA substrate to detect 5'-->3' single stranded DNA dependent exonuclease and phosphatase contaminating activities in some critical steps of the purification process of the restriction enzyme Kpn I.

Am J Physiol, 1996 Jan, 270(1 Pt 1), L141 - 51
Glucocorticoids regulate glutamine synthetase expression in lung epithelial cells; Abcouwer SF et al.; During septic states efflux of glutamine from the lung increases, a response sustained by an increase in glutamine synthetase (IGS) activity . We have used a cell culture model employing a rat epithelial cell line of pulmonary origin (L2 cells) to study the effect of several hormones and cytokines which mediate the septic shock response on GS expression . We found that GS mRNA and GS protein contents increased rapidly and severalfold in response to physiologically relevant levels of the synthetic glucocorticoid dexamethasone (Dex) . In contrast, GS expression was not markedly induced by Escherichia coli lipopolysaccharide (LPS), cytokines, activated complement C5a, or prostaglandins . Dex did not alter the kinetics of GS mRNA decay in the presence of actinomycin D . The increase in GS mRNA in response to Dex was completely blocked by RU-38486 and by actinomycin D, but not by cycloheximide (CHX) . CHX together with Dex caused a superinduction of GS mRNA . GS mRNA decay kinetics suggested that this superinduction is at least in part caused by an approximately twofold increase in GS mRNA half-life caused by CHX . In addition, actinomycin D was found to increase GS mRNA half-life by approximately 50% . Actinomycin D plus CHX acted synergistically to cause a profound inhibition of GS mRNA decay . Our results are consistent with regulation of lung GS expression via a direct glucocorticoid receptor-mediated response . In addition, GS mRNA decay in L2 cells seems to be regulated by two independent mechanisms, one which is sensitive to CHX and one which is sensitive to actinomycin D.

Microbios, 1996, 86(346), 23 - 6
The recA gene and cadmium toxicity in Escherichia coli K12; Shapiro N et al.; The influence of the recA gene on cadmium toxicity was studied in Escherichia coli K12 strains . Those strains mutant in the recA gene showed a 1,000-fold loss of viability upon exposure to cadmium, but recovered and started growing approximately 16-20 h following the initial exposure to cadmium . In contrast to previous studies with E . coli B strains, the E . coli K12 strains carrying a functional recA gene showed little or no loss of viability upon exposure to cadmium . The cells also exhibited a significant lag period in which no net growth occurred and then began growing 16-20 h after initial exposure to cadmium . These results indicate the importance of the recA gene to cell survival during exposure to metals, and also support the hypothesis that cadmium causes DNA damage.

Protein Sci, 1996 Jan, 5(1), 178 - 80
Two domains of superfamily I helicases may exist as separate proteins; Koonin EV et al.; DNA and RNA helicases of superfamily I are characterized by seven conserved motifs . The five N-terminal motifs are separated from the two C-terminal ones by a spacer that is highly variable in both sequence and length, suggesting the existence of two distinct domains . Using computer methods for protein sequence analysis, we show that PhoH, an ATP-binding protein that is conserved in Escherichia coli and Mycobacterium leprae, is homologous to the putative N-terminal domain of the helicases, whereas the putative E . coli protein YjhR is homologous to the C-terminal domain . These findings suggest that the N-and C-terminal domains of superfamily I helicases have distinct activities, with only the N-terminal domain having the ATPase activity . It is speculated that PhoH and YjhR have evolved from helicases through deletion of the portions of the helicase genes coding for the C- and N-terminal domain, respectively.

Protein Sci, 1996 Jan, 5(1), 170 - 3
An alternative topological model for Escherichia coli OmpA; Stathopoulos C; The current topological model for the Escherichia coli outer membrane protein OmpA predicts eight N-terminal transmembrane segments followed by a long periplasmic tail . Several recent reports have raised serious doubts about the accuracy of this prediction . An alternative OmpA model has been constructed using (1) computer-aided predictions developed specifically to predict topology of bacterial outer membrane porins, (2) the results of two reports that identified sequence homologies between OmpA and other peptidoglycan-associated proteins, and (3) biochemical, immunochemical, and genetic topological data on proteins of the OmpA family provided by numerous previous studies . The new model not only agrees with the varied experimental data concerning OmpA but also provides an improved understanding of the relationship between the structure and the multifunctional role of OmpA in the bacterial outer membrane.

Protein Sci, 1996 Jan, 5(1), 154 - 61
Identification of arginine 331 as an important active site residue in the class II fructose-1,6-bisphosphate aldolase of Escherichia coli; Qamar S et al.; Treatment of the Class II fructose-1,6-bisphosphate aldolase of Escherichia coli with the arginine-specific alpha-dicarbonyl reagents, butanedione or phenylglyoxal, results in inactivation of the enzyme . The enzyme is protected from inactivation by the substrate, fructose 1,6-bisphosphate, or by inorganic phosphate . Modification with {7-14C} phenylglyoxal in the absence of substrate demonstrates that enzyme activity is abolished by the incorporation of approximately 2 moles of reagent per mole of enzyme . Sequence alignment of the eight known Class II FBP-aldolases shows that only one arginine residue is conserved in all the known sequences . This residue, Arg-331, was mutated to either alanine or glutamic acid . The mutant enzymes were much less susceptible to inactivation by phenylglyoxal . Measurement of the steady-state kinetic parameters revealed that mutation of Arg-331 dramatically increased the K(m) for fructose 1,6-bisphosphate . Comparatively small differences in the inhibitor constant Ki for dihydroxyacetone phosphate or its analogue, 2-phosphoglycolate, were found between the wild-type and mutant enzymes . In contrast, the mutation caused large changes in the kinetic parameters when glyceraldehyde 3-phosphate was used as an inhibitor . Kinetic analysis of the oxidation of the carbanionic aldolase-substrate intermediate of the reaction by hexacyanoferrate (III) revealed that the K(m) for dihydroxyacetone phosphate was again unaffected, whereas that for fructose 1,6-bisphosphate was dramatically increased . Taken together, these results show that Arg-331 is critically involved in the binding of fructose bisphosphate by the enzyme and demonstrate that it interacts with the C-6 phosphate group of the substrate.

Protein Sci, 1996 Jan, 5(1), 98 - 105
Human protoporphyrinogen oxidase: expression, purification, and characterization of the cloned enzyme; Dailey TA et al.; Protoporphyrinogen oxidase (E.C.1.3.3.4) catalyzes the oxygen-dependent oxidation of protoporphyrinogen IX to protoporphyrin IX . The enzyme from human placenta has been cloned, sequenced, expressed in Escherichia coli, purified to homogeneity, and characterized . Northern blot analysis of eight different human tissues show evidence for only a single transcript in all tissue types and the size of this transcript is approximately 1.8 kb . The human cDNA has been inserted into an expression vector for E . coli and the protein produced at high levels in these cells . The protein is found in both membrane and cytoplasmic fractions . The enzyme was purified to homogeneity in the presence of detergents using a metal chelate affinity column . The purified protein is a homodimer composed of subunits of molecular weight of 51,000 . The enzyme contains one noncovalently bound FAD per dimer, has a monomer extinction coefficient of 48,000 at 270 nm and contains no detectable redox active metals . The apparent K(m) and Kcat for protoporphyrinogen IX are 1.7 microM and 10.5 min-1, respectively . The enzyme does not use coproporphyrinogen III as a substrate and is inhibited by micromolar concentrations of the herbicide acifluorfen . Protein database searches reveal significant homology between protoporphyrinogen oxidase and monoamine oxidase.

Protein Sci, 1996 Jan, 5(1), 52 - 61
The crystal structure of trypanothione reductase from the human pathogen Trypanosoma cruzi at 2.3 A resolution; Zhang Y et al.; Trypanothione reductase (TR) is an NADPH-dependent flavoprotein unique to protozoan parasites from the genera Trypanosoma and Leishmania and is an important target for the design of improved trypanocidal drugs . We present details of the structure of TR from the human pathogen Trypanosoma cruzi, the agent responsible for Chagas' disease or South American trypanosomiasis . The structure has been solved by molecular replacement, using as the starting model the structure of the enzyme from the nonpathogenic Crithidia fasciculata, and refined to an R-factor of 18.9% for 53,868 reflections with F > or = sigma F between 8.0 and 2.3 A resolution . The model comprises two subunits (968 residues), two FAD prosthetic groups, two maleate ions, and 419 water molecules . The accuracy and geometry of the enzyme model is improved with respect to the C . fasciculata enzyme model . The new structure is described and specific features of the enzyme involved in substrate interactions are compared with previous models of TR and related glutathione reductases from human and Escherichia coli . Structural differences at the edge of the active sites suggest an explanation for the differing specificities toward glutathionylspermidine disulfide.

Protein Sci, 1996 Jan, 5(1), 34 - 41
A model for the regulation of D-3-phosphoglycerate dehydrogenase, a Vmax-type allosteric enzyme; Grant GA et al.; Escherichia coli D-3-phosphoglycerate dehydrogenase (ePGDH) is a tetramer of identical subunits that is allosterically inhibited by L-serine, the end product of its metabolic pathway . Because serine binding affects the velocity of the reaction and not the binding of substrate or cofactor, the enzyme is classified as of the Vmax type . Inhibition by a variety of amino acids and analogues of L-serine indicate that all three functional groups of serine are required for optimal interaction . Removing or altering any one functional group results in an increase in inhibitory concentration from micromolar to millimolar, and removal or alteration of any two functional groups removes all inhibitory ability . Kinetic studies indicate at least two serine-binding sites, but the crystal structure solved in the presence of bound serine and direct serine-binding studies show that there are a total of four serine-binding sites on the enzyme . However, approximately 85% inhibition is attained when only two sites are occupied . The three-dimensional structure of ePGDH shows that the serine-binding sites reside at the interface between regulatory domains of adjacent subunits . Two serine molecules bind at each of the two regulatory domain interfaces in the enzyme . When all four of the serines are bound, 100% inhibition of activity is seen . However, because the domain contacts are symmetrical, the binding of only one serine at each interface is sufficient to produce approximately 85% inhibition . The tethering of the regulatory domains to each other through multiple hydrogen bonds from serine to each subunit apparently prevents the body of these domains from undergoing the reorientation that must accompany a catalytic cycle . It is suggested that part of the conformational change may involve a hinge formed in the vicinity of the union of two antiparallel beta-sheets in the regulatory domains . The tethering function of serine, in turn, appears to prevent the substrate-binding domain from closing the cleft between it and the nucleotide-binding domain, which may be necessary to form a productive hydrophobic environment for hydride transfer . Thus, the structure provides a plausible model that is consistent with the binding and inhibition data and that suggests that catalysis and inhibition in this rare Vmax-type allosteric enzyme is based on the movement of rigid domains about flexible hinges.

Genetics, 1996 Jan, 142(1), 25 - 37
Two enzymes, both of which process recombination intermediates, have opposite effects on adaptive mutation in Escherichia coli; Foster PL et al.; Reversion of a lac- frameshift allele carried on an F' episome in Escherichia coli occurs at a high rate when the cells are placed under lactose selection . Unlike Lac+ mutations that arise during nonselective growth, the production of these adaptive mutations requires the RecA-RecBCD pathway for recombination . In this report, we show that enzymes that process recombination intermediates are involved in the mutagenic process . RuvAB and RecG, E . coli's two enzymes for translocating Holliday junctions, have opposite effects: RuvAB is required for RecA-dependent adaptive mutations, whereas RecG inhibits them.

Biotechniques, 1996 Jan, 20(1), 122 - 9
Synthesis of a new substrate for detection of lacZ gene expression in live Drosophila embryos; Minden JS; Escherichia coli lacZ, which encodes beta-galactosidase, has become a widely used reporter gene to study the developmental regulation of gene expression in a variety of organisms . To detect the presence of the beta-galactosidase, the sample must be fixed and appropriately stained . This sort of analysis yields rather crude estimates of the spatial-temporal changes in gene expression patterns . In addition, one cannot recover interesting specimens for propagation . A novel fluorogenic beta-galactosidase substrate for use in live Drosophila melanogaster embryos has been designed and synthesized . This compound provides a means to determine gene dosage in live embryos so that one can unambiguously determine the genotype of a living embryo . This will be useful for detailed analysis of cellular and morphogenetic behavior changes in live embryos that are homozygous for embryonic lethal mutations . In the course of testing this compound, a new beta-galactoside hydrolytic activity, different from the previously identified beta-galactosidase, has been discovered to reside in macrophages and the intervitelline space.

Biophys J, 1996 Jan, 70(1), 135 - 45
Hypothesis: hypersensitive plasmid copy number control for ColE1; Ehrenberg M; Initiation of replication of the plasmid ColE1 is primed by the cis-acting RNA II . Copy numbers are regulated by inhibition of RNA II by the antisense RNA I, whose concentration is proportional to the plasmid concentration . This inhibition is enhanced by a protein . Rom, and takes place during a time set by the transcription of 250 bases of the gene for RNA II . When this transcription is dominated by several steps of about equal duration, the probability for RNA II to prime DNA replication is approximately determined by e-constant{RNA I} . For large values of the "constant" small changes in {RNA I} give large variations in the priming probability . It is shown, first, that this type of mechanism can reduce the rate of plasmid loss and enable single copies of ColE1 to duplicate at a well-defined time in the cell cycle; second, that when the rate of initiation of transcription of RNA II increases, plasmid losses decrease and the distribution of single copy duplication times becomes narrower; third, that the action of Rom may further reduce plasmid losses and further narrow the distribution of duplication times in the single-copy case.

Am J Physiol, 1996 Jan, 270(1 Pt 2), H7 - 15
Endotoxin pretreatment enhances portal venous contractile response to endothelin-1; Pannen BH et al.; To test whether endotoxin pretreatment modulates the portal hemodynamic response to endothelin (ET)-1 and phenylephrine (PE), two potent vasoconstrictors in the portal circulation of the normal liver, rats received intraperitoneal injections of Escherichia coli lipopolysaccharide (LPS; 1 mg/kg body wt) or saline . Livers were isolated after 6 or 24 h and perfused with Krebs buffer containing 5% autologous erythrocytes . Analyses of portal pressure-flow (P-Q) relationships and epifluorescence video microscopy were performed before and after ET-1 (10(-9) M) or PE (10(-5) M) administration . LPS pretreatment increased total portal resistances (Rt), zero-flow pressures (PQ = 0), and linear regression slopes of P-Q relationships, and decreased the sinusoidal diameters (Ds) and sinusoidal volumetric flow (Qv) . The response to ET-1 was enhanced 6 and 24 h after LPS administration, leading to greater increases in Rt, PQ = 0, and slope and more pronounced decreases in Dx, red blood cell velocity (VRBC), and Qv . In contrast, PE effects were similar (PQ = 0, slope, Ds) or even attenuated (Rt, VRBC, Qv) in livers from LPS-treated compared with control animals . Thus endotoxin pretreatment increased the portal contractile response to ET-1 but not to PE . This enhanced ET-1 response appeared to occur at sinusoidal and presinusoidal levels and may contribute to endotoxin-induced hepatic microcirculatory failure.

Morfologiia, 1996, 109(1), 51 - 6
{The repopulation and regranulation of tissue basophils at the site of acute inflammation}; Klimenko NA et al.; Regularities of rat mast cells (MC) repopulation and regranulation in late reparative stage of inflammation compared to that in conditions approximating to MC natural renewal (after their osmiophilic elimination) and MC restoration after the inflammation caused during their absence were established on the model of E.coli induced acute infectious peritonitis . MC number in peritoneal fluid and mesenterium restores by 90th day in typical development of the inflammation, with peritoneal MC regranulation yielding to that in mesenterium and remaining unfinished by 120th day . MC repopulation in peritoneal fluid and mesenterium forestalls the one observed after the osmotic MC elimination . Inflammation, induced during MC absence retards peritoneal MC regranulation and promotes that of mesenterial ones . MC repopulation and regranulation on the site of inflammation combines their degranulation and high content of free histamine in peritoneal fluid and mesenterium.

Vet Res, 1996, 27(3), 267 - 71
Serum sensitivity and apathogenicity for chickens and chick embryos of Escherichia coli J5 strain; Abdul-Aziz TA et al.; Escherichia coli J5 strain was assessed for its serum resistance and pathogenicity for both chickens and chick embryos . Pathogenicity for chickens was assessed by intravenous inoculation into three-week-old broiler chickens . Pathogenicity for chick embryos was determined by inoculation of 12-day-old chick embryos into the allantoic cavity . Chicken serum was used to determine the serum resistance status . E coli J5 strain was found to be serum-sensitive and apathogenic for chickens and chick embryos . No mortality and no gross lesions occurred in the chickens inoculated with this strain . The chick embryos inoculated with this strain remained alive . It is worth trying this strain as a live vaccine to immunize chickens against infection from different serotypes of E coli.

Eur Biophys J, 1996, 24(6), 371 - 80
Solution studies of the SH2 domain from the fyn tyrosine kinase: secondary structure, backbone dynamics and protein association; Pintar A et al.; The SH2 domain from Fyn tyrosine kinase, corresponding to residues 155-270 of the human enzyme, was expressed as a GST-fusion protein in a pGEX-E . coli system . After thrombin cleavage and removal of GST, the protein was studied by heteronuclear NMR . Two different phosphotyrosyl-peptides were synthesized and added to the SH2 domain . One peptide corresponded to the regulatory C-terminal tail region of Fyn . Sequence-specific assignment of NMR spectra was achieved using a combination of 1H-15N-correlated 2D HSQC, 15N-edited 3D TOCSY-HMQC, and 15N-edited 3D NOESY-HMQC spectra . By analysis of the alpha-proton chemical shifts and NOE intensities, the positions of secondary structural elements were determined and found to correspond closely to that seen in the crystal structure of the, homologous, Src-SH2 domain . To investigate the internal dynamics of the protein backbone, T1 and T2 relaxation parameters were measured on the free protein, as well as on both peptide complexes . Analytical ultracentrifugation and dynamic light scattering were employed to measure the effect of concentration and peptide-binding on self-association . The results suggest that, at NMR-sample concentrations, the free protein is present in at least dimeric form . Phosphopeptide binding and lower concentration significantly, but not completely, shift the equilibrium towards monomers . The possible role of this protein association in the regulation of the Src-family tyrosine kinases is discussed.

EXS, 1996, 75, 425 - 9
Lytic transglycosylases; Holtje JV; Although cleaving the same glycosidic bond between MurNAc and GlcNAc in murein, lytic transglycosylases differ from lysozymes by catalyzing an intramolecular transglycosylation of the glycosyl-bond onto the C6 hydroxyl group of the muramic acid residue yielding 1.6-anhydromuramic acid-carrying products . The three dimensional structure of the soluble lytic transglycosylase Slt70 of E . coli revealed a doughnut-like shape that would allow the protein to encircle the polysaccharide strands of the murein . Despite the absence of significant sequence homology, the catalytic center shows structural similarity to lysozymes, although the catalytic aspartate is missing . All lytic transglycosylases which have been characterized up until now turned out to be processive exo-glycosylases.

Life Sci, 1996, 59(9), 719 - 30
Studies on the immunoglobulin-E system of the common marmoset in comparison with human data; Schmidt S et al.; In the common marmoset (Callithrix jacchus jacchus) immunoglobulin E (IgE) serum levels and IgE synthesis of peripheral blood mononuclear cells (PBMC) in vitro were investigated in order to look for homologies to the human system . While IgE was not found in marmoset blood plasma with three commercial antihuman IgE-kits with monoclonal antibodies (mAbs), two other kits using polyclonal antibodies against human IgE revealed detectable IgE concentrations of up to 10 kU/liter in plasma samples of 19 out of 21 marmosets . In accord with human data, rhIL-4 showed biological functions under in vitro conditions in PBMC of the New World monkey . Proliferation, measured by 3H-thymidine incorporation, of isolated PBMC of marmosets could be induced by rhIL-4 . FACScan analysis showed an enhanced expression of the low affinity IgE receptor CD23 (Fc epsilon RII) on CD20+ B lymphocytes after incubation with rhIL-4 . Furthermore, PBMC from marmosets could be stimulated by IL-4 alone or in combination with dexamethasone as well as with lipopolysaccharide (E . coli) to produce IgE in culture . The results indicate that Callithrix jacchus is using an IgE system that is rather similar to that of humans, although not completely identical . Antihuman mAbs and rhIL-4 can be used to investigate IgE regulation in vitro of marmoset PBMC . These data encourage the development of a primate animal model for studying possible modifications of the IgE system under pathological conditions to find new therapeutic strategies in atopic diseases.

Life Sci, 1996, 59(8), 649 - 57
Endotoxin depletes ascorbate in the guinea pig heart . Protective effects of vitamins C and E against oxidative stress; Rojas C et al.; The effect of acute endotoxin-induced septic shock on myocardium oxidative stress after low or high vitamin C and/or E dietary supplementation was studied in guinea pigs, laboratory animals which, like human, do not have capacity for ascorbate synthesis . Neither the antioxidant enzymes or GSH were modified by endotoxin and vitamin treatments . Vitamin E showed a strong capacity to protect the myocardium against both enzymatic and non-enzymatic lipid peroxidation even in the presence of endotoxin . Vitamin C supplementation increased heart ascorbate whereas endotoxic shock totally depleted the heart ascorbate of vitamin C supplemented animals without changing vitamin E . Endotoxin significantly increased myocardium uric acid, a marker of ischemia induced oxidative stress, in animals fed with low vitamin C levels . This increase was totally prevented in vitamin C supplemented, but not in vitamin E supplemented animals . Strongly depressed levels of plasma vitamin C have been recently described in sepsis in human patients . The results suggest that ascorbate is a primary antioxidant target in the heart of endotoxin treated mammals lacking the capacity to synthesize ascorbate and that ascorbate can have a protective value against endotoxin-induced free radical damage in the myocardium . Implications of these results for the possible preventive role of vitamin C in humans during sepsis are discussed.

J Tongji Med Univ, 1996, 16(1), 14 - 7
Construction of eukaryotic expression vector pBlacz and its expression both in vitro and in vivo; Qu S et al.; A novel eukaryotic expression vector pBlacZ was constructed, which was transfected into the cell lines of NIH/3T3, COS-1, CHO and the primary culture of murine dermatic fibroblasts in vitro, and also into the murine subcutaneous layer and skeletal muscles of rats in vivo . It was detected that the gene expression vector could encode the E . Coli beta-galactosidase effectively in all these histocytes . The results suggested that pBlacZ, as a novel expression vector, might have certain value of application.

Zhonghua Yi Xue Za Zhi, 1996 Jan, 76(1), 34 - 7
{Enhancing immunogenicity of pres antigen of hepatitis B virus by fusing genetically it with interleukin-2}; Chen Z et al.; OBJECTIVE: To combine the bioactivities of human interleukin-2 (IL-2) with entire preS antigen of hepatitis B virus (HBV), and search for specific immunotherapeutic agent against chronic hepatitis B . METHODS: A chimeric gene composed of preS gene from HBV DNA and human IL-2 cDNA was constructed by using polymerase chain reaction and genetic engineering methods, and a novel type of chimeric protein (IL-2-preS) was expressed with high efficiency in E . coli transformed by the chimeric gene clone . RESULTS: It was confirmed that the chimeric protein retained the full bioactivities of natural IL-2 and preS molecules, such as maintaining CTLL cells to proliferate, with the specific activity being about 10(7)u/mg protein, and binding with monoclonal antibodies against preS1 and preS2 and polymerized human serum albumin (PHSA), etc . It was shown that the titer of antibody against preS antigen in mice induced by IL-2-preS was 9, 11 and 13 times more than those induced by a mixture of IL-2 with preS antigen, MS-2-preS chimeric protein and preS antigen alone, respectively . CONCLUSION: IL-2-preS potentiates immunogenecity of preS antigen and enhances immune responses of human bodies against preS antigen . In addition, IL-2-preS is of double targetting effect in human bodies, and may be used as a new generation of immunotherapeutic agent for chronic hepatitis B and hepatocellular carcinoma.

Graefes Arch Clin Exp Ophthalmol, 1996 Jan, 234(1), 47 - 54
Evidence of cross-link formation of vitreous collagen during experimental ocular inflammation; Hikichi T et al.; PURPOSE: To determine the mechanisms of vitreous changes during ocular inflammation . METHODS: We investigated vitreous changes, with special emphasis on collagen, in an experimental model of ocular inflammation induced by intravitreal injection of endotoxin (Escherichia coli) in rabbits . RESULTS: Inflammation caused gel contraction and loss of elasticity, accompanied by release of a water-like liquid from the gel, and increases in the amount of insoluble material and high-molecular-weight components of vitreous collagen, presumably due to extensive cross-links of the collagen molecules . Those changes were partially inhibited by intravitreal injection of superoxide dismutase . CONCLUSIONS: The cross-links of vitreous collagen may promote vitreous gel contraction and release of a water-like liquid from the gel . Superoxide anion may play a role in this process.

J Clin Microbiol, 1996 Jan, 34(1), 165 - 9
Cloning of Brucella abortus gene and characterization of expressed 26-kilodalton periplasmic protein: potential use for diagnosis; Rossetti OL et al.; Brucella spp . are the causative agents of brucellosis in many different hosts, including humans . Most of the serological methods of diagnosis are based on the detection of antilipopolysaccharide antibodies, which makes the differentiation of vaccinated animals from infected animals difficult . By using molecular biology techniques, a gene that encodes a 26-kDa protein (BP26) was isolated from a Brucella abortus S19 genome lambda gt11 library . This protein is in the periplasm of B . abortus and in transformed Escherichia coli . It is exported to the periplasm via a preprotein of 29 kDa with a signal sequence of 28 amino acids . The nucleotide and amino acid sequences of this gene and protein did not show any similarity with those of previously sequenced genes . The use of this protein in Western blotting allowed the differentiation between vaccinated bovines from infected bovines and the detection of infected rams: on the other hand, sera from human patients with active brucellosis were positive, while sera from human patients with chronic brucellosis or without clinical signs were nonreactive . BP26 might be of value as an antigen for serological diagnosis of brucellosis in different mammals.

J Clin Microbiol, 1996 Jan, 34(1), 144 - 8
Genotypic and phenotypic characterization of Escherichia coli isolates from dogs manifesting attaching and effacing lesions; Beaudry M et al.; Thirteen Escherichia coli isolates from dogs manifesting attaching and effacing lesions were characterized genetically with respect to the presence of the following virulence determinants associated with human enteropathogenic E . coli (EPEC): eaeA, encoding the outer membrane protein intimin; eaeB, which is necessary for inducing signal transduction; bfpA, encoding the bundle-forming pilus; and the EAF (stands for EPEC adherence factor) plasmid . These isolates were also analyzed phenotypically with respect to adherence to mammalian cells in vivo and in vitro . Nine of these 13 isolates were found to be eaeA positive by PCR: four of these nine were eaeB positive . The 5' end, but not the 3' end, of the eaeA gene was amplified by PCR when primers derived from the eaeA gene of EPEC were used . Six and eight of these 13 isolates were found to be bfpA positive and EAF positive, respectively . The bfpA gene and EAF locus were found on high-molecular-weight plasmids, whereas the eaeA and eaeB genes were chromosomally located when present . Only one canine E . coli isolate, 4221, which was positive for eaeA, eaeB, bfpA, and EAF, adhered to HEp-2 cells in a localized manner and was positive in the fluorescence actin staining test . The nine eaeA-positive isolates adhered to the mucosal surface of piglet ileal explants and induced some microvillus effacement . However, when tested in experimentally inoculated gnotobiotic piglets, isolate 4221 did not induce attaching and effacing lesions at any level of the intestinal tract . Our results indicate that canine E . coli isolates associated with attaching and effacing lesions share some properties with human EPEC but form a heterogeneous group.

J Clin Microbiol, 1996 Jan, 34(1), 119 - 25
Antigenic properties and diagnostic potential of puumala virus nucleocapsid protein expressed in insect cells; Vapalahti O et al.; Puumala virus (PUU) is a member of the genus Hantavirus in the family Bunyaviridae and the causative agent of nephropathia epidemica, a European form of hemorrhagic fever with renal syndrome . Sera of nephropathia epidemica patients react specifically with PUU nucleocapsid (N) protein . In order to safely provide large quantities of antigen for diagnostic purposes, PUU Sotkamo strain N protein was expressed by using the baculovirus system in Sf9 insect cells to up to 30 to 50% of the total cellular protein . The recombinant N protein (bac-PUU-N) was solubilized with 6 M urea, dialyzed, and purified by anion-exchange liquid chromatography . In an immunoglobulin M mu-capture assay purified and unpurified bac-PUU-N antigen showed identical results compared with the results of a similar assay based on native PUU antigen grown in Vero E6 cells . An immunoglobulin G monoclonal antibody-capture assay based on unpurified bac-PUU-N also showed results identical to those of an assay with native PUU-N antigen . Moreover, a panel of monoclonal antibodies reactive with eight different epitopes showed identical reactivity patterns with both natural and bac-PUU-N antigen, while two epitopes in PUU-N expressed as a fusion protein in Escherichia coli were not recognized . Puumala hantavirus N protein expressed by the baculovirus system offers a safe and inexpensive source of specific antigen for large-scale diagnostic and seroepidemiological purposes.

Res Vet Sci, 1996 Jan, 60(1), 88 - 91
Quantitative determination of bovine serum haptoglobin in experimentally induced Escherichia coli mastitis; Salonen M et al.; A high performance liquid chromatography (HPLC) method was developed, validated and used to analyse haptoglobin concentrations in serum samples taken from eight cows which had been challenged twice intramammarily with Escherichia coli . The results of the HPLC were compared with those from a photometric assay . The kinetics of the haptoglobin response were analysed with pharmacokinetic computer software . In contrast with the photometric assay, the HPLC was sufficiently sensitive to detect normal background levels of bovine serum haptoglobin . The serum haptoglobin concentrations of healthy cows ranged from 22 to 47 mg litre-1 . As the concentration of haptoglobin increased, the results of the two methods correlated well (r = 0.96) . A 52-fold increase in serum haptoglobin was detected after the first challenge with E coli . The mean pharmacokinetic parameters of the response after the first challenge were: lag phase 12 hours, t2/1increase 20 hours, tmax 72 hours, t1/2decrease 46 hours, and mean residence time 112 hours . The second challenge three weeks later resulted in a significantly lower haptoglobin response, the area under curve being 35 per cent of that after the first challenge . The clinical signs and inflammatory changes in the milk did not differ significantly between the challenges.

Methods Enzymol, 1996, 266, 295 - 322
Protein sequence comparison at genome scale; Koonin EV et al.; An adequate set of computer procedures tailored to address the task of genome-scale analysis of protein sequences will greatly increase the beneficial impact of the genome sequencing projects on the progress of biological research . This is especially pertinent given the fact that, for model organisms, one-half or more of the putative gene products have not been functionally characterized . Here we described several programs that may comprise the core of such a set and their application to the analysis of about 3000 proteins comprising 75% of the E . coli gene products . We find that the protein sequences encoded in this model genome are a rich source of information, with biologically relevant similarities detected for more than 80% of them . In the majority of cases, these similarities become evident directly from the results of BLAST searches . However, methods for motif analysis provide for a significant increase in search sensitivity and are particularly important for the detection of ancient conserved regions . As a result of sequence similarity analysis, generalized functional predictions can be made for the majority of uncharacterized ORF products, allowing efficient focusing of experimental effort . Clustering of the E . coli proteins on the basis of sequence similarity shows that almost one-half of the bacterial proteins have at least one paralog and that the likelihood that a protein belongs to a small or a large cluster depends on the function of this particular protein.

Methods Enzymol, 1996, 266, 131 - 41
Applications of network BLAST server; Madden TL et al.; The sequence databases continue to grow at an extraordinary rate . Contributions come from both small laboratories and large-scale projects, such as the Merck EST project . This growth has placed new demands on computational sequence comparison tools such as BLAST . Even now it is no longer practical to evaluate some BLAST reports manually; it is necessary to filter the output by, for example, organism, source, or degree of annotation . The new network BLAST service makes such tools possible . It is also possible to present BLAST output in different formats, such as BLANCE . Perhaps most important of all, it becomes simple to call BLAST from another application, making it one step within an integrated system . This makes the automated preparation of sequence evaluations that include BLAST runs possible . In the near future we expect to see a number of applications that use the network BLAST interface to help molecular biologists search against a database that is growing not only in size but in biological richness.

Methods Enzymol, 1996, 267, 52 - 68
Phage display of proteases and macromolecular inhibitors; Wang CI et al.; We describe methods for displaying the protease trypsin and the macromolecular protease inhibitor ecotin on the surface of filamentous phage . Our strategy for selecting variant ecotins against target proteases is also described . We believe that the two proteins that have been displayed serve as ideal models for studying molecular recognition in detail . The ability to search efficiently through a large number of variant proteins for desired properties using phage display technology and the in vitro selection methods described opens a new avenue for studying protein-ligand interactions, as well as creating proteins with novel functions.

Tsitol Genet, 1996 Jan-Feb, 30(1), 42 - 8
{Heterologous protein expression in the baculovirus vector system: the nuclear polyhedrosis virus of Malacosoma neustria in Antheraea pernyi cells}; Strokovskaia LI et al.; The results of the creation of new expressive system on Malacosoma neustria nuclear polyhedrosis virus and cell line originated from Antheraea pernyi tissues was demonstrated . The expression of bacterial beta-galactosidase-coding gene was analyzed in this system.

J Struct Biol, 1996 Jan-Feb, 116(1), 17 - 24
A new generation of the IMAGIC image processing system; van Heel M et al.; One of the aims of modern microscopy is to quantify two-, three-, or even four-dimensional phenomena in biology, medicine, and material sciences . The requirements imposed on software by such data processing are exemplified by the design considerations of the IMAGIC-5 software system . This system includes facilities for multivariate statistical analysis of large data sets, for correlation averaging of two-dimensional crystals, and for three-dimensional reconstruction of macromolecular structures . The molecules may be arranged as two-dimensional crystals, as helices, or as single particles with arbitrary pointgroup symmetry . IMAGIC's novel angular reconstitution approach allows for the rapid determination of three-dimensional structures of uncrystallized molecules to high resolution . The general organization, user interaction strategy, file structure, and extendibility of IMAGIC are discussed and illustrated with some practical examples.

Cytokine, 1996 Jan, 8(1), 6 - 13
Expression and co-cytokine function of murine thioredoxin/adult T cell leukaemia-derived factor (ADF); Blum H et al.; Human ADF (adult T cell leukaemia-derived factor), an isoform of thioredoxin, promotes proliferation of certain human lymphoid cell lines and is involved in many thiol-dependent reducing reactions . To study functional aspects of the murine homologue, we established inducible overexpression of murine ADF in E . coli and a purification method which led to an apparently homogeneous 14 kDa protein . This recombinant ADF was tested in proliferation assays with murine Th2 cells (D10.G4.1) and CTLL-2 cells . In synergy with IL-2, IL-4, IL-7 and IL-9 ADF displayed co-cytokine activity . These proliferative effects were neutralized by an affinity-purified polyclonal rabbit anti-ADF antiserum . The effects of ADF were critically dependent on the presence of 2-mercaptoethanol . Bacterial thioredoxin had similar effects on the proliferation of murine T cells . Thus, the thiol-related reducing capacity of these proteins is essential for their growth promoting activity . As investigated at the levels of mRNA and protein in several murine cell clones and lines as well as in mouse tissues ADF is expressed ubiquitously . Finally it could be demonstrated by competitive PCR that in contrast to cytokine mRNAs (e.g . IL-4 and IL-13) the expression of ADF mRNA in murine Th2 clones and spleen cells is not influenced by stimulation of these cells through the T cell receptor complex . Murine ADF therefore represents a protein constitutively expressed in a wide variety of cells with the capacity to enhance the proliferative effect of several cytokines on murine T cells.

Bioconjug Chem, 1996 Jan-Feb, 7(1), 126 - 30
On-off switching of enzymatic reaction by recombinant calmodulin on a solid-phase matrix; Kobatake E et al.; A fusion protein consisting of human calmodulin (CaM) and glutathione S-transferase (GST) was produced by gene fusion . The fusion protein was overexpressed in Escherichia coli as a soluble form and purified with one-step affinity chromatography using glutathione-Sepharose . The protein had the modulating activity of CaM and the binding capability to glutathione of GST . Phosphodiesterase, which is a CaM dependent enzyme, was activated by the fusion protein, with the Ca2+ level equal to the level equivalent to a native CaM . Furthermore, CaM could be immobilized on a solid-phase matrix through the use of GST moiety while its modulating activity was retained . Phosphodiesterase activity was switched on and off by the immobilized CaM with or without Ca2+, and repeated use of CaM was demonstrated.

Parasitol Res, 1996, 82(4), 291 - 6
Parasite vaccine development: large-scale recovery of immunogenic Taenia ovis fusion protein GST-45W(B/X) from Escherichia coli inclusion bodies; Dempster RP et al.; Genetically modified Escherichia coli expressing the Taenia ovis fusion protein GST-45W(B/X) as inclusion bodies were grown in volumes ranging up to 1000 l . Bacteria were inactivated by heat or chemical treatment without affecting immunogenicity . The fusion protein was recovered in a highly immunogenic form from washed inclusion bodies and from urea-solubilised inclusion bodies . The fusion protein was found to be stable in solution after storage at 4 degrees C for up to 2 years . Vaccines formulated with fusion protein from urea-soluble inclusion bodies gave consistently high protection (89-100%) against challenge infection . The methods described enabled the production of sufficient vaccine for large field trials . These trials generated the data required for product registration and manufacture of a vaccine to prevent T . ovis infection in sheep.

Pflugers Arch, 1996, 431(6 Suppl 2), R233 - 4
Searching for new TNF-alpha analogs having potential application in cancer therapy; Menart V et al.; Two new TNF-alpha analogs were prepared and tested for their anti-tumor activity on fibrosarcoma SA-1 tumor model in vivo . In analog LK-801 two histidines (His107His108) were introduced into the surface loop thus enabling efficient purification by metal-affinity chromatography . This analog showed less side effects and can serve as a lead compound to look for other useful mutations . Another analog LK-802 was designed by introduction of additional pair of mutations (Cys95Cys148) into LK-801 in order to prepare disulfide linked TNF trimers . Cytotoxicity on mouse cell line L929 was comparable to TNF-alpha, but effect on tumor growth was quite reduced . Pharmacokinetic study revealed that serum levels of LK-802 were quite low in comparison to native TNF-alpha . This at least partially explains why anti-tumor activity of LK-802 is reduced and also illustrates the problems in designing the analogs with desired in vivo biological properties.

Pflugers Arch, 1996, 431(6 Suppl 2), R227 - 8
Human interleukin-2 (IL-2) expressed by transfected mammalian cells; Knezevic M et al.; cDNA for human interleukin-2 (IL-2) was cloned into the pRc/RSV vector for expression in animal cells . Baby hamster kidney (BHK-21) cells and Chinese hamster ovary (CHO) cells were transfected several times using calcium phosphate and electroporation methods with the construct pRc/RSV SIGIL2 . Different transfection efficiencies were obtained . The biological test on CTLL-2 (mouse cytotoxic lymphocytes) showed that the kinetics of cell proliferation were different from those of rIL-2 (recombinant IL-2) expressed in bacteria and in BHK cells . When high concentrations of rIL-2 were applied, an inhibitory effect on CTLL-2 was observed when bacterial product was used, whilst rBHK interleukin caused no inhibition . Recombinant BHK IL2 induced a slower response of CTLL-2 cells at the beginning of the cultivation, however, prolonged activity was detected at the later stage of the experiment.

Brain Res Bull, 1996, 40(3), 151 - 6
Endotoxin administration induced differential neurochemical activation of the rat brain stem nuclei; Molina-Holgado F et al.; Lipopolisaccharide (LPS) is a potent activator of the immune system, but also activates the hypothalamic-pituitary-adrenocortical (HPA) axis and cerebral catecholamine systems . In the present study, the effect of peripheral LPS administration on catecholaminergic and serotonergic neurotransmission in discrete brainstem nuclei was examined . Two hours following systemic administration of LPS (1, 10, or 100 micrograms/kg) norepinephrine (NE) content in the locus coeruleous (LC) was significantly increased in a dose related manner . An increased dopamine (DA) turnover as reflected by the 3,4-dihydroxyphenylacetic (DOPAC) + Homovanillic acid (HVA)/DA ratio, {DO-PAC + HVA}/{DA}, was also observed at the LC with the medium and high doses of LPS administered . Endotoxin caused the main effects in the nucleus of the tractus solitarii (NTS) in which (a) it was found NE content increased in a dose related fashion, (b) DA turnover index was elevated with 10 and 100 micrograms/kg LPS doses, and (c) levels of serotonin (5-HT) and its catabolite, 5-hydroxyindole-3-acetic acid (5-HIAA), were also significantly elevated following the injection of 10 or 100 micrograms/kg LPS . By contrast, a consistent lack of catecholaminergic and serotonergic responses to endotoxin treatment was observed at the level of midbrain Raphe nuclei (MRN) . These results demonstrate that differential neurochemical changes may occur in the brainstem region with a rank order of activation by LPS that was NTS > LC > MRN, suggesting different neural substrate for central effects of peripheral immune activation.

Peptides, 1996, 17(3), 363 - 6
Release of serotonin from RBL-2H3 cells by the Escherichia coli peptide toxin STb; Harville BA et al.; Heat-stable enterotoxin b (STb) of Escherichia coli is a 48-amino acid basic, disulfide-bonded peptide that causes intestinal secretion in experimental animal models . Recent evidence suggests that the in vivo mechanism of STb action involves release of 5-hydroxytryptamine (5-HT) and production of prostaglandin E2 (PGE2) . Here we show STb-mediated release of 5-HT from rat basophilic leukemic cells (RBL-2H3), a mast cell line model used extensively to study 5-HT release . Increasing concentrations of biologically active STb resulted in a dose-dependent release of 5-HT from RBL-2H3 cells . In contrast to these results, reduced and alkylated STb had no effect on 5-HT release . Release of 5-HT from RBL-2H3 cells was independent of extracellular calcium ions and did not involve changes in the intracellular concentration of free Ca2+ . In addition, pertussis toxin treatment completely blocked 5-HT release, indicating a role for a pertussis toxin-sensitive G-protein in the mechanism of 5-HT release from this cell type.

Pathol Biol (Paris), 1996 Jan, 44(1), 14 - 24
Role of DNA repair enzymes in the cellular resistance to oxidative stress; Laval J; Oxidative stress occurs in cells when the equilibrium between prooxidant and antioxidant species is broken in favor of the prooxidant state . It is due to reactive oxygen species (ROS) generated either by the cellular metabolism such as phagocytosis, mitochondrial respiration, xenobiotic detoxification, or by exogenous factors such as ionizing radiation or chemical compounds performing red-ox reactions . Some ROS are extremely reactive and interact with all the macromolecules including lipids, nucleic acids and proteins . Cells have numerous defence systems to counteract the deleterious effects of ROS . Proteins and small molecules specifically eliminate ROS when they are formed . There are three species of superoxyde dismutases which transform the superoxyde anion O2- in hydrogen peroxyde H2O2 which in turn will be destroyed by peroxysomal catalase or by various peroxydases . There are numerous small molecules in the cell such as glutathion, alpha-tocopherol, vitamines A and C, melanine, etc . which are antioxydant molecules . ROS escaping destruction generate various lesions in DNA such as base modifications, degradation products of deoxyribose, chain breaks . These various lesions have been characterized and it is possible to quantitate them in the DNA of cells which have been irradiated or treated by free radical generating systems . The biological properties of the bases modified by ROS have been established . For example C8-hydroxyguanine (8-oxoG) is promutagenic since, if present in DNA during replication, it leads to incorporation of dAMP residues, leading to transversion mutation (GC-->TA) . Purines whose imidazole ring is opened (Fapy residues) are stops for the DNA polymerase during DNA replication and are therefore potentially lethal lesions for the cell . Oxidized pyrimidines have comparable coding properties . Efficient DNA repair mechanisms remove these oxidized bases . In Escherichia coli cells, endonuclease III (NTH protein) and endonuclease VIII (NEI protein) excise many oxidized pyrimidines, whereas the FPG protein (formamidopyrimidine-DNA-glycosylase) eliminates 8-oxoG and Fapy lesions . Besides its DNA glycosylase activity, the protein FPG has a beta-lyase activity incising DNA at abasic site by a beta-delta elimination mechanism, and a dRPase activity . The FPG protein has a zinc finger motive which is mandatory for the recognition of its substrate . Mammalian cells have similar DNA repair proteins and it should be emphazized that there is conservation of the different functions and in most cases a remarquable homology of the amino acids sequences from E . coli to man.

J Comp Pathol, 1996 Jan, 114(1), 11 - 21
Comparison of the effects of infectious bronchitis and infectious laryngotracheitis on the chicken respiratory tract; Nakamura K et al.; In infectious bronchitis (IB) virus infection of the chicken the upper and lower respiratory tracts were damaged, but infectious laryngotracheitis (ILT) virus caused lesions only in the upper respiratory tract . Secondary infection with Escherichia coli was apparent in the trachea of birds inoculated with either virus but was more striking in those given IB virus . Serum alpha 1-acid glycoprotein, an acute-phase protein, occurred in higher concentrations in chickens inoculated with IB virus than in those given ILT virus.

Perit Dial Int, 1996, 16 Suppl 1, S489 - 91
Successful pregnancy complicated by peritonitis in a 35- year old CAPD patient; Tison A et al.; A 35-year old woman conceived six months after initiating continuous ambulatory peritoneal dialysis (CAPD) . A medical plan was developed to give the patient adequate dialysis for a 1.5 g/kg/day protein intake . In addition, alterations in calcium, magnesium, and erythropoietin administration were required to reach the objectives set by the obstetrical/renal team . Three weeks prior to delivery, an amniotic leak developed, and vaginal cultures were positive for Escherichia coli . Oral amoxicillin was administered (500 mg per os q.i.d.) until the day of delivery . A 1545-g baby girl was delivered by cesarean section at 32 weeks . Five days postpartum the patient developed severe peritonitis, which subsequently grew E . coli . The patient fully recovered from the peritonitis, but catheter removal was required . Successful pregnancy can be expected on CAPD, and adequacy can be achieved with aggressive dialysis . Cesarean section delivery should probably be accompanied by full peritonitis therapy.

Hokkaido Igaku Zasshi, 1996 Jan, 71(1), 55 - 68
{Influence of total parenteral nutrition in immature rats: effect of glutamine infusion on bowel integrity}; Okawa Y; Total parenteral nutrition (TPN) is accepted important therapeutic adjunct in spite of many complications for management of pediatric patients who aren't allowed to eat . Recently, bacterial translocation was added to the complications of TPN . The purpose of this study was to examine the influence of TPN on the gut in immature rats . 65g rats were randomized to one of four groups: Group C (control) received food and water ad libitum . Group P (TPN) received standard TPN solution . Group G (TPN+GLT) received glutamine (7g/400ml of TPN) . Group E (TPN with enhanced protein) received TPN solution with enhanced protein (18.5g/400ml), without GLT . CFU of E . coli in mesenteric lymphnodes was significantly higher in group P than in other 3 groups at 5 days . Hepatic glutathione was significantly higher in group G than in group P and group E at 7 days . Weight of wet intestine was the highest in group C in all groups, and significantly higher in group G than in group P and group E at 3 and 7 days . Mucosal protein of group C was the highest of those of 4 groups . That of group G was significantly higher than those of group P and G in 5 and 7 days . Mucosal thickness and villous height were the highest in group C in 4 groups . Mucosal thickness was significantly higher in group G than in group P and group E at 5 and 7 days . Villous height was significantly higher in group G P and group E at all days . These results suggest that TPN promotes intestinal atrophy from early days after TPN in immature rats, that glutamine might play a role in maintenance of structural integrity of intestine, and that glutamine would prevent the bacterial translocation.

Biochimie, 1996, 78(1), 14 - 25
Production, analysis and bioactivity of recombinant vasoactive intestinal peptide analogs; Raingeaud J et al.; Recombinant vasoactive intestinal polypeptide (VIP) analogs were expressed in Escherichia coli as a fusion protein containing tandemly repeated multiple copies of a synthetic VIP gene joined to glutathione S-transferase . The encoded protein contains VIP units separated by a linker peptide, potentially excisable by a double cleavage with endoprotease factor Xa and hydroxylamine . Expression of different polyVIP genes, from 1 to 32 units, was detected and the production of a 16 VIP polymer was performed . MonoVIP analogs appended by 5 or 10 amino acids at their C terminus were released by factor Xa from this polymerized product . They were then submitted to hydroxylamine cleavage to remove the linker sequence to finally obtain a recombinant VIP analog devoid of any amino acid extension . The biological activity of the recombinant polyVIP and VIP analogs was tested . Although less efficient than the natural neuropeptide, some of these components bound to VIP receptor, activated adenylate cyclase in human colonic adenocarcinoma cells and displayed a relaxation activity on guinea pig tracheal rings.

J Mol Recognit, 1996 Jan-Feb, 9(1), 39 - 51
Comparative interaction kinetics of two recombinant Fabs and of the corresponding antibodies directed to the coat protein of tobacco mosaic virus; Chatellier J et al.; Two recombinant Fab fragments, 57P and 174P, recognizing peptide 134-146 of the coat protein of tobacco mosaic virus have been cloned, sequenced and expressed in Escherichia coli . They differ by 15 amino acid changes in the sequence of their variable region . The interaction kinetics of the Fabs with the wild-type and four mutant peptides have been compared using a BIAcoreTM biosensor instrument . The recombinant Fab 174P had the same reactivity as the Fab fragment obtained by enzymatic cleavage of monoclonal antibody 174P . The two recombinant Fabs recognized the various peptides in the same ranking order but Fab 174P consistently dissociated somewhat faster from the peptides compared to Fab 57P . The two whole antibodies showed the same relative differences in reactivity as the two recombinant Fabs . The location of amino acid changes was visualized on a model structure of the Fab . Differences in dissociation rates of the two antibodies are most likely due to changes located at the periphery of the antigen-combining site and/or at the interface between the light and heavy chain domains . Our results demonstrate the feasibility of detecting very small differences in binding affinity by the biosensor technology, which is a prerequisite for assessing the functional effect of limited structural changes.

J Mol Recognit, 1996 Jan-Feb, 9(1), 13 - 22
Lac repressor-operator interaction: N-terminal peptide backbone 1H and 15N chemical shifts upon complex formation with DNA; Artz PG et al.; When the lac repressor tetramer is bound to its DNA operator, methylation protection shows the nearly symmetric operator half-sites are contracted asymmetrically . This asymmetric binding results from the DNA sequence/structure . The reported structure of lac repressor N-terminal fragment and an 11 base-pair operator left half-site provides no information concerning the effect of asymmetric binding, from left operator half-site to right half-site, upon the polypeptide backbone . We isolated uniformly 15N labeled 56 amino acid wild-type (HP56WT) and 64 residue mutant {Pro3 > Tyr3} (HP64tyr3) lac repressor N-terminal DNA binding fragments for 1H/15N NMR studies with the left and right operators separately . Spectral coincidence of these longer fragments, indicating structural similarity with a protease derived 51 amino acid fragment for which the amide correlations are assigned, allows for assignment of the common amide resonances . For both HP56WT and HP64tyr3, spectral overlap of the amide correlation peaks reveals the polypeptide backbones of the uncomplexed polypeptides are structurally similar . Likewise the complexes of the peptides to the 11 base-pair lac left operator half-site are similar . On the other hand, complexes of HP56WT and the left compared to the right lac operator half-site show different residues of the polypeptide are affected by binding different half-sites of the operator . Thus, the DNA sequence/structure transmits asymmetry to the polypeptide backbone of the interacting protein.

DNA Seq, 1996, 6(3), 179 - 84
Cloning and sequencing of the dnaK locus in Streptomyces coelicolor A3(2); Brans A et al.; The dnaK operon of Streptomyces coelicolor A3(2) was cloned by the DNA-probing method using synthetic oligonucleotides designed on the basis of two of the most conserved regions in 30 different DnaK proteins (HSP70) . The isolated insert-a BamHI 5.6-kb fragment-was sequenced and shown to contain three open-reading frames organized in an operon and coding for proteins analogous to DnaK, GrpE and DnaJ, successively.

DNA Seq, 1996, 6(3), 127 - 35
Characterization of cDNA encoding for phosphoglucose isomerase of rice (Oryza sativa L.); Nozue F et al.; Two types of genes (Pgi-a and Pgi-b) encoding phosphoglucose isomerase (PGI; EC 5.3.1.9) were cloned from cDNA libraries of rice cultured cells (Oryza sativa L.) . Pgi-a and Pgi-b consisted of 2132 and 2030 nucleotides, respectively . The homology between these genes was 93.0% at nucleotide level . The homology scores between these genes in protein coding region and 3' non-coding region were 95.6% and 79.4%, respectively . PGI proteins encoded by Pgi-a and Pgi-b consisted of 567 and 568 amino acid, respectively, sharing 95.8% homology at amino acid sequences . Of 11 PGI genes from other plant species and organisms whose amino acid sequences had been determined, a dicotyledonous plant Clarkia lewisii PGI showed the highest homology (about 80%) with rice PGIs . GC contents at the third position of rice PGI genes were about 40% . In order to confirm the enzyme activity of the protein encoded by the rice cDNA, Pgi-a was subcloned into an expression vector, pBluescript II SKp, which was introduced into Escherichia coli . The transformant had an additional PGI activity from Pgi-a.

Scand J Gastroenterol Suppl, 1996, 215, 11 - 5
Prevention of Helicobacter pylori infection; Lee A; The only successful strategy for large-scale eradication of an infectious disease from whole populations has been through immunization . In societies fortunate enough to have a healthcare infrastructure that allows mass vaccination, diseases such as poliomyelitis, whooping cough and diphtheria have been virtually eliminated . But what about infection with Helicobacter pylori, which has now been proved to be a major worldwide pathogen? Infection by H . pylori is lifelong, despite a vigorous immune response . How, then, could immunization be feasible? Recent investigations using a mouse model of H . pylori infection have shown that protective immunity can indeed be induced with an oral vaccine . Furthermore, protection was lifelong in the animal model . Also, more importantly, the vaccine actually cured existing infection, raising the exciting possibility of therapeutic immunization . Stimulated by the medical need, the race is now on to produce the first vaccine . However, many critical questions remain to be answered before the first patients are immunized with the complete vaccine . For example, what is the best antigen to target? Urease is currently the favourite antigen, but others will be tested . What about the adjuvant? LT, the heat labile enterotoxin of diarrhoeaogenic strains of Escherichia coli, is the most likely candidate, but we need to examine alternatives . Is there any risk of immunopotentiation? The answer is probably no, but this has to be proved . A vaccine against H . pylori will be developed, and infection with H . pylori will be prevented--the question remains 'how long will it take'?

Mikrobiologiia, 1996 Jan-Feb, 65(1), 10 - 4
{Distribution of polyamines in Escherichia coli and their role in potassium exchange between the cell and the environment during aerobiosis-anaerobiosis transitions}; Tkachenko AG et al.; The transition of E . coli cells to anaerobiosis is accompanied by the onset of two cellular cation flows in different directions: potassium is released into the environment, and putrescine enters the cells . If aerobic conditions are reestablished, the cation flows change directions . Under anaerobiosis, the cell components bind putrescine . Investigation of the chemical interactions resulting in putrescine binding and metabolic conversion under anaerobic conditions revealed DNA to be the main target in this process . The driving forces and mechanisms of cation transfer are discussed, and the involvement of putrescine in topological changes in the DNA and the development of adaptive cell responses to anaerobiosis is considered.

Brain Res Mol Brain Res, 1996 Jan, 35(1-2), 227 - 36
A herpes simplex virus-1 vector containing the rat tyrosine hydroxylase promoter directs cell type-specific expression of beta-galactosidase in cultured rat peripheral neurons; Oh YJ et al.; A defective herpes simplex virus-1 (HSV-1) vector system was used to study cell type-specific expression of the tyrosine hydroxylase (TH) gene . HSV-1 particles containing 663 bp (pTHlac 663), 278 bp (pTHlac 278), or 181 bp (pTHlac 181) of the rat TH promoter driving E . coli LacZ were used to infect superior cervical ganglia (SCG: TH-expressing tissue) and dorsal root ganglia (DRG:non-TH-expressing tissue) cultures . One day after infection, expression of beta-galactosidase was visualized by X-gal cytochemistry . Following viral transduction with pTHlac 663 at a multiplicity of infection of 0.2, 14.4% of the SCG neurons were X-gal positive whereas only about 0.9% of DRG neurons were X-gal positive . Infection with either pTHlac278 or 181 resulted in 3-fold more X-gal-positive DRG neurons . These results suggest that (i) the defective HSV-1 vector system may be useful in defining regulatory promoter motifs; (ii) 663 bp of the rat TH promoter contains sufficient information for cell type-specific expression in peripheral nervous system neurons; and (iii) sequences between -278 and -663 contain an element(s) that represses gene expression in non-catecholamingeric neurons.

Arch Virol, 1996, 141(6), 987 - 99
Accumulation kinetics of CMV RNA 3-encoded proteins and subcellular localization of the 3a protein in infected and transgenic tobacco plants; Vaquero C et al.; The complete nucleotide sequence of RNA 3 of a Spanish isolate of cucumber mosaic virus (CMV-24) has been determined . The encoded putative cell-to-cell movement protein (3a protein) and the coat protein are 279 and 218 amino acids long, respectively . The 3a protein was expressed in Escherichia coli using the vector pT7-7 and was used to raise an immunoserum . We have followed the time course of accumulation of the 3a protein, in parallel to that of the coat protein, and its subcellular localization as a function of time after CMV-24 infection on tobacco plants . The maximum accumulation level of the 3a protein was reached at early stages of infection, being detected in the cytosolic and the cell wall fractions . At later stages of infection, a decline in accumulation levels of the 3a protein was observed, and the protein was essentially associated with the cell wall fractions . These data were corroborated by immunocytochemistry performed in both infected and 3a-expressing transgenic tobacco plants.

Anesth Analg, 1996 Jan, 82(1), 167 - 72
Identification of human liver cytochrome P-450 3A4 as the enzyme responsible for fentanyl and sufentanil N-dealkylation; Tateishi T et al.; Alfentanil, sufentanil, and fentanyl are synthetic opioids that are metabolized by oxidative N-dealkylation in the liver . We have previously shown that cytochrome P-450 3A4 (CYP3A4) contributes significantly to human liver microsomal alfentanil oxidation . Since identification of specific drug-metabolizing enzymes allows prediction of the variables affecting drug metabolism, the purpose of the present study was to identify the P-450 enzymes responsible for sufentanil and fentanyl metabolism in human liver microsomes . Microsomal preparations fortified with a reduced nicotinamide-adenine dinucleotide phosphate-generating system were incubated with 0.25 microM 3H-fentanyl or 3H-sufentanil . Rates of N-dealkylated metabolite formation significantly correlated with nifedipine oxidation activity (a marker of CYP3A4 activity) for fentanyl and sufentanil (r = 0.93 and 0.87, n = 18, respectively), but not with the oxidation activity for ethoxyresorufin (CYP1A2), S-mephenytoin (CYP2C19), bufuralol (CYP2D6), or chlorzoxazone (CYP2E1) . Gestodene and troleandomycin (chemical inhibitors of CYP3A4) and antibody to CYP3A4 inhibited N-dealkylation of fentanyl and sufentanil . Chemical inhibitors of CYP2C, 2E1, and 2D6 did not inhibit N-dealkylation of fentanyl and sufentanil . Recombinant CYP3A4 expressed in Escherichia coli showed N-dealkylation activity of fentanyl and sufentanil, while expressed CYP1A2, 2C10, and 2E1 enzymes did not . We conclude that CYP3A4 is responsible for fentanyl and sufentanil N-dealkylation in vitro.

Vet Res Commun, 1996, 20(2), 183 - 90
The effects of glibenclamide, a blocker of K+ ATP-sensitive potassium channels, on diaphragmatic fatigue during endotoxaemia in pigs; Aguggini G et al.; An in vivo porcine model of endotoxaemia was used to study the effects of glibenclamide, a K+ ATP-sensitive potassium channel blocker . Escherichia coli lipopolysaccharides (LPS, 70 micrograms/kg, i.v., as a bolus) were infused into anaesthetized, mechanically ventilated, indomethacin-treated pigs . After 120 min of endotoxaemia, glibenclamide was administered (10 mg/kg, i.v., over 5 min) to half the pigs . The strength at different frequencies of stimulation (10, 20, 30, 50 Hz, 20 V,) 1 s) and the endurance capacity (10 Hz, 20 V, 30 s) of the diaphragm were evaluated after 120 min of endotoxaemia and 5, 10, 20 and 30 min after drug infusion . Glibenclamide transiently increased the blood pressure without changing the decreased cardiac output and at the same time further impaired the diaphragmatic activity . The reduced ability of the diaphragm to generate force in response to different electrical stimulations was shown by a significant reduction in strength . The endurance index decreased 5 min after glibenclamide infusion, returning to the pre-glibenclamide values by 150 min . These results indicate that glibenclamide modifies the activity of vascular smooth muscle and of the diaphragm.

Gene, 1996, 173(1 Spec No), 33 - 8
FACS-optimized mutants of the green fluorescent protein (GFP); Cormack BP et al.; We have constructed a library in Escherichia coli of mutant gfp genes (encoding green fluorescent protein, GFP) expressed from a tightly regulated inducible promoter . We introduced random amino acid (aa) substitutions in the twenty aa flanking the chromophore Ser-Tyr-Gly sequence at aa 65-67 . We then used fluorescence-activated cell sorting (FACS) to select variants of GFP that fluoresce between 20-and 35-fold more intensely than wild type (wt), when excited at 488 nm . Sequence analysis reveals three classes of aa substitutions in GFP . All three classes of mutant proteins have highly shifted excitation maxima . In addition, when produced in E . coli, the folding of the mutant proteins is more efficient than folding of wt GFP . These two properties contribute to a greatly increased (100-fold) fluorescence intensity, making the mutants useful for a number of applications.

Gene, 1996, 173(1 Spec No), 19 - 23
Dual color microscopic imagery of cells expressing the green fluorescent protein and a red-shifted variant; Yang TT et al.; The green fluorescent protein (GFP) from the jellyfish, Aequorea victoria, has become a versatile reporter for monitoring gene expression and protein localization in a variety of cells and organisms . GFP emits bright green light (lambda max = 510 nm) when excited with ultraviolet (UV) or blue light (lambda max = 395 nm, minor peak at 470 nm) . The chromophore in GFP is intrinsic to the primary structure of the protein, and fluorescence from GFP does not require additional gene products, substrates or other factors . GFP fluorescence is stable, species-independent and can be monitored noninvasively using the techniques of fluorescence microscopy and flow cytometry {Chalfie et al., Science 263 (1994) 802-805; Stearns, Curr . Biol . 5 (1995) 262-264} . The protein appears to undergo an autocatalytic reaction to create the fluorophore {Heim et al., Proc . Natl . Acad . Sci . USA 91 (1994) 12501-12504} in a process involving cyclization of a Tyr66 aa residue . Recently {Delagrave et al., Bio/Technology 13 (1995) 151-154}, a combinatorial mutagenic strategy was targeted at aa 64 through 69, which spans the chromophore of A . victoria GFP, yielding a number of different mutants with red-shifted fluorescence excitation spectra . One of these, RSGFP4, retains the characteristic green emission spectra (lambda max = 505 nm), but has a single excitation peak (lambda max = 490 nm) . The fluorescence properties of RSGFP4 are similar to those of another naturally occurring GFP from the sea pansy, Renilla reniformis {Ward and Cormier, Photobiochem . Photobiol . 27 (1978) 389-396} . In the present study, we demonstrate by fluorescence microscopy that selective excitation of A . victoria GFP and RSGFP4 allows for spectral separation of each fluorescent signal, and provides the means to image these signals independently in a mixed population of bacteria or mammalian cells.

Exp Gerontol, 1996 Jan-Apr, 31(1-2), 145 - 57
Expression of senescence-induced protein WS3-10 in vivo and in vitro; Grigoriev VG et al.; In our efforts to characterize cellular senescence we have shown that the mRNA encoding WS3-10 protein is overexpressed in senescent human diploid fibroblasts (HDF) when compared with their younger counterparts, and that forced expression of the WS3-10 cDNA in young HDF results in suppression of calcium-dependent membrane currents, presumably due in part to the presence of a calcium binding domain within the WS3-10 protein . We have now expressed this protein in E . coli and have obtained affinity purified antibodies . Western blot analysis utilizing these antibodies showed that WS3-10 protein is also overexpressed in senescent HDF when compared to young HDF, and in normal fetal lung HDF when compared to SV40-transformed fetal lung HDF . HeLa cells do not express WS3-10 protein . In addition, we looked for WS3-10-related species in a variety of rat tissues . Analysis of WS3-10 immunologically related proteins in rat tissue extracts revealed two WS3-10 homologs, sized 22 kDa and 20 kDa . The latter presumably result from proteolytic removal of the C-terminal end of the 22 kDa polypeptide . The ratio between these polypeptides varies in a tissue-specific manner . Two proteins immunologically related to WS3-10 with sizes of 39 kDa and 91 kDa were present in rat spleen and skeletal muscle, respectively.

Exp Gerontol, 1996 Jan-Apr, 31(1-2), 135 - 44
Impaired expression of hematopoietic growth factors: a candidate mechanism for the hematopoietic defect of aging; Buchanan JP et al.; Aged humans and aged experimental animals exhibit a diminished ability to upregulate hematopoiesis, but the mechanism responsible for this is unknown . The purpose of the studies was to test the hypothesis that this disregulation might be attributable to altered expression of hematopoietic growth factors . We studied the in vitro ability of cells from aged humans or mice to release bioactive colony stimulating activity (CSA) and to accumulate mRNA for defined growth factors . Human light density leukocytes from the aged released less CSA than cells from the young . GM-CSF accounted for 72-100% of the CSA released by young humans, as judged by antibody inhibition studies . In contrast, GM-CSF accounted for 0-42% of CSA from aged humans . The observation of diminished expression of GM-CSF by cells from aged humans was further accompanied by a reduced accumulation of mRNA for GM-CSF . In other in vitro studies, splenic cells from aged mice released less CSA than cells from young mice, and in vivo experiments showed that splenic cells from mice challenged with E . coli accumulated less mRNA for M-CSF than young mice . These data demonstrate a similar defect in the expression of hematopoietic growth factors in aged man and mice . We propose that this diminished production of bioactive and identifiable hematopoietic growth factors may be mechanistically important in explaining the disordered hematopoiesis of aging.

Eur Cytokine Netw, 1996 Jan-Mar, 7(1), 67 - 9
Interleukin-10 pharmacokinetics in intact and nephrectomized mice; Chiu PJ et al.; The influence of kidney function on interleukin-10 (IL-10) pharmacokinetics was assessed by comparing the disappearance of IL-10 from the circulation in the intact and acutely nephrectomized mice over 1 h following a single bolus injection of E . coli-derived recombinant h IL-10 at 250 micrograms/kg i.v . The intact mice demonstrated a C(max) of 1,172 ng/ml and an AUC(tf) of 385 n g.h/ml . By comparison, there was a 4-fold elevation in C(max) and a 7-fold increase in AUC(tf) in the anephric mice . The serum IL-10 concentration at 1 h post-injection was 87 ng/ml in the intact vs 1,684 ng/ml in the anephric mice . The results imply that IL-10, in common with other cytokines, is eliminated through the kidney.

Microbios, 1996, 85(345), 205 - 12
Recovery of soluble protein after expression in Escherichia coli depends on cellular disruption conditions; Gonzalez C et al.; Proteins overproduced in Escherichia coli are frequently recovered from bacterial extracts as insoluble material . The influence of different cell disruption procedures on the recovery of soluble protein, after recombinant protein expression in E . coli, was assessed using two beta-tubulin derivatives . Nonionic detergents such as Triton X-100 and Nonidet P-40 promote aggregation when present in the lysis buffer . The effect of Triton X-100 is reversed by the addition of 1 M NaCl in the lysis buffer indicating that the recombinant protein aggregation is probably caused by interactions with membrane proteins . The importance of the cellular disruption method on the recovery of potentially soluble recombinant proteins is discussed.

J Cancer Res Clin Oncol, 1996, 122(8), 453 - 7
Distribution of Gs-alpha activating mutations in human thyroid tumors measured by subcloning; Gorelov VN et al.; In the search for the prevalence and distribution pattern of Gs-alpha gene mutations in differentiated thyroid tumors we examined 66 tumor tissue samples for the presence of mutations at "hot-spot" codons 201 and 227 using methods based on the polymerase chain reaction, subcloning and sequencing . The prevailing type of single-base substitution at codon 201 (71.4%) corresponded to the replacement of the wild-type sequence CGT (Arg) with TGT (Cys) . The fragments of the Gs-alpha gene, including codon 201 or 227 from five follicular carcinomas and one follicular adenoma, were subcloned in Escherichia coli and it was found that the proportion of alleles with mutated codon 201 varied from 3.2% to 43% . Sequencing of the corresponding region has confirmed preliminary data indicating that the single-base changes CGT (Arg) to TGT (Cys) or CGT to CAT (His) occurred . There was only a weak correlation between the prevalence of cells bearing a mutation in the Gs-alpha gene and the level of Gs-alpha protein expression in the corresponding thyroid tumors.

J Appl Bacteriol, 1996 Jan, 80(1), 81 - 90
Rapid estimation of Escherichia coli in live marine bivalve shellfish using automated conductance measurement; Dupont J et al.; Conductance measurement for quantitative estimations of Escherichia coli in live bivalve shellfish was evaluated as an alternative to the conventional most probable number (MPN) method used in France . The sensitivity of the two techniques was comparable . A single regression line (r = -0.968, P < 10(-6)) between log10 MPN and detection time (DT) was used to estimate E . coli concentrations for all shellfish examined . Estimation accuracy was +/- 0.92 log unit . Repeatability was better for DT than the log10 of MPN estimations (average coefficients of variation 2.7 and 9.3%, respectively) . The conductance signal was attributable to E . coli in 96% of cases, and only 0.7% of E . coli cultures failed to exhibit a signal within 20 h . The conductance method reduces analysis handling time and is much easier to use than the MPN method . Moreover, results can be obtained within 5-9 h compared to 3 d for the MPN method.

J Comput Biol, 1996 Spring, 3(1), 191 - 212
Integrated access to metabolic and genomic data; Karp PD et al.; The EcoCyc system consists of a knowledge base (KB) that describes the genes and intermediary metabolism of Escherichia coli, and a graphical user interface (GUI) for accessing that knowledge . This paper addresses two problems: How can we create a GUI that provides integrated access to metabolic and genomic data? We describe the design and implementation of visual presentations that closely mimic those found in the biology literature, and that offer hypertext navigation among related entities, and multiple views of the same entity . We employ a frame knowledge representation system (FRS) called HyperTHEO to manage the EcoCyc knowledge base . Among the advantages of FRSs are an expressive data model for capturing the complexities of biological information, and schema-evolution capabilities that facilitate the constant schema changes that biological databases tend to undergo . HyperTHEO also includes rule-based inference facilities that are the foundation of expert systems, a constraint language for maintaining data integrity, and a declarative query language . A graphic KB editor and browser allow the EcoCyc developers to interactively inspect and modify this evolving KB.

Plasmid, 1996 Jan, 35(1), 67 - 70
Plasmid pDGO100 contains a second integron with the trimethoprim resistance gene dfrA7 as the inserted cassette; Burnside JM et al.; Southern hybridization analysis of the IncC plasmid pDGO100 showed that, in addition to the well-characterized integron In7, there is a second integron which is located on a 3.6-kb BamHI fragment . This integron also possesses the qacE delta l and sulI genes typically found as part of the 3'-conserved segment of integrons . The 3.6-kb BamHI fragment, when cloned into pUC19 to form pDGO301, conferred resistance to trimethoprim as well as sulfamethoxazole . The DNA sequence of the trimethoprim resistance gene in pDGO301 was determined and it was shown that the gene was precisely inserted as a cassette in an integron with 100% identity to the trimethoprim resistance gene dfrA7.

Plasmid, 1996 Jan, 35(1), 58 - 61
Use of a DNA polymerase III bypass mutant of Escherichia coli, pcbA1, to isolate potentially useful mutations of a complex plasmid replicon; Banerjee S et al.; The essential replicon region of plasmid pCU1 has, within 1.2 kb, two origins of replication that can function in the absence of Escherichia coli DNA polymerase I and one that requires this polymerase . To isolate mutants in the replicon pathway that uses the PolI-dependent origin in the presence of the two other origins, we examined the feasibility of exploiting E . coli strains carrying a polymerase c bypass mutation (pcbA) and which can survive and form colonies with the polymerase activity of polC inactivated at 42 degrees C . The selection scheme that is described was successful and resulted in the isolation of a mutant replicon that is not maintained at 42 degrees C in a PcbA-PolC+tsPolA+ strain . Nucleotide sequencing indentified the mutated region to be within the origin (OriV) that was known to be polA-dependent . Electron microscopy of mutant plasmid molecules replicating in a Pcb+ strain confirmed that OriV is inactivated.

Microb Pathog, 1996 Jan, 20(1), 41 - 55
Molecular characterization of a surface-exposed superoxide dismutase of Mycobacterium avium; Escuyer V et al.; Mycobacterium avium is an intracellular pathogen capable of growing inside the phagosomal compartment of macrophages . In this work, we characterized the superoxide dismutase of M . avium, as a putative candidate to resist the oxidative stress . The gene sodA encoding superoxide dismutase (SOD:EC1.15.1.1) from Mycobacterium avium TMC724 was cloned and sequenced . It encodes a 23 kDa protein (207 aminoacids) showing identity with the Mycobacterium leprae SOD (91%) and the M . tuberculosis SOD (83%) . This enzyme was functionally expressed in both Escherichia coli and Mycobacterium smegmatis, and identified as a manganese (Mn) SOD on the basis of sequence comparison with other MnSODs from different organisms, and by activity inhibition studies . By indirect immunogold labeling of M . avium with a mAb directed against M . leprae SOD, the enzyme was found to be exposed at the cell surface of M . avium . It was also shown that SOD was released in supernates of M . avium TMV724 during exponential growth, suggesting a role of this enzyme during interactions with the environment . When SOD was expressed in the non-pathogenic M . smegmatis, it was also exposed at the surface of bacteria and released in supernates, but this was not sufficient to protect this recombinant mycobacterium from the killing mechanisms of macrophages.

Bioorg Med Chem, 1996 Jan, 4(1), 131 - 41
Overexpression, one-step purification and characterization of UDP-glucose dehydrogenase and UDP-N-acetylglucosamine pyrophosphorylase; De Luca C et al.; Two enzymes of the Leloir pathway, UDP-GlcNAc pyrophosphorylase and UDP-Glc dehydrogenase, which are involved in the synthesis of activated sugar nucleotides have been cloned, overexpressed in Escherichia coli, and purified to homogeneity in only one step by chelation-affinity chromatography . The gene KfaC of E . coli K5 was thus demonstrated to encode UDP-Glc DH . Some properties of the cloned enzymes, such as stability, pH dependence, and substrate kinetics, were studied in order to facilitate the use of these enzymes in carbohydrate synthesis, especially in the synthesis of hyaluronic acid.

Eur Surg Res, 1996, 28(1), 63 - 9
Changes of bile duct mucosa after choledochoduodenostomy in rats; Kuo K et al.; This study investigated the changes of bile duct mucosa in rats after choledochoduodenostomy . Wistar rats were divided into three groups: group I (n = 6) was treated with sham operation as control; group II (n = 10) was treated with common bile duct ligation without choledochoduodenostomy, and group III (n = 12) had both common bile duct ligation and choledochoduodenostomy creation 4 days after common bile duct ligation . From our observations, retrograde induced cholangitis due to enteric reflux into the common bile duct is the possible cause of chronic inflammation after choledochuduodenostomy . At the end of the study, Choledocholithiasis developed in 5 of 12 rats . Severe dysplasia was present in the bile duct mucosa in 6 of 12 rats in group III, but not in group I or II . A technique for silver staining of nucleolar organizing regions (AgNOR) was applied . This technique demonstrated differences in AgNOR counts between normal mucosa and dysplasia . Under AgNOR stain, the number of AgNOR was significantly greater than in the normal or benign counterparts and gradually increased from the normal bile duct mucosa group to the severe dysplasia group (group I 2.1 +/- 0.8, group II 3.2 +/- 1.0, group III 5.3 +/- 1.1) . All of these observations suggest that 'sump syndrome' and bile stasis could occur after choledochoduodenostomy in rats and can result in chronic inflammation of the bile duct . It has been well established that calcium bilirubinate is the major type of choledocholithiasis in Orientals . beta-Glucuronidase from bacteria, such as Escherichia coli present in the biliary tree, hydrolyzes bilirubin diglucuronide to bilirubin . Bilirubin combines with calcium in the bile flow to form calcium bilirubinate . Bacterial infection plays a key role in calcium bilirubinate stone formation . Since choledochoduodenostomy did increase reflux cholangitis, and bacterial infection would increase mucin overproduction, bile deconjugation and eventually new calcium bilirubinate stones would be formed . The dysplastic changes in the bile duct mucosa could possibly be related to the prolonged exposure to the biochemically altered infected bile . Thus choledochoduodenostomy might not be the perfect choice in treating calcium bilirubinate choledocholithiasis in Orientals.

Biokhimiia, 1996 Jan, 61(1), 89 - 99
{Isolation and certain properties of mutant alkaline phosphatase of Escherichia coli}; Nesmeianova MA et al.; Natural and mutant alkaline phosphatases with amino acid substitutions in the processing site and N-terminal domain of the mature polypeptide chain Val for Ala(-1), Gln for Glu (+4) and simultaneously Gln for Glu (+4) and Ala for Arg (+1) have been isolated from the periplasm and cultural fluid of E . coli . It has been found that these substitutions have little effect on the dependence of the enzyme activity on pH, ionic strength and temperature but influence its isoenzymic spectrum and decrease (almost twofold) the maximal rate of the enzyme-catalyzed reaction . Extracellular enzymes display, in contrast with periplasmic ones, other catalytic properties (Vmax) and binding activity (Km) . After translocation through the outer membrane all the enzymes display decreased Vmax and increased Km . These changes are especially well-pronounced in case of the mutant protein PhoA46 which contains an uncleaved signal peptide due to the impossibility of processing resulting from the substitution of Val for Ala(-1) . The Vmax for this protein is decreased 20 times, while the Km is increased 4-fold . The protein also shows a higher (in comparison with other proteins) sensitivity towards proteolytic enzymes and is less resistant upon storage . The experimental data suggest that the changes in the N-end of alkaline phosphatase located at a long distance from its active center influence the enzyme function.

Biokhimiia, 1996 Jan, 61(1), 152 - 9
{Physicochemical properties of recombinant luciferase from the firefly Luciola mingrelica and its mutant forms}; Dement'eva EI et al.; Physico-chemical properties of the recombinant L . mingrelica luciferase synthesized by E . coli cells have been studied . The catalytic and spectral properties of recombinant luciferase were similar to those of the native enzyme but the former was less stable in the presence of the additional Cys residue . The mutant forms of L . mingrelica firefly luciferase with point mutations Cys-82-->Ala, Cys-260-->Ala, Cys-393-->Ala and Thr-204-->Asp, have been constructed using the method of site-specific mutagenesis . Mutations Cys-82,260,393-->Ala changed slightly the Km values for ATP and luciferin but did not influence kcat . The Cys-393-->Ala mutant appeared to be more stable in comparison with the native enzyme . Mutation Thr-204-->Asp resulted in a 8-fold increase in the ATP binding constant and in a 2-fold increase in the kcat, thus indicating that Thr-204 may be located in the ATP-binding region of luciferase . Dithiothreitol, ethylene glycol, bovine serum albumin and trehalose had a stabilizing effect on the native, recombinant and mutant luciferases.

Biokhimiia, 1996 Jan, 61(1), 100 - 9
{Disruption of processing of alkaline phosphatase as a result of single amino acid changes affects the composition and metabolism of phospholipids from Escherichia coli, secreting mutant proteins}; Kalinin AE et al.; Amino acid substitutions in the cleavage site of the E . coli alkaline phosphatase signal peptide Val for Ala(-1) or Pro for Arg(+1) result in the block of the enzyme processing . In cells secreting such mutant proteins the relative content and rate of turnover of anionic phospholipids (phosphatidylglycerol and cardiolipin) are increased . The rise of the transfer of the phosphoglycerol residue from phosphatidylglycerol to periplasmic membrane derived oligosaccharides or to the model substrate, arbutin performed by the activity of phosphoglycerol transferase I testifies to phosphatidylglycerol accumulation on the outer surface of the cytoplasmic membrane . The results suggest of phosphatidylglycerol interaction with the alkaline phosphatase precursor and their subsequent joint translocation through the cytoplasmic membrane of E . coli.

Vet Immunol Immunopathol, 1996 Jan, 49(4), 359 - 73
Macrophage function in deer; Cross ML et al.; Macrophage inflammatory and immune functions were characterised in red deer (cervus elaphus), for use as a model for natural infection with bovine tuberculosis . Highly enriched populations of deer macrophages were obtained from 14 day cultures of plastic-adherent peripheral blood mononuclear cells . Cervine macrophages produced superoxide anion in response to respiratory burst stimuli (serum-opsonised zymosan and phorbol myristic acetate), but nitric oxide production could not be detected under the conditions tested . The lysosomal enzymes acid phosphatase and lysozyme were detected at the intercellular and extracellular level . Stimulation with bacterial lipopolysaccharide extract (Escherichia coli LPS) enhanced the production of superoxide and acid phosphatase with a peak increase in activity observed after 2h . Production of interleukin 1 (IL-1) and tumour necrosis factor (TNF), determined using cytokine-sensitive cell lines and mRNA analysis (Northern blotting), indicated maximal secretion of both cytokines after 24 h stimulation with LPS, preceded by a peak in message accumulation at 2-6 h post-stimulation . Cervine macrophages stimulated proliferative responses in T cell-enriched lymphocyte populations derived from the peripheral blood of autologous animals that had been primed to mycobacterial antigens (Mycobacterium bovis Bacille Calmette-Guerin, BCG) . Macrophages were able to stimulate responses after pulsing with particulate (BCG) or soluble (purified protein derivative) mycobacterial antigens . These results indicate that macrophage inflammatory and immune responses in red deer are similar to those in other mammalian species, and that macrophages may play an important role in resistance to mycobacterial infection.

Microbios, 1996, 85(344), 161 - 77
Sensitization to acid induced by sodium ions in Escherichia coli: dependence of (p)ppGpp and cAMP and suppression of the relA-associated defect by mutations in envZ; Rowbury RJ et al.; NaCl-induced acid sensitivity is not a response to high osmotic pressure but is triggered by a high internal sodium ion concentration, evidenced by the finding that conditions which enhance Na+ influx or reduce Na+ efflux, led to increased internal Na+ and allowed induction at lower external sodium ion concentrations and that induction could occur with no osmotic upshock . NaCl-induced acid sensitivity was not observed in Escherichia coli strain MC4100 and its relA lesion was investigated as a possible cause . Transformation of this strain and two other relA mutants to relA+ allowed sensitization by NaCl and whereas strain CF1648 (relA+) was sensitized, its relA deletion derivative was not . Additionally, transduction of two relA+ strains to relA produced derivatives which were not sensitized by salt . Although MC4100 was not sensitized by NaCl, envZ derivatives of it were sensitized . Lesions in fur and tonB did not prevent sensitization by NaCl, although extent of sensitization was slightly increased by fur and considerably increased by tonB . Acetate strongly inhibited sensitization, which was also subject to glucose repression, reversible by cAMP . The possibility was discussed that (p)ppGpp and cAMP both positively affect transcription of the genes encoding the acid sensitization components; in accord with this, high concentrations of cAMP suppressed the effect of the relA lesion of sensitization . Salt-induced organisms are more sensitive to acid damage to DNA, to acid inhibition of enzyme synthesis and DNA transfer and slightly less able to repair acid-damaged transforming DNA.

J Dairy Sci, 1996 Jan, 79(1), 71 - 5
alpha-Tocopherol concentrations in milk and plasma during clinical Escherichia coli mastitis; Hogan JS et al.; Eighteen cows were challenged by intramammary infusion with Escherichia coli 727 to determine the effects of acute clinical mastitis on alpha-tocopherol concentrations in plasma and milk . Cows were fed diets supplemented with 1000 IU of vitamin E/d from calving through the experimental period . At challenge, geometric mean DIM was 33 d . Each mammary quarter was diagnosed with an IMI and clinical mastitis at 24 and 48 h after challenge . The alpha-tocopherol concentrations in milk from challenged quarters were approximately 60% greater by 24 and 48 h after challenge than concentrations at prechallenge and 168 h postchallenge . Plasma alpha-tocopherol concentrations did not change after intramammary challenge . The alpha-tocopherol in plasma and milk was correlated at 48 and 168 h postchallenge but not at prechallenge or 24 h postchallenge . Milk alpha-tocopherol and SCC were correlated positively across all sample periods . Milk fat and milk alpha-tocopherol concentrations were correlated at each sample period except 24 h postchallenge . Increases in milk alpha-tocopherol during clinical mastitis were not correlated to milk production, DMI, or BSA concentration in milk . Changes in milk alpha-tocopherol concentration during clinical mastitis were similar to the dynamics of milk SCC.

Mutagenesis, 1996 Jan, 11(1), 69 - 73
Aflatoxin B1 induced lacI mutation in liver and kidney of transgenic mice C57BL/6N: effect of phorone; Autrup H et al.; Transgenic C57BL/6N mice containing a lambda shuttle vector carrying a lacI target and an alpha-lacZ reporter gene have been used to study the modulating effect of phorone, a glutathione-depleting agent, on the mutagenic activity of aflatoxin B1 (AFB1) in vivo . Animals were treated with AFB1 (8 mg/kg) for four consecutive days and the animals sacrificed 21 days after the last treatment . Treatment with AFB1 alone did not result in a significant increase in mutation frequency in the liver and kidney . When the animals were treated with phorone 4 h prior to treatment with AFB1 a significant increase in mutation frequency was observed in the liver (4-fold) and kidney (1.5-fold) . Phorone treatment did not increase the AFB1-induced mutation frequency in the lung and intestine . DNA sequence analyses of 30 independent clones isolated from the liver of AFB1-treated animals showed that G:C --> T:A transversion (60%) was the predominant mutational event . Mutations within the lacI gene could not be detected in seven of 30 mutants . The mutations were randomly distributed throughout the coding sequences of the lacI gene and no hotspots for the mutations were observed . However, codons 86 and 928 appeared to be major sites for mutation . The study shows that the transgenic mouse in vivo mutagenesis model can be used to study the influence of effect-modifying compounds on the mutagenic activity of known carcinogens.

Mutagenesis, 1996 Jan, 11(1), 43 - 8
Mutagenicity of high fat diets in the colon and small intestine of transgenic mice; Zhang XB et al.; Dietary fat has been implicated as a cause of colon cancer by epidemiological studies . Unfortunately, these studies are compatible with high fat as either initiator or promoter . Since initiators are normally mutagenic, we have tested the mutagenicity of high fat diets in the intestinal epithelium at two loci . The Dlb-1 assay, used in the small intestine, detects a wide spectrum of mutations . The lacI assay (Big Blue Mouse assay) is not as sensitive to some types of mutation as is Dlb-1 but was used in the colonic epithelium . Mice suitable for both assays were fed isocaloric high fat diets and subsequently assayed for somatic mutation . The diets consisted of: (i) a mixture of beef tallow, butter and lard totalling to 31% w/w of the diet (AIN-76A) up to 17 weeks; and (ii) corn oil, beef tallow, lard or butter individually, at 31% w/w of the diet for 5 and 9 weeks . These diets provided 50% of the calories from fat . The weights of the experimental and control mice were similar throughout the experiment . No significant increases in mutant frequencies were observed on any high fat diet compared to controls, so we conclude that uncooked fats are not mutagenic and are not initiators of carcinogenesis in the intestinal epithelium.

Eur Biophys J, 1996, 24(4), 195 - 201
Comparative analysis of the amino- and carboxy-terminal domains of calmodulin by Fourier transform infrared spectroscopy; Fabian H et al.; Fourier transform infrared spectra were obtained for mammalian calmodulin and two of its fragments produced by limited proteolysis with trypsin TR1C (1-77) and TR2C (78-148) . Experiments were done in H2O, D2O and D2O/trifluoroethanol (TFE) mixtures . Information about secondary structure was obtained from analysis of the amide I and II bands; while characteristic absorbances for tyrosine, phenylalanine and carboxylate groups were analyzed for changes in tertiary structure . Our data indicate that the secondary and tertiary structure is preserved in the two half molecules of CaM, both in the apo- and Ca(2+)-saturated state . Addition of the structure-inducing solvent TFE causes marked changes only in the apo-TR1C domain . The maximum wavenumber for the amide I band of the two domains of CaM in D2O was markedly different (1642 cm-1 for TR1C versus 1646/1648 cm-1 for Ca2+ and apo-TR2C) . This renders the amide I band for the intact protein very broad in comparison to that in other proteins and is indicative of a distribution of alpha-helices with slightly different hydrogen bonding patterns.

Cell Transplant, 1996 Jan-Feb, 5(1), 77 - 91
Arterial delivery of genetically labelled skeletal myoblasts to the murine heart: long-term survival and phenotypic modification of implanted myoblasts; Robinson SW et al.; The ability to replace damaged myocardial tissue with new striated muscle would constitute a major advance in the treatment of diseases that irreversibly injure cardiac muscle cells . The creation of focal grafts of skeletal muscle has been reported following the intramural injection of skeletal myoblasts into both normal and injured myocardium . The goals of this study were to determine whether skeletal myoblast-derived cells can be engrafted into the murine heart following arterial delivery . The murine heart was seeded with genetically labeled C2C12 myoblasts introduced into the arterial circulation of the heart via a transventricular injection . A transventricular injection provided access to the coronary and systemic circulations . Implanted cells were characterized using histochemical staining for beta-galactosidase, immunofluorescent staining for muscle-specific antigens, and electron microscopy . Initially the injected cells were observed entrapped in myocardial capillaries . One week after injection myoblasts were present in the myocardial interstitium and were largely absent from the myocardial capillary bed . Implanted cells underwent myogenic development, characterized by the expression of a fast-twitch skeletal muscle sarcoendoplasmic reticulum calcium ATPase (SERCA1) and formation of myofilaments . Four months following injection myoblast-derived cells began to express a slow-twitch/cardiac protein, phospholamban, that is normally not expressed by C2C12 cells in vitro . Most surprisingly, regions of close apposition between LacZ labeled cells and native cardiomyocytes contained structures that resembled desmosomes, fascia adherens junctions, and gap junctions . The cardiac gap junction protein, connexin43, was localized to some of the interfaces between implanted cells and cardiomyocytes . Collectively, these findings suggest that arterially delivered myoblasts can be engrafted into the heart, and that prolonged residence in the myocardium may alter the phenotype of these skeletal muscle-derived cells . Further studies are necessary to determine whether arterial delivery of skeletal myoblasts can be developed as treatment for myocardial dysfunction.

Urol Int, 1996, 56(2), 90 - 5
Increase in lectin binding sites on epithelial cells by chronic bladder infection in rats; Nakagawa T et al.; Using lectin histochemistry we assessed whether chronic bladder infection modifies carbohydrate residues of glycoconjugates on uroepithelial cells in rats . The bladder infection was produced by implanting a knotted silk thread with Escherichia coli into the bladder . One or 4 weeks after the implantation the bladder was excised, incubated with sixteen biotinylated lectins and stained . The bladder epithelia as a whole stained more strongly positive for eight lectins in the infected rats than in the control rats having a sterile silk thread in the bladder . In the infected rats, the superficial epithelial layer that stained negative for Arachis hypogaea (PNA) in the controls became strongly positive for PNA, whereas the middle and deep epithelial layers increased in staining for Canavalia ensiformis and six other lectins . These results indicate that chronic bladder infection increases carbohydrate residues on uroepithelial cells and may facilitate bacterial adherence to uroepithelial cells.

Neurol Neurochir Pol, 1996 Jan-Feb, 30(1), 39 - 48
{Blood-cerebrospinal fluid barrier in purulent cerebrospinal meningitis}; Przyjalkowski W et al.; Our investigations concerned the blood-brain barrier (b.b.b.) in patients with acute bacterial purulent meningitis . For that purpose concentrations of proteins, which are synthesized beyond the central nervous system and in normal cerebrospinal fluid (CSF) exist only in slight amounts, were determined in CSF and in blood serum . Albumin was examined in the CSF of 59 patients and in the serum of 35 of them, transferrin of 40 and 32 patients, respectively . Etiological verification was obtained in 84.7% of patients . The control group consisted of 20 persons . Quantitative analytical tests were carried out by means of immunochemical, turbidimetric methods . High levels of albumin and transferrin in CSF and low in serum of patients with meningitis were observed . The obtained results, confirmed by statistical analysis, demonstrate that in the acute phase of purulent meningitis b.b.b is impaired, what leads to the transfer of the proteins from the blood serum into the cerebrospinal fluid and that transferrins a better indicator of the damage to blood-brain barrier than albumin.

SAAS Bull Biochem Biotechnol, 1996, 9, 77 - 82
Circularizing ribozymes and decoy-competitors by autocatalytic splicing in vitro and in vivo; Puttaraju M et al.; An Anabaena group I intron was circularly permuted at loop 5, loop 6 and loop 8, and tested for self-splicing activity . Precursor RNAs from these constructs spliced efficiently and produced circular exons in vitro . Using group I permuted-intron-exon sequences, circular forms of the HDV ribozyme, the RNA component of RNaseP from B . subtilis, the HIV TAR and a short HIV Rev-binding element were generated and tested for activity and stability . The activity of circular ribozymes is comparable to the linear counterparts with similar core sequences . Circular forms of the TAR and Rev-binding element showed specific binding to Tfr-38 and Rev(35-50) peptide, respectively . To explore the potential for using this methodology to express circular RNA in vivo, circular forms of the HDV ribozyme and RNaseP RNA were produced in E . coli . Analysis of total RNA indicated that the precursor RNA spliced efficiently and accurately to produce circular ribozymes . The activity of in vivo expressed circular ribozymes could be demonstrated indicating that they fold into active conformation . These results suggest that self-splicing group I PIE sequences could prove useful for expressing small stable circular ribozyme/decoy-competitor or antisense RNAs in cells.

Bioorg Khim, 1996 Jan, 22(1), 14 - 9
{Recombinant proteins containing oxytocin oligomeric sequences}; Gurevich AI et al.; Expression plasmids were constructed with genes encoding the ILOX3, ILOX6, and ILOX9 recombinant proteins, which contain the C-terminal fragments of trimer, hexamer, or nonamer of oxytocinoyl-Lys . Upon expression in E . coli, all three genes yielded inclusion bodies containing protein products of similar length and heterogeneous in the C-terminal region . It is likely that in the case of the ilox3 gene, the obtained protein mixture includes the full-length product of translation with the C-terminal lysine . In the case of the ilox6 and ilox9 genes, the protein products are formed as the result of a site-specific proteolysis in the regions between the second and the fourth oxytocin units.

Genetika, 1996 Jan, 32(1), 42 - 52
{Unequal crossing over in Escherichia coli: genetic and physical mapping of duplications isolated in conjugational matings}; Sukhodolets VV et al.; We previously demonstrated that stable heterozygous duplications deoA deoB :: Tn5/deoC deoD can be isolated among Deo+ recombinants obtained in conjugational matings between Escherichia coli strains HfrH deoA deoB :: Tn5 and HfrH deoC deoD . In this work, 30 duplications were tested by transduction for the inclusion of a set of genetic markers adjoining the deo operon (99.5 min) at the genetic map of E . coli: cycA :: Tn10 (96 min), zji :: Tn10 (98.2 min), thr :: Tn9 (100/0 min), car :: Tn10 (1 min), leu :: Tn9 (2 min), and proAB :: Tn10 (6 min) . The results obtained indicate that only three out of 30 duplications could have originated from unequal crossing over between the rrn operons . In eight strains, duplications were chosen for physical mapping by the use of Not I restriction, pulsed-field electrophoresis, and Southern blot hybridization with DNA of the deo operon . In all these strains, the presence of duplications (once a triplication) was confirmed by corresponding changes in the set of Not I digests, although some strains had additional genetic rearrangements . The order of operon copies in a tandem was determined, and the length of a duplicated chromosomal segment was calculated as equal to 25, 46, 80, 150.5, and 175 kb in duplication D49, D4, D5, D9, and D18, respectively . Thus, the use of the conjugational Hfr x Hfr system allows the generation of unique rearrangements of the E . coli genetic material.

Genetika, 1996 Jan, 32(1), 23 - 34
{Physical mapping of Escherichia coli genome rearrangements using pulsed-field gel electrophoresis and the PCR method}; Zaporozhets DA et al.; Physical mapping of Tn10-induced chromosomal rearrangements in Escherichia coli was carried out by means of pulse-field electrophoresis, Southern hybridization, and PCR analysis . Mutants with chromosomal rearrangements were obtained by a method of selection of strains with constitutive udp-gene expression elaborated earlier . Screenings were performed in two E . coli strains that had a udp gene orientation diametrical to oriC and carried in metE :: Tn10- insertion and mutation in the dam gene encoding DNA-methylase . Selection of mutants with constitutive uridine phosphorylase synthesis was aimed at placing the udp-gene under the control of a novel, strong promoter via genome rearrangements . Physical mapping indicated that chromosomal rearrangements, in all cases studied, involved regions of operons encoding ribosomal RNA synthesis and placed the udp gene under the control of an rrn promoter . The majority of rearrangements are inversions of the chromosomal segment between the IS10-element, located in the metE gene, and various sites in different rrn operons . It is shown that the involvement of a particular operon in inversion depends on the direction of rrn operon and udp gene transcription . The rearrangement frequency increases 8-10 times in the presence of dam mutation.

Arch Virol, 1996, 141(3-4), 751 - 61
Kinetics of humoral immune response to the major structural proteins of the porcine reproductive and respiratory syndrome virus; Loemba HD et al.; The kinetics of appearance of antibodies directed to the major structural proteins N, M and E of porcine reproductive and respiratory syndrome virus (PRRSV) was followed in pigs naturally- and experimentally-exposed to the virus . Specific IgM antibody titers were first detected by indirect immunofluorescence (IIF) at the end of the first week of PRRSV infection, peaked by day 14 to 21 post-inoculation (p.i.), then rapidly decreased to undetectable levels by day 35 to 42 p.i . On the other hand, specific IgG antibody titers peaked by day 21 to 28 p.i . and remained unchanged to the end of the 6- or 9-week observation period; in addition, a persistent viremia was observed . Virus neutralizing (VN) antibody titers > 8 were not detected until 3 to 4 weeks p.i . Taken together, the results obtained by Western blotting analyses using purified virus and E . coli-expressed ORFs 5 to 7 gene products, suggested that antibodies directed against the envelope E protein appear by day 7 p.i., whereas antibodies directed against the nucleocapsid N and membrane M proteins can only be detected by the end of the second week p.i . No correlation could be demonstrated between VN and IIF antibody titers, viremia, and viral protein specificities of circulating antibodies at various times p.i.

Arch Virol, 1996, 141(3-4), 685 - 99
RNA binding properties of core protein of the flavivirus Kunjin; Khromykh AA et al.; Kunjin virus (KUN) C is a typical flavivirus core protein which is truncated in vivo to a mature form of 105 residues enriched in lysine and arginine . In order to study the possible association of KUN C with RNA in vitro, we prepared several recombinant C proteins with specific deletions, each fused at the amino-terminus to glutathione-S-transferase (GST) and expressed in E . coli . They were reacted with KUN RNA probes transcribed in vitro from cDNA representing the 5' untranslated region (5' UTR, 93 to 96 nucleotides), the 3' UTR (624 nucleotides), and the 5' UTR plus most of the C coding region (5' core, 440 nucleotides) . Fusion protein C107 (incorporating mature C) bound strongly to all KUN RNA probes with apparent specificity, being completely resistant to inhibition by 800 mM NaCl, and to competition by a large excess of tRNA . In reactions with labelled KUN RNA probes putative binding sites were identified in the isolated amino-terminal (32 residues) and carboxy-terminal (26 residues) basic amino acid domains; this binding was strongly competed by unlabelled KUN UTR probes but weakly or not at all by tRNA . These small domains probably acted co-operatively in binding of mature C to KUN RNA probes . The KUN RNA-core protein binding reactions are similar to those reported with other viral coat or capsid proteins and viral RNAs.

Arch Virol, 1996, 141(3-4), 671 - 83
Rabies virus M protein expressed in Escherichia coli and its regulatory role in virion-associated transcriptase activity; Ito Y et al.; Rabies virus M protein was expressed in Escherichia coli in the form of a fusion protein with maltose binding protein (MBP) and purified by amylose affinity column chromatography after extraction . In order to investigate the possible regulatory role of M protein in viral transcription, an assay system for rabies virion-associated transcriptase activity was established by using the ribonucleoprotein (RNP) cores prepared from purified virions . Analysis of the products of the transcription assay system showed that the products are sensitive to RNase and are positive-strand RNA . Addition of the fusion protein to the system after cleavage with a proteinase Factor Xa (FXa), which cleaves the fusion protein into the M protein and MBP, resulted in an efficient and dose-dependent inhibition of the transcription . Furthermore, addition to the system of anti-M protein monoclonal antibody significantly restored the transcription . Control experiments with the same transcription assaying system using rabies virus nucleoprotein expressed as a fusion protein with MBP and cleaved with FXa did not result in an inhibition of the transcription . These results suggest that the M protein of rabies virus has the property to down-regulate virion-associated transcription.

Somat Cell Mol Genet, 1996 Jan, 22(1), 21 - 9
Targeted retroviral gene transfer into the rat biliary tract; Cabrera JA et al.; The ability to induce proliferation by temporary duct ligation suggested an hypothesis that retrovirus-mediated gene transfer into cells of the biliary tract could be accomplished . The time course of histologic changes, incorporation of 3H-thymidine and immunofluorescent staining with a monoclonal antibody to cytokeratin-19 (a marker for differentiated bile ducts) was studied in male Fischer F344 rats . A recombinant Gibbon ape leukemia virus (GALV), containing a gene encoding Escherichia coli beta-galactosidase was next introduced into 24 hr obstructed bile ducts . Gene transfer was maximal when virus was exposed to the obstructed duct for 12 hr (approximately 0.1%) . The majority of X-gal positive cells were in cytokeratin-19 negative peribiliary tissues, which had the appearance of newly forming bile ducts . The data suggest that cells targeted by retroviral infection of the obstructed rat bile duct may be a precursor of mature, fully differentiated biliary epithelium.

Arch Microbiol, 1996 Jan, 165(1), 69 - 72
Nickel incorporation into hydrogenase 3 from Escherichia coli requires the precursor form of the large subunit; Binder U et al.; A mutant derivative of hycE, the gene for the large subunit of hydrogenase 3 from Escherichia coli, was constructed that lacks the 3'-terminal part encoding the C-terminal portion of the HycE polypeptide, which is proteolytically removed during maturation of the hydrogenase . The truncated gene was transferred to the in situ position on the chromosome . Although the mutant possessed HycE in its "mature" form, it was devoid of hydrogenase 3 activity . The activity was not restored by high nickel concentrations in the medium . The mutated HycE was not associated with detectable radioactivity when the strain was grown in the presence of 63Ni2+ . These results indicate that the C-terminal extension in the precursor form of the large subunit keeps the protein in a conformation required for the coordination of the metal.

Environ Mol Mutagen, 1996, 27(1), 30 - 3
Mutagenesis of AS52 cells by low concentrations of lead(II) and mercury(II)
Ariza ME, Williams MV.
Little is known at the molecular level concerning the genotoxic effects following the acute exposure of eukaryotic cells to low concentrations of lead (II) or mercury (II) . There have been conflicting reports concerning the mutagenic potential of these heavy metals, and there have not been any studies performed to determine the molecular mechanism(s) by which these metals are mutagenic . The Chinese hamster ovary cell line, AS52, contains a stably integrated single functional copy of the Escherichia coli xanthine-guanine phosphoribosyltransferase (gpt) gene . Mutations in the gpt gene confer resistance to 6-thioguanine (TG) . There was little effect on viability, as measured by relative cloning efficiency, of AS52 cells exposed to lead (II) or mercury (II) up to concentrations of 0.5 microM and 0.3 microM, respectively . However, higher concentrations of the metals caused a significant increase in cell death . There was also a dose-dependent increase in the isolation of mutants resistant to TG in treated cells when compared to non-treated controls . Concentrations of the metals as low as 0.1 microM caused a significant increase in the number of mutants resistant to TG when compared to the number of spontaneous mutants obtained in nontreated controls . While the molecular mechanism(s) by which lead and mercury (II) are genotoxic is unknown, the results of this study demonstrate that low concentrations of lead (II) and mercury (II) are mutagenic in eukaryotic cells.

Domest Anim Endocrinol, 1996 Jan, 13(1), 91 - 103
Physiological responses to repeated endotoxin challenge are selectively affected by recombinant bovine somatotropin administration to calves; Elsasser TH et al.; The study determined 1) whether the pretreatment of calves with recombinant bovine somatotropin (bST, sometribove) would alter the change in packed cell volume (PCV), rectal temperature (RT), and the plasma concentrations of Ca2+, Fe2+, glucose (G), urea N (PUN), nonesterified free fatty acids (NEFA), albumin (ALB), and blood cell populations after endotoxin challenge (EC) and 2) whether the natural development of physiologic tolerance to repeated EC was affected by bST . Twelve steer beef calves were assigned to either control (-bST) or +bST treatment in equal numbers . Calves were injected intramuscularly with either HCO3(-)-buffered saline or bST (0.1 mg/kg) daily for 5 d . On Day 6, the first EC was administered (Escherichia coli, 055:B5, 0.2 microgram/kg, intravenous bolus in pyrogen-free saline) . Saline or bST injections were continued from Day 7 up to the repeat of EC on Day 11 . RT and PCV were measured hourly through 12 and 6 hr, respectively . Jugular blood was obtained at 0, +1, 2, 3, 4, 6, 8, 12, and 24 hr relative to each EC . bST had no effect on the increase in RT, the hyperglycemic phase of the G response, the biphasic change in Fe2+, or increases in NEFA and PUN . PCV increased after each EC only in -bST . The mean decrease in G during the hypoglycemic phase was less in +bST . Hypocalcemic responses were significantly less in +bST . ALB concentrations decreased after each challenge; the response was unaffected by bST . CD2+, CD4+, and CD8+ T-lymphocyte populations were unaffected by bST and EC . Overall, the magnitude of change in all plasma variables was less after the second EC compared with the first, either in terms of magnitude or duration . The data suggest that the treatment of calves with bST diminishes the magnitude of hypoglycemic, hypocalcemic, and PCV changes after EC and does not compromise fever response, changes in blood cell populations, or tolerance to repeated EC.

Vet Res, 1996, 27(1), 33 - 44
Reconstitution of mastitic milk by adding blood plasma and leukocytes into low cell count milk; Fang W et al.; Milk from inflamed quarters is high in somatic cells, proteolytic activity and lysosomal enzyme activity . Addition of homologous blood plasma and leukocytes into low cell count milk did not increase the plasmin activity, total proteolytic activity or N-acetyl-beta-D-glucosaminidase (NAGase) as anticipated, even if the reconstituted milk samples were stimulated by endotoxin or opsonized zymosan . Addition of urokinase activated the latent proteolytic system in plasma-supplemented milk . The reactive sulfhydryls in milk samples with different treatments decreased significantly after incubation, indicating the presence of an oxidative mechanism in milk (eg, sulfhydryl oxidase) . The high background resazurin reduction of fresh milk might be due, in part, to the cellular activity of somatic cells . This study indicates that a more complex system including inflammatory mediators and complex cellular interactions is required for expression of plasmin-activator activity and for full activation of the proteolytic and lysosomal enzymes in mastitic milk.

Plant Mol Biol, 1996 Jan, 30(2), 373 - 9
Characterization of a cDNA encoding cysteine proteinase inhibitor from Chinese cabbage (Brassica campestris L . ssp . pekinensis) flower buds; Lim CO et al.; A cDNA encoding a new phytocystatin isotype named BCPI-1 was isolated from a cDNA library of Chinese cabbage flower buds . The BCPI-1 clone encodes 199 amino acids resulting in a protein much larger than other known phytocystatins . BCPI-1 has an unusually long C-terminus . A BCPI-1 fusion protein expressed in Escherichia coli strongly inhibits the enzymatic activity of papain, a cysteine proteinase . Genomic Southern blot analysis revealed that the BCPI gene is a member of a small multi-gene family in Chinese cabbage . Northern blot analysis showed that it is differentially expressed in the flower bud, leaf and root.

Plant Mol Biol, 1996 Jan, 30(2), 269 - 79
Cloning and characterization of a maize cDNA encoding phytoene desaturase, an enzyme of the carotenoid biosynthetic pathway; Li ZH et al.; To study regulation of the plastid-localized maize carotenoid biosynthetic pathway, a cDNA encoding phytoene desaturase (PDS) was isolated and characterized . The DNA sequence of the maize Pds cDNA was determined and compared with available dicot Pds genes . The deduced PDS protein, estimated at 64.1 kDa (unprocessed), had a dinucleotide binding domain and conserved regions characteristic of other carotene desaturases . Alignment of available PDS sequences from distantly related organisms suggests that Pds has potential as a phylogenetic tool . By use of heterologous complementation in Escherichia coli, maize PDS was shown to catalyze two desaturation steps converting phytoene to zeta-carotene . RFLP (restriction fragment length polymorphism) mapping was used to place Pds on chromosome 1S near viviparous5 (vp5), and RT-PCR (reverse-transcriptase polymerase chain reaction) analysis indicated reduced Pds transcript in vp5 mutant relative to normal endosperm . Other phytoene-accumulating mutant endosperms, vp2 and white3 (w3), showed no difference in Pds transcript accumulation as compared with normal endosperm counterparts . RT-PCR analysis of Pds transcript accumulation in developing endosperm showed Pds was constitutively expressed . Therefore, endosperm carotenogensis is not regulated by increasing the level of Pds transcripts.

Plant Mol Biol, 1996 Jan, 30(1), 51 - 64
Isolation, characterization and expression of cDNA clones encoding a mitochondrial malate translocator from Panicum miliaceum L; Taniguchi M et al.; Three cDNA clones that hybridize to a partial rice cDNA that show similarity to bovine mitochondrial 2-oxoglutarate/malate translocator were isolated from leaves of Panicum miliaceum L . (proso millet), an NAD-malic enzyme-type C4 plant . The nucleotide sequences of the clones resemble each other, and some of the isolated cDNAs contained extra sequences that seemed to be introns . The predicted proteins encoded by the cDNAs have 302 amino acids and molecular weights of 32211 and 32150 . The hydrophobic profile of the amino acid sequence predicted the existence of six transmembrane alpha-helices that is a common property of members in the mitochondrial transporter family . The predicted amino acid sequence showed the highest similarity with that of the 2-oxoglutarate/malate translocator from mammalian mitochondria . An expression plasmid containing the coding region of the cDNAs was used to over-express recombinant protein with a C-terminal histidine tag Escherichia coli, which was affinity purified . The antibody against the recombinant protein cross-reacted with proteins of 31-32 kDa in the membrane fraction from P . miliaceum mitochondria, but not with the chloroplast fraction . The recombinant protein reconstituted in liposomes efficiently transported malate, citrate, and 2-oxoglutarate.

Plant Mol Biol, 1996 Jan, 30(1), 39 - 50
Carboxyl-terminal processing protease for the D1 precursor protein: cloning and sequencing of the spinach cDNA; Inagaki N et al.; A previous study has demonstrated that the carboxyl-terminal (C-terminal) processing protease in spinach for the D1 precursor protein (pDl) of the photosystem II reaction center is a monomeric protein of about 45 kDa . Based on the amino acid sequence data of the purified protease, a cDNA clone encoding the enzyme has been identified and sequenced, from a spinach green leaf cDNA library . In order to determine the 5' end of the transcript, the rapid amplification of cDNA end (5'-RACE) technique was applied . By these analyses, the full-length transcript was established to consist of 1906 nucleotides and a poly(A) tail, containing an open reading frame (ORF) corresponding to a protein with 539 amino acid residues . By comparing the amino acid sequence of the purified protease with that deduced from nucleotide sequence of the cDNA clones, the enzyme was shown to be furnished with an extra amino-terminal extension characteristic of both a transit peptide and a signal sequence . This suggests that the protease is synthesized in the cytosol and translocated into the lumenal space of thylakoids . The mature part of the enzyme consists of 389 amino acid residues and exhibits a significant sequence homology with two groups of proteins as demonstrated by a computer homology search, i.e . (1) the deduced sequence of a protein proposed to be the C-terminal processing protease for pD1 in Synechocystis sp . PCC 6803, based on genetic experiments and (2) proteases for C-terminal cleavage identified in Escherichia coli and Bartonella bacilliformis.

J Antibiot (Tokyo), 1996 Jan, 49(1), 81 - 5
Improved production of phenomycin by a genetically engineered Escherichia coli; Nagano Y et al.; We established an improved production of an antitumor polypeptide anti biotic, phenomycin (PHM), by using a genetically engineered Escherichia coli . Phenomycin consists of 89 natural amino acids without intramolecular disulfide bridge . PHM gene was synthesized as a fusion gene in which PHM at the C-terminus and the residues 1 approximately 20 of Hirudin variant 1 (HV1) at the N-terminus connected by a factor Xa recognition sequence (Ile-Glu-Gly-Arg) . E coli JM 109 transformed with a plasmid containing the synthesized gene expressed a fusion protein and the trypsinization of the fusion protein purified by ultrafiltration and ion-exchange chromatography gave efficiently recombinant PHM at a final yield of 50 mg/liter of culture . This PHM yield was six times higher than that obtained by a natural PHM producing strain of Streptoverticillium baldacci . Recombinant PHM was not distinguishable from natural PHM in all aspects observed.

Food Chem Toxicol, 1996 Jan, 34(1), 63 - 72
Interaction of lipopolysaccharide endotoxin produced from Escherichia coli with D-tubocurarine at the nicotinic2 receptor and adenosine 3':5' cyclic monophosphate during physiological contraction in skeletal muscle; Tomera JF; In this report the murine model of endotoxicosis was used to evaluate hyposensitivity to the neuromuscular relaxant D-tubocurarine (dTC) . This hyposensitivity was expressed in terms of a decreased potency to dTC . A rightward shift of the dose-response curve due to endotoxin was observed . Mice were subjected to cumulative intraperitoneal doses of Escherichia coli endotoxin over a 2-wk period . The interaction between endotoxin and dTC was examined during an acute (1 wk) and chronic (2 wk) period of endotoxicosis . Muscle twitch analyses were performed and samples of gastrocnemius muscle were assayed for adenosine 3':5' cyclic monophosphate (cAMP) by {125I}radioimmunoassay . A parallel shift in the dose-response curve occurred in the endotoxin group subjected to doses corresponding to one-third the dose evoking 50% lethality for 2 wk . Both skeletal muscle tension and cAMP levels decreased as cumulative endotoxin doses increased . A relationship between decreasing cAMP levels and increasing dTC and effective dose required to achieve 50% muscle paralysis values was thought to be evoked by the agonistic activity of E . coli endotoxin leading to desensitizing of adenylate cyclase . The perturbations of the classical second messenger cAMP system by endotoxin may be responsible for skeletal muscle dysfunction observed in immunocompromised patients.

Int J Radiat Biol, 1996 Jan, 69(1), 99 - 105
Thiols, recA induction and radiosensitivity in Escherichia coli; Naslund M et al.; Induction by gamma-radiation, UV radiation or hydroxyurea of RecA gene product synthesis in Escherichia coli, monitored as beta-D-galactosidase in recA-lacZ fusion strains, was shown to be inhibited if 2-mercaptoethylamine (MEA) was added before treatment with the inducing agents . If cysteine (Cys) at low concentrations was added at the same time as MEA it counteracted the action of MEA . The effect of MEA may be described as a competitive inhibition of an inducing or conducting effect of Cys . In E . coli GE499 (uvrA+), complete inhibition by 30-mmol dm-3 MEA of recA induction was associated with about five times higher radio-resistence . Both of these effects of MEA were completely reversed by 0.3-mmol dm-3 Cys . As shown in parallel experiments with E . coli GE500 (uvrA-), these effects of MEA and Cys were shown to be independent of excision-repair proficiency . Treatment of bacteria with MEA and/or Cys was shown not to lead to increased intracellular concentrations of these thiols . Instead, treatment with them appeared to provoke conspicuous increases in glutathione levels, which are, however, probably not directly involved in the studied action of MEA and Cys.

Biosens Bioelectron, 1996, 11(1-2), 81 - 90
Development of CCD-based direct observation equipment for biological samples; Miyamoto S et al.; Equipment for the direct observation of biological samples has been developed . The equipment comprises a charge coupled device (CCD) unit and a light-emitting diode (LED), which is placed above the light-sensing face of the CCD . The biological sample is positioned just onto the CCD with no lens system interposed . Because of its small size, the equipment can be operated in an incubator, allowing the continuous observation of biological samples under appropriate temperature, humidity and gas concentration conditions . The equipment is operated periodically at 5-minute periods to reduce the influence of heat generated by the equipment and to maintain a constant sample temperature . E . coli colony formation is observed continuously for 70 hours without any adverse effects occurring during the observation . Hepatocyte morphological change and hepatocyte pattern formation are also observed.

FEMS Microbiol Lett, 1996 Jan 1, 135(1), 57 - 63
Expression and sequence analysis of a Treponema pallidum gene, tpn38(b), encoding an exported protein with homology to T . pallidum and Borrelia burgdorferi proteins; Stamm LV et al.; An Escherichia coli clone containing recombinant plasmid C19 was identified from a Treponema pallidum genomic DNA library by in situ immunoassay . E . coli maxicells containing pC19 synthesized a treponemal protein doublet of 39.2 and 38.2 kDa, designated TpN38(b) . Pulse-chase and protein processing studies showed that TpN38(b) is synthesized with a cleavable amino-terminal signal peptide . A 2.0-kb fragment of pC19 containing the tpn38(b) gene was subcloned and sequenced . The tpn38(b) gene is 1029 nucleotides long and encodes a protein of 343 amino acids with a calculated molecular mass of 37.9 kDa . The deduced amino acid sequence of TpN38(b) has homology with the T . pallidum TpN35 lipoprotein and the Borrelia burgdorferi BmpA, BmpB, BmpC, and BmpD proteins.

FEMS Microbiol Lett, 1996 Jan 1, 135(1), 45 - 50
Sigma s regulates pLS1 maintenance in stationary-phase Escherichia coli; Espinosa-Urgel M et al.; We have studied the influence of sigma s on the stability and number of copies of the promiscuous plasmid pLS1 in Escherichia coli . Our results indicate that pLS1 is less stable and has a lower number of copies in a rpoS mutant than in a wild-type strain during stationary phase . This behaviour does not seem to be due to differences in the expression of pLS1 replication regulators, but to be related to plasmid topology.

FEMS Microbiol Lett, 1996 Jan 1, 135(1), 17 - 22
The hydrophobic surface protein layer of enteroaggregative Escherichia coli strains; Wai SN et al.; The surface of three strains of enteroaggregative Escherichia coli (EAggEC) and three strains of enteropathogenic E . coli (EPEC) were examined using the freeze-substitution technique of electron microscopy and as a result an electron dense surface layer was found only on EAggEC strains but not on EPEC strains . The analysis of the outer membrane proteins by polyacrylamide gel electrophoresis revealed the existence of a unique 38 kDa protein in EAggEC strains . The protein could be easily extracted from the bacterial surface with 5 M LiCl treatment at room temperature . The antiserum raised in mice with 38 kDa protein extracted from the electrophoresed gel was immunoreacted with the surface of the bacteria of EAggEC by immunoelectron microscopy . The hydrophobic surface character of the EAggEC strains was lost after the extraction of the protein layer by LiCl . We thus conclude that the surface protein layer therefore plays an important role in the expression of the aggregative phenotype in EAggEC strains.

FEMS Microbiol Lett, 1996 Jan 1, 135(1), 111 - 6
High pressure represses expression of the malB operon in Escherichia coli; Sato T et al.; The formation of plaques by lambda phage in Escherichia coli was prevented by elevated hydrostatic pressure; phage plaques were not detected at 30 MPa . Furthermore, using promoter fragments derived from the malB operon, we showed that gene expression initiated from both promoters (malK-lamB and malEFG) was repressed by elevated hydrostatic pressure . Our findings suggest that high pressure affects gene expression directed by the malB regulatory interval, and this may cause a decrease in the quantities of lambda receptor protein, LamB.

Nucleic Acids Res . 1996 Jan 1;24(1):40.
Genes and proteins of Escherichia coli (GenProtEc); Riley M et al.; GenProtEc is a database of Escherichia coli genes and their gene products, classified by type of function and physiological role and with citations to the literature for each . Also present are data on sequence similarities among E.coli proteins with PAM values, percent identity of amino acids, length of alignment and percent aligned . The database is available as a PKZip file by ftp from mbl.edu/pub/ecoli.exe . The program runs under MS-DOS on IMB-compatible machines . GenProtEc can also be accessed through the World Wide Web at URL http://mbl.edu/html/ecoli.html.

Nucleic Acids Res, 1996 Jan 1, 24(1), 32 - 9
EcoCyc: an encyclopedia of Escherichia coli genes and metabolism; Karp PD et al.; The encyclopedia of Escherichia coli genes and metabolism (EcoCyc) is a database that combines information about the genome and the intermediary metabolism of E.coli . It describes 2034 genes, 306 enzymes encoded by these genes, 580 metabolic reactions that occur in E.coli and the organization of these reactions into 100 metabolic pathways . The EcoCyc graphical user interface allows query and exploration of the EcoCyc database using visualization tools such as genomic map browsers and automatic layouts of metabolic pathways . EcoCyc spans the space from sequence to function to allow investigation of an unusually broad range of questions . EcoCyc can be thought of as both an electronic review article, because of its copious references to the primary literature, and as an in silico model of E.coli that can be probed and analyzed through computational means.

Nucleic Acids Res, 1996 Jan 1, 24(1), 29 - 31
Compilation of DNA sequences of Escherichia coli K12 (ECD and ECDC; update 1995); Kroger M et al.; We have compiled the DNA sequence data for Escherichia coli available from the GenBank and EMBL data libraries and independently from the literature . Unlike the previous updates of our E.coli databases, we provide the most recent version preferentially via the World Wide Web System (use URL: +/html/ecdc.html) . Our database includes an assembled set of contiguous sequences . Each of these contigs compiles all available sequence information, including those derived from a variety of elder sequences . The organization of the database allows one to find the exact physical location of each individual gene or regulatory region, even regarding discrepancies in nomenclature . The WWW program allows access into the original EMBL and SWISSPROT datafiles . A FASTA and BLAST search may be performed online . Besides the WWW format a flat file version may be obtained via ftp . The complete compilation, including a full set of genetic map data and the E.coli protein index, can be obtained in machine readable form from the EMBL data library as a part of the CD-ROM issue of the EMBL sequence database, released and updated every three months . After deletion of all detected overlaps a total of 3 333 878 individual bp was determined by the end of September 1995 . This corresponds to a total of 71.71% of the entire E.coli chromosome consisting of about 4720 kbp . About 94 kbp (2%) are available additionally, but have not yet been definitely mapped.

Nucleic Acids Res, 1996 Jan 1, 24(1), 180 - 1
The SWISS-2DPAGE database of two-dimensional polyacrylamide gel electrophoresis, its status in 1995; Appel RD et al.; SWISS-2DPAGE is a database of proteins identified on two-dimensional polyacrylamide gel electrophoresis (2-D PAGE) . The current release contains 343 entries of human, yeast (Saccharomyces cerevisiae) and Escherichia coli origin, as well as virtual entries for each of the protein sequences in the SWISS-PROT database.

Nucleic Acids Res, 1996 Jan 1, 24(1), 169 - 71
The 23S Ribosomal RNA Mutation Database (23SMDB); Triman KL; The 23S Ribosomal RNA Mutation Database (23SMDB), provides a list of mutated positions in 23S ribosomal RNA from Escherichia coli and the identity of each alteration . Information provided for each mutation includes: (i) a brief description of the phenotypes(s) associated with each mutation, (ii) whether a mutant phenotype has been detected by in vivo or in vitro methods, and (iii) relevant literature citations . The database is available via ftp and on the World Wide Web.

Nucleic Acids Res, 1996 Jan 1, 24(1), 166 - 8
The 16S ribosomal RNA mutation database (16SMDB); Triman KL; The 16S ribosomal RNA mutation database (16SMDB) provides a list of mutated positions in 16S ribosomal RNA from Escherichia coli and the identity of each alteration . Information provided for each mutation includes: (i) a brief description of the phenotype(s) associated with each mutation; (ii) whether a mutant phenotype has been detected by in vivo or in vitro methods; (iii) relevant literature citations . The database is available via ftp and on the World Wide Web.

Bioessays, 1996 Jan, 18(1), 63 - 70
Unusual evolution of 11beta- and 17beta-hydroxysteroid and retinol dehydrogenases; Baker ME; 11beta-hydroxysteroid dehydrogenases regulate glucocorticoid concentrations and 17beta-hydroxysteroid dehydrogenases regulate estrogen and androgen concentrations in mammals . Phylogenetic analysis of the sequences from two 11beta-hydroxysteroid dehydrogenases and four mammalian 17beta-hydroxysteroid dehydrogenases indicates unusual evolution in these enzymes . Type 1 11beta- and 17beta-hydroxysteroid dehydrogenases are on the same branch; Type 2 enzymes cluster on another branch with beta-hydroxybutyrate dehydrogenase,11-cis-retinol dehydrogenase and retinol dehydrogenase; Type 3 17beta-hydroxysteroid dehydrogenase is on a third branch; while the pig dehydrogenase clusters with a yeast multifunctional enzyme on a fourth branch . Pig 17beta-hydroxysteroid dehydrogenase appears to have evolved independently from the other three 17beta-hydroxysteroid dehydrogenases; in which case, the evolution of 17beta-hydroxysteroid dehydrogenase activity is an example of functional convergence . The phylogeny also suggests that independent evolution of specificity toward C11 substituents on glucocorticoids and C17 substituents on androgens and estrogens has occurred in Types 1 and 2 11beta- and 17beta-hydroxysteroid dehydrogenases.

Transgenic Res, 1996 Jan, 5(1), 37 - 48
Spatial and temporal regulation of a lacZ reporter transgene in a binary transgenic mouse system; Gardner DP et al.; The transgenic mouse system is a powerful tool for the study of gene function . However, when the analysis involves genes that are critical for the normal developmental process, the usefulness of transgenic mouse systems is limited (for review see Hanahan, 1989; Westphal and Gruss, 1989; Byrne et al., 1991) . This is due to potential transgene interference with development in case of ectopic or high level expression . As a result, establishing permanent transgenic mouse lines expressing these types of genes has proven difficult . To circumvent these difficulties, a binary transgenic mouse system has been established, termed the Multiplex System (Byrne and Ruddle, 1989) . This is a two-tiered gene activation system in which expression of the gene of interest occurs only in offspring carrying transgenes encoding both components: transactivator and transresponder . Transactivator lines contain the gene encoding the VP16 protein of herpes simplex virus . Transresponder lines harbour the gene of interest linked to the IE promoter which includes recognition sequences for the VP16 transactivator . Previously, the inducibility of a chloramphenicol acetyltransferase reporter gene in newborn offspring that carried both a transactivator and transresponder transgene (Byrne and Ruddle, 1989) has been shown . Moreover, it has been demonstrated that expression of the VP16 protein was not detrimental to development and that transactivation appeared to be tissue specific . Here, the potential of the system for the expression of transgenes in early mouse embryogenesis was examined, using the Escherichia coli beta-galactosidase gene as a reporter in the transresponder mouse strain . To direct expression of VP16, the murine Hoxc-8 promoter, which is known to be active during early development, was used . Embryos from crosses of transactivators to transresponders were isolated at different stages of development and stained for beta-galactosidase activity . Transactivation, as demonstrated by strong beta-galactosidase staining, could be detected as early as eight days of development . At all stages examined, the pattern of lacZ transresponder gene expression accurately reflected the activity of the Hoxc-8 promoter controlling VP16 expression . It is demonstrated that the Multiplex System can be used to express transresponder transgenes in a spatially and temporally defined manner in multiple cell types early during mouse embryogenesis.

Lett Appl Microbiol, 1996 Jan, 22(1), 57 - 61
Exposure of Escherichia coli to acid habituation conditions sensitizes it to alkaline stress; Rowbury RJ et al.; Escherichia coli transferred from pHo 7.0 to pHo 5.5 or 6.0 became alkali-sensitive by a rapidly induced phenotypic response . Alkali sensitization was reduced at pHo 5.0 and virtually abolished at pHo 6.5 . The response was triggered by cytoplasmic rather than external or periplasmic acidification and de novo protein synthesis was needed . Alkali sensitivity failed to appear at pHo 5.5 plus DNA gyrase inhibitors and was markedly reduced by himA, himD, hns, ompC and nhaA lesions . A tonB deletion mutant showed alkali sensitivity at pHo 7.0 . Alkali sensitivity induction was not subject to catabolite repression nor was it appreciably affected by a relA lesion . Acid-induced cells were more sensitive to alkali damage to both DNA and beta-galactosidase and to alkali inhibition of beta-galactosidase induction . Alkali sensitization induced at pHo 5.5 may involve NhaB loss.

Phytochemistry, 1996 Jan, 41(1), 65 - 9
Spinach chloroplast ATP-dependent endopeptidase: Ti-like protease; Benesova M et al.; The soluble fraction of spinach chloroplast was used for purification and characterization of an ATP-dependent protease . Purification included Q Sepharose Fast Flow, hydroxylapatite and FPLC Superose 6 column chromatography . The isolated enzyme requires ATP and Mg2+ for stimulation and represents a ubiquitin independent serine protease, containing essential sulphydryl group(s) . By using fluorogenic peptides a similarity of chloroplast protease to Escherichia coli Ti protease was observed . The chloroplast protease is immunochemically cross-reactive with the bacterial protease Ti.

Plant Physiol, 1996 Jan, 110(1), 43 - 9
Molecular analysis of a new member of the opium poppy tyrosine/3,4-dihydroxyphenylalanine decarboxylase gene family; Maldonado-Mendoza IE et al.; An aromatic amino acid decarboxylase DNA fragment was generated from opium poppy (Papaver somniferum L.) genomic DNA by the PCR using primers designed from conserved amino acid sequences of other aromatic amino acid decarboxylase genes . Using this fragment as a probe, a genomic clone was isolated that encodes a new member of the opium poppy tyrosine/3,4-dihydroxyphenylalanine decarboxylase gene family (TyDC5) . The predicted TyDC5 amino acid sequence shares extensive identity with other opium poppy tyrosine/3,4-dihydroxyphenylalanine decarboxylases (84%), and when expressed in Escherichia coli, it is active against tyrosine and to a lesser extent against 3,4-dihydroxyphenylalanine . Ribonuclease protection assays indicate that TyDC5 is expressed primarily in the roots of mature poppy plants . A peak of TyDC5 expression was also observed during germination, coincident with the emergence of the radicle from the seed coat . Parallel results were obtained in transgenic tobacco using a TyDC5 promoter fragment (-2060) translationally fused to the beta-glucuronidase reporter gene (GUS) . IN TyDC5::GUS tobacco, GUS activity transiently appeared in all parts of the seedling during germination, but was limited to the roots in older plants . These results indicate that TyDC5 expression is transcriptionally regulated and suggest that the TyDC5 enzyme may play an important role in providing precursors for alkaloid synthesis in the roots and germinating seedlings of opium poppy.

Plant Physiol, 1996 Jan, 110(1), 203 - 10
Two novel thioesterases are key determinants of the bimodal distribution of acyl chain length of Cuphea palustris seed oil; Dehesh K et al.; The seed oil of Cuphea palustris has an unusual fatty-acyl composition, whereby the principal fatty-acyl groups, myristate (64%) and caprylate (20%), differ by more than two methylenes . We have isolated two thioesterase (TE) cDNAs from C . palustris, encoding proteins designated Cp FatB1 and Cp FatB2, which, when expressed in Escherichia coli, have TE activities specific for 8:0/10:0- and 14:0/16:0-acyl carrier protein substrates, respectively . The specific activities of the recombinant affinity-purified enzymes indicate that Cp FatB2 is kinetically superior to Cp FatB1 . This result is consistent with the predominance of 14:0 in the seed oil, despite apparently equal mRNA abundance of the two transcripts in the seed . In C . palustris the expression of both sequences is confined to the seed tissues . Based on these findings we propose that these two enzymes are major factors determining the bimodal chain-length composition of C . palustris oil . Analysis of the immature and mature seed oil by reverse-phase high-performance liquid chromatography confirmed that the principal triglycerides contain both 8:0 and 14:0 . This result indicates that both fatty acids are synthesized at the same time and in the same cells at all developmental stages during oil deposition, suggesting that the two TEs act together in the same fatty acid synthesis system.

Plant Physiol, 1996 Jan, 110(1), 195 - 202
An insecticidal N-acetylglucosamine-specific lectin gene from Griffonia simplicifolia (Leguminosae); Zhu K et al.; Griffonia simplicifolia II, an N-acetylglucosamine-specific legume lectin, has insecticidal activity when fed to the cowpea weevil, Callosobruchus maculatus (F.) . A cDNA clone encoding G . simplicifolia II was isolated from a leaf cDNA library, sequenced, and expressed in a bacterial expression system . The recombinant protein exhibited N-acetylglucosamine-binding and insecticidal activity against cowpea weevil, indicating that glycosylation and multimeric structure are not required for these properties . These results support the hypothesis that genes of the legume lectin gene family encode proteins that function in plant defense against herbivores.

Photochem Photobiol, 1996 Jan, 63(1), 68 - 73
Role of Fapy glycosylase and UvrABC excinuclease in the repair of UVA (320-400 nm)-mediated DNA damage in Escherichia coli; Shennan MG et al.; In contrast to the damage caused by far-UV, the damage caused by UVA (320-400 nm) is largely oxygen dependent, suggesting near-UV-mediated DNA damage involves reactive oxygen species . The DNA repair enzymes that recognize oxidized bases may, therefore, be an important part of the cell's near-UV defense repertoire . To evaluate the relative importance of Fpg (Fapy) glycosylase (an enzyme known to remove oxidized bases) and the DNA damage-inducible UvrABC excinuclease in recovery from near-UV-induced stress, we have constructed fpg- and uvrA- derivatives of Escherichia coli and tested the response (survival) of these strains to both UVA and far-UV radiation . Relative to control strains, the fpg- derivatives were found to be consistently more sensitive to the lethal effects of UVA, but not far-UV radiation . In contrast, uvrA- mutants were more sensitive than control strains to both UVA and far-UV radiation . Thymine dimers, known to be produced by far-UV and corrected by UvrABC, were not generated by the UVA fluences used in this study, suggesting that some other UVA-induced lesion(s) is recognized and repaired by this excinuclease.

Chromosoma, 1996, 104(5), 332 - 40
Introduction of a DNA methyltransferase into Drosophila to probe chromatin structure in vivo; Wines DR et al.; The dam DNA methyltransferase gene from Escherichia coli was introduced into Drosophila in order to probe chromatin structure in vivo . Expression of the gene caused no visible defects or developmental delay even at high levels of active methylase . About half of each target site was found to be methylated in vivo, apparently reflecting a general property of chromatin packaged in nucleosomes . Although site-specific differences were detected, most euchromatic and heterochromatic sites showed comparable degrees of methylation, at least at high methylase levels . Methylase accessibility of a lacZ reporter gene subject to position-effect variegation throughout development was only slightly reduced, consistent with studies of chromatin accessibility in vitro . Silencing of lacZ during development differed from silencing of an adjacent white eye pigment reporter gene in the adult, as though chromatin structure can undergo dynamic alterations during development.

Appl Environ Microbiol, 1996 Jan, 62(1), 55 - 60
Combined effects of the signal sequence and the major chaperone proteins on the export of human cytokines in Escherichia coli; Berges H et al.; We have studied the export of two human proteins in the course of their production in Escherichia coli . The coding sequences of the granulocyte-macrophage colony-stimulating factor and of interleukin 13 were fused to those of two synthetic signal sequences to direct the human proteins to the bacterial periplasm . We found that the total amount of protein varies with the signal peptide-cytokine combination, as does the fraction of it that is soluble in a periplasmic extract . The possibility that the major chaperone proteins such as SecB and the GroEL-GroES and DnaK-DnaJ pairs are limiting factors for the export was tested by overexpressing one or the other of these chaperones concomitantly with the heterologous protein . The GroEL-GroES chaperone pair had no effect on protein production . Overproduction of SecB or DnaK plus DnaJ resulted in a marked increase of the quantity of human proteins in the periplasmic fraction, but this increase depends on the signal peptide-heterologous protein-chaperone association involved.

Appl Environ Microbiol, 1996 Jan, 62(1), 109 - 14
Effect of signal peptide alterations and replacement on export of xylanase A in Streptomyces lividans; Page N et al.; Starting from its translation initiation site, the Streptomyces lividans xylanase A signal peptide consists of 41 amino acids . This signal peptide was deleted and successively replaced with one of six signal peptides from other enzymes secreted by S . lividans and by a signal peptide from the outer membrane protein (LamB) of Escherichia coli . Deletion of the xylanase A signal peptide or modification of its cleavage site abolished secretion of the enzyme . Replacement with the signal peptides of either xylanase B, cellulase A, mannanase, or acetylxylan esterase produced equivalent amounts of xylanase A, while the signal peptides of cellulase B, xylanase C, and LamB secreted less enzyme than did the wild type . All the clones exhibited the same transcription levels, which indicated that the variations in xylanase production were due to the natures of the signal sequences.

J Allergy Clin Immunol, 1996 Jan, 97(1 Pt 1), 95 - 103
Induction of IgE antibodies with predefined specificity in rhesus monkeys with recombinant birch pollen allergens, Bet v 1 and Bet v 2; Ferreira FD et al.; BACKGROUND: Recombinant birch pollen allergens Bet v 1 and Bet v 2 (birch profilin) have been characterized in vitro previously . OBJECTIVE: To establish a close-to-man model of type I allergy, recombinant birch pollen allergens were injected into rhesus monkeys . METHODS: The allergens were expressed in Escherichia coli, purified to homogeneity and injected into rhesus monkeys with aluminium hydroxide as adjuvans . The development of type I allergy was monitored by measurement of specific IgE, in vitro histamine release tests, cellular proliferation assays, skin testing, and bronchial provocation tests . RESULTS: Immunized rhesus monkeys displayed symptoms of type I allergy comparable to those of allergic patients, and cross-reactivity of IgE antibodies with Bet v 1 and Bet v 2 homologous allergens was observed . Systemic application of corticosteroids during secondary immunizations suppressed specific antibody responses . CONCLUSION: Recombinant birch pollen allergens (Bet v 1 and Bet v 2) were effective to establish a close-to-man model of natural type I allergy in rhesus monkeys, allowing study of specific IgE regulation in vivo.

Gut, 1996 Jan, 38(1), 28 - 32
Oral administration of protease inhibits enterotoxigenic Escherichia coli receptor activity in piglet small intestine; Mynott TL et al.; The virulence of enterotoxigenic Escherichia coli (ETEC) is attributed to their ability to adhere via fimbrial adhesins to specific receptors located on the intestinal mucosa . A novel approach to preventing ETEC induced diarrhoea would be to prevent attachment of ETEC to intestine by proteolytically modifying the receptor attachment sites . This study aimed to examine the effect of bromelain, a proteolytic extract obtained from pineapple stems, on ETEC receptor activity in porcine small intestine . Bromelain was administered orally to piglets and K88+ ETEC attachment to small intestine was measured at 50 cm intervals using an enzyme immunoassay . K88+ ETEC attachment to intestinal sections that were not treated with bromelain varied appreciably between sampling sites . Variability in receptor activity along the intestinal surface is though to be caused by the localised effects of endogenous proteases . Oral administration of exogenous protease inhibited K88+ ETEC attachment to pig small intestine in a dose dependent manner (p < 0.05) . Attachment of K88+ ETEC was negligible after treatment, resembling the levels of attachment of K88 to piglets of the genetically determined non-adhesive phenotype, which are resistant to K88+ ETEC infection . Serum biochemical analysis and histopathological examination of treated piglets showed no adverse effects of the bromelain treatment . It is concluded that administration of bromelain can inhibit ETEC receptor activity in vivo and may therefore be useful for prevention of K88+ ETEC induced diarrhoea.

FASEB J, 1996 Jan, 10(1), 20 - 6
Protein folding in the cell: competing models of chaperonin function; Ellis RJ et al.; The long-standing view that polypeptide chains newly synthesized inside cells fold spontaneously to their functional conformations in an energy-independent fashion derives from the observation that many pure denatured proteins will refold spontaneously in vitro when the denaturant is removed . This view is being challenged by the alternative proposal that in vivo many chains need to be helped to fold correctly by preexisting proteins acting as molecular chaperones, some of which hydrolyse ATP . The need for molecular chaperones arises because of the high concentrations of transiently interacting protein surfaces inside cells permit the formation of incorrect nonfunctional structures . The best-studied family of molecular chaperones are called the chaperonins, the archetypal examples being the GroEL and GroES proteins of Escherichia coli . The chaperonins increase the yield of correctly refolded polypeptide chains, both by decreasing their propensity to aggregate with one another and by allowing polypeptides kinetically trapped in incorrect conformations to make fresh attempts to refold into the functional conformations . The mechanisms by which the chaperonins achieve these remarkable results are currently under debate . This review surveys competing models for chaperonin action, and emphasizes the importance when evaluating these models of considering the intracellular environment in which the chaperonins have evolved to function.

FASEB J, 1996 Jan, 10(1), 148 - 52
In vivo protein folding: suppressor analysis of mutations in the groES cochaperone gene of Escherichia coli; Zeilstra-Ryalls J et al.; Our previous work has shown that the Escherichia coli groES14 and groES15 mutations result in reduced GroE chaperone machine function . By selecting for restoration of the ability of those mutant groES alleles to suppress the thermosensitivity of bacteria bearing the dnaA46 mutation, we isolated a number of intra- and extragenic suppressors that increase in vivo GroE chaperone function . One of the intragenic suppressors has been mapped to a segment that codes for the GroES mobile loop, previously shown to be indispensable for proper GroES/GroEL interaction . Two extragenic suppressors have been mapped to a groEL segment, previously identified by mutational analysis as coding for an important functional region of the GroEL protein . Our results should contribute to our eventual understanding of the structure-function relationships of the universally conserved GroE chaperone machine.

FASEB J, 1996 Jan, 10(1), 102 - 9
The molten globule state of alpha-lactalbumin; Kuwajima K; The molten globule state of alpha-lactalbumin is the best-characterized folding intermediate of globular proteins and has been studied intensively by various spectroscopic and physiochemical techniques, including stopped-flow CD and fluorescence spectroscopies, a hydrogen-exchange technique, 1H-NMR spectroscopy, disulfide-exchange chemistry, site-directed mutagenesis, and calorimetric techniques . This review summarizes recent studies . Major findings about the structure of the molten globule state are: 1) It is highly heterogeneous, having a highly structured alpha-helical domain with the beta-sheet domain being significantly unfolded; and 2) it is not a nonspecific, collapsed polypeptide but already has a native-like tertiary fold . These structural characteristics are essential to fully understand the thermodynamic properties of the molten globule state which are described in connection with a recently proposed computational approach to predict the structure of the molten globule state of a protein . Mutant proteins in which the stability of the molten globule state was changed were constructed . Studies of the equilibrium unfolding and kinetic refolding of the mutant proteins will provide further insight into the molten globule state as a folding intermediate . In spite of an initial expectation that the structure recognized by an Escherichia coli chaperone, GroEL, is the molten globule, the interaction of GroEL with alpha-lactalbumin in the molten globule state is much weaker than the interaction with more unfolded states of alpha-lactalbumin, a disulfide-reduced form, and disulfide rearranged species.

Exp Neurol, 1996 Jan, 137(1), 43 - 8
A herpes simplex virus vector overexpressing the glucose transporter gene protects the rat dentate gyrus from an antimetabolite toxin; Dash R et al.; The use of herpes simplex virus vectors offers an attractive means for the in vitro and in vivo transfer of novel genes into postmitotic neurons . Such an approach allows for the introduction of genes with the potential to protect neurons from necrotic insults . Toward that end, we have previously constructed a bicistronic herpes viral vector expressing the gene for the Glut-1 rat brain glucose transporter (GT), along with the Escherichia coli lacZ reporter gene . We observed that this vector enhances glucose uptake both in primary hippocampal cultures and in the hippocampus itself . Moreover, we have found that this vector will protect a variety of types of cultured neurons from necrotic insults and protect hippocampal neurons in vivo from seizure-induced damage . In the present report, we further demonstrate the neuroprotective potential of this GT-expressing vector . 3-Acetylpyridine, an electron transport uncoupler which is preferentially toxic to the dentate gyrus, was microinfused into the dorsal hippocampus of rats . Infection of dentate neurons with GT vectors at the time of exposure to the toxin significantly decreased damage, whereas infection with a physiologically neutral control vector did not . Moreover, there was a window of opportunity for this intervention, as microinfusion of the GT-expressing vector up to 1 h, but not 4 h, after the insult was still neuroprotective.

Development, 1996 Jan, 122(1), 205 - 14
The eve stripe 2 enhancer employs multiple modes of transcriptional synergy; Arnosti DN et al.; Previous studies have provided a detailed model for the regulation of even-skipped (eve) stripe 2 expression in the Drosophila embryo . The bicoid (bcd) regulatory gradient triggers the expression of hunchback (hb); these work synergistically to activate the stripe in the anterior half of the embryo, bcd also coordinates the expression of two repressors, giant (gt) and Kruppel (Kr), which define the anterior and posterior borders of the stripe, respectively . Here, we report the findings of extensive cis- and trans- complementation analyses using a series of defective stripe 2 enhancers in transgenic embryos . This study reaches two primary conclusions . First, the strip 2 enhancer is inherently 'sensitized' for repression by gt . We propose that gt specifies the sharp anterior stripe border by blocking two tiers of transcriptional synergy, cooperative binding to DNA and cooperative contact of bound activators with the transcription complex . Second, we find that the synergistic activity of hb and bcd is 'promiscuous' . For example, a maternally expressed Gal4-Sp1 fusion protein can functionally replace hb in the stripe 2 enhancer . This finding challenges previous proposals for dedicated hb and bcd interactions in the segmentation process.

Development, 1996 Jan, 122(1), 113 - 20
Defective bone formation in Krox-20 mutant mice; Levi G et al.; Endochondral ossification is the prevalent mode of vertebrate skeleton formation; it starts during embryogenesis when cartilage models of long bones develop central regions of hypertrophy which are replaced by bony trabeculae and bone marrow . Although several transcription factors have been implicated in pattern formation in the limbs and axial skeleton, little is known about the transcriptional regulations involved in bone formation . We have created a null allele in the mouse Krox-20 gene, which encodes a zinc finger transcription factor, by in frame insertion of the E . coli lacZ gene and shown that hindbrain segmentation and peripheral nerve myelination are affected in Krox-20-/- embryos . We report here that Krox-20 is also activated in a subpopulation of growth plate hypertrophic chondrocytes and in differentiating osteoblasts and that its disruption severely affects endochondral ossification . Krox-20-/- mice develop skeletal abnormalities including a reduced length and thickness of newly formed bones, a drastic reduction of calcified trabeculae and severe porosity . The periosteal component to bone formation and calcification does not appear to be affected in the homozygous mutant suggesting that the major role for Krox-20 is to be found in the control of the hypertrophic chondrocyte-osteoblast interactions leading to endosteal bone formation.

Mol Carcinog, 1996 Jan, 15(1), 64 - 9
ras effector loop mutations that dissociate p120GAP and neurofibromin interactions; Stang S et al.; ras proteins are positively regulated by nucleotide exchange factors and negatively regulated by GTPase-activating proteins (GAPs) . Two GAPs have been found in mammalian cells, p120GAP and neurofibromin, the product of the type 1 neurofibromatosis (NF1) gene . A library of substitutions in the effector loop region of ras in an Escherichia coli plasmid expression system was screened for c-Ha-ras species with altered GAP interactions . Several substitutions preferentially disrupted the interaction of ras with p120GAP as compared with the interaction with the recombinant GAP-related domain of neurofibromin (NF1-GRD) . The most extreme example, Tyr32His, encoded a ras species that was unaffected by p120GAP but was stimulated normally by NF1-GRD . Tyr32His was weakly transforming in Rat2 cells . Tyr32His ras was primarily GDP-bound in quiescent Rat2 cells, although it rapidly associated with GTP after treatment of cells with epidermal growth factor . These results show that the NF1 product has less stringent requirements than p120GAP for ras effector domain structure and that negative regulation of ras can be achieved in rat fibroblasts by the product of NF1.

Scand J Immunol, 1996 Jan, 43(1), 122 - 5
Interleukin 10 release during endotoxaemia in chimpanzees: role of platelet-activating factor and interleukin 6; van der Poll T et al.; Interleukin (IL-)10 has been demonstrated to inhibit endotoxin-induced production of a number of pro-inflammatory cytokines . The present study sought to compare the appearances in the circulation of IL-10, IL-6 and IL-8, and to assess the roles of endogenously produced platelet-activating factor (PAF) and IL-6 in IL-10 release during endotoxaemia in chimpanzees . Intravenous injection of endotoxin (lot EC-5, 4 ng/kg, n = 8) induced a transient rise in serum IL-10 concentrations, peaking after 2 h (213 +/- 70 pg/ml; P < 0.05) . No correlations existed between peak IL-10 levels, and peak IL-6 and IL-8 levels . Neither infusion of the specific PAF antagonist TCV-309 (n = 4), nor infusion of a neutralizing anti-IL-6 monoclonal antibody (n = 4) influenced endotoxin-induced IL-10 release . IL-10 release elicited by injection of endotoxin is not mediated by PAF or IL6.

J Gen Virol, 1996 Jan, 77 ( Pt 1), 49 - 59
Assessment of antigenicity and genetic variation of glycoprotein B of murine cytomegalovirus; Xu J et al.; An analysis of linear antibody-binding sites of the glycoprotein B (gB) molecule of murine cytomegalovirus (MCMV) and of genetic variation within these regions was performed . To achieve this, a series of overlapping fragments spanning the entire coding sequence of the gB gene of the K181 strain of MCMV was expressed in E . coli as fusion proteins with glutathione S-transferase (GST) using the pGEX expression system . Four antibody-binding regions were mapped to locations spanning amino acid residues 17-79 (BS), 155-278 (BE2), 809-926 (SS) and 347-508 (BB and EE), based on reactivity in Western blot analysis of GST-gB fusion proteins with murine polyclonal antiserum raised against MCMV . Only the antibody-binding region BE2 (155-278) elicited an antiserum that exhibited complement-dependent neutralizing activity, and immunization of mice with the fusion protein BE2 led to moderate but significant reductions in the level of MCMV replication in the spleen . Polyclonal antisera raised against the GST-gB fusion proteins detected purified virion proteins of 105 kDa (anti-BS and anti-BE2) and 52 kDa (anti-SS) and are therefore likely to recognize the N-terminal and C-terminal portions of the gB molecule, respectively . The antibody-binding region within amino acid residues 17-79 was found to be MCMV strain-specific, whereas antibody-binding regions within residues 155-278 and 809-926 were found to be conserved among MCMV field isolates . Comparative sequence analysis of the corresponding regions of MCMV gB revealed a level and extent of sequence of sequence heterogeneity consistent with these findings.

J Gen Virol, 1996 Jan, 77 ( Pt 1), 129 - 37
Identification of a short domain within the non-structural protein NS2 of epizootic haemorrhagic disease virus that is important for single strand RNA-binding activity; Theron J et al.; The role that a conserved amino acid motif, found in the non-structural protein NS2 of orbiviruses, plays in the interaction of this protein with single stranded (ss) RNA was investigated by mutation analysis of the NS2 of epizootic haemorrhagic disease virus . An NS2 mutant in which this motif (amino acids 75 to 83) was deleted was expressed in Spodoptera frugiperda cells by a recombinant baculovirus and found to be unable to bind to poly(U)-Sepharose . The deletion mutant also differed from wild-type NS2 in that it did not appear to be complexed with ssRNA in cells infected with the baculovirus recombinant . Furthermore, the deletion exerted an adverse effect on the ability of NS2 to form inclusion bodies in the cytoplasm of baculovirus-infected insect cells . To further characterize the role of this motif in RNA-binding, specific residues within the region were substituted by site-directed mutagenesis and the mutants were expressed in Escherichia coli as fusion proteins . Analysis of the different mutant proteins indicated that in each case ssRNA-binding was impaired relative to that of the wild-type NS2 control . The degree of impairment corresponded to the number of amino acid substitutions and the largest effects were associated with non-conserved substitutions . It is suggested that the conserved motif is an important structural determinant in the interaction of NS2 with ssRNA.

Infect Immun, 1996 Jan, 64(1), 55 - 60
Vaccination with genetically modified Shiga-like toxin IIe prevents edema disease in swine; Bosworth BT et al.; Escherichia coli strains producing Shiga-like toxin II variant (SLT-IIe, formerly called SLT-IIv) cause edema disease in weaned pigs . Vaccination of pigs with a genetically modified form of Shiga-like toxin IIe, SLT-IIe(E167Q), has been previously shown to be nontoxic and to induce antibodies to SLT-IIe (V.M . Gordon . S.C . Whipp, H.W . Moon, A.D . O'Brien, and J.E . Samuel, Infect, Immun . 60:485-502, 1992) . Fifty micrograms of SLT-IIe(E167Q) toxin was used to vaccinate suckling pigs at 1 and 2 weeks of age . Both vaccinated and nonvaccinated pigs were orally inoculated with an SLT-IIe-producing strain of E . coli after weaning (3 to 4 weeks of age) . Pigs fed a low-protein diet that were not vaccinated with SLT-IIe(E167Q) developed subclinical edema disease, histologically evident as vascular necrosis . Pigs fed a high-protein diet that were not vaccinated with SLT-IIe(E167Q) developed clinical edema disease manifested as vascular necrosis, reduced weight gain, ataxia, palpebral edema, lateral recumbency, and death . Pigs vaccinated with SLT-IIe(E167Q) had a reduction in the incidence of subclinical edema disease and never developed clinical edema disease . These data demonstrate that vaccination with a genetically modified form of SLT-IIe prevents edema disease and are consistent with the notion that diet influences susceptibility to edema disease.

Infect Immun, 1996 Jan, 64(1), 332 - 42
Characterization of 20K fimbria, a new adhesin of septicemic and diarrhea-associated Escherichia coli strains, that belongs to a family of adhesins with N-acetyl-D-glucosamine recognition; Bertin Y et al.; Bovine septicemic Escherichia coli 31A agglutinates bovine, rabbit, and human erythrocytes and adheres in vitro to the brush border of bovine or ovine intestinal epithelial cells and to the human colon carcinoma Caco-2 cell line . The adhesion and hemagglutination of E . coli 31A are mediated by a chromosome-encoded fimbrial adhesin serologically distinct from known fimbrial adhesins found in enterotoxigenic and septicemic bovine E . coli strains . By electron microscopy studies the fimbriae designated 20K were observed as fine flexible filaments (diameter, 3 nm) and the purified major fimbrial subunit appeared with an apparent molecular mass of 20,000 Da . Western blot (immunoblot) analysis, N-terminal sequence alignment, and amino acid composition revealed a high homology with the N-acetyl-D-glcosamine-specific G fimbria of human uropathogenic E . coli and with fimbriae belonging to the F17 family produced by bovine enterotoxigenic and invasive E . coli strains . Immunological study revealed that 20K fimbria was closely related to G fimbria and represents a serological variant of F17 fimbria . Hemagglutination and adhesion inhibition assays demonstrated that 20K, G, and F17 fimbriae bind to an N-acetyl-D-glucosamine-containing receptor, but each probably binds to different oligosaccharide sequences or different receptors on host tissues . 20K fimbriae were produced by a limited group of clonally related strains with the unusual m-inositol-positive phenotype and appeared highly associated with the plasmid-mediated virulence factor . An examination of natural occurrence of 20K fimbriae among a large collection of human and animal pathogenic E . coli showed that 20K fimbria is the prominent adhesin among bovine septicemic E . coli isolated from European countries.

Infect Immun, 1996 Jan, 64(1), 326 - 31
Primary structure of the variable region of monoclonal antibody 2B10, capable of inducing anti-idiotypic antibodies that recognize the C-terminal region of MSA-1 of Plasmodium falciparum; Su S et al.; Previously, we reported on the properties of a monoclonal antibody, 2B10, which has the same determinant on the human erythrocyte as MSA-1 of Plasmodium falciparum (FCR3 strain); the binding of both ligands to erythrocyte receptors was totally sialic acid dependent . In this work, rabbit anti-2B10 idiopathic antibodies were generated . The anti-idiotypic antibodies recognized both the erythrocyte binding site of 2B10 and the C-terminal region of MSA-1 (amino acids 1047 to 1640); they were able to inhibit 2B10 and MSA-1 binding to erythrocytes and partially prevent P . falciparum merozoites from invading erythrocytes . The utility of 2B10 in the study of the interaction between MSA-1 and human erythrocytes prompted us to determine the nucleotide and deduced amino acid sequences of its VH and VL regions . The data show that the 2B10 VH region is part of the J558 family and is especially homologous to BALB/c anti-nitrophenyl monoclonal antibody 21.1.43; the VL region belongs to the VK1 subgroup and comes from the same genomic locus as (NZB x W)F1 anti-DNA and C57BL anti-dextran monoclonal antibodies BXW-14 and 42.48.12.2, respectively . Most of the differences among the VH and VL segments are located in CDR1 and -3 . The binding site of 2B10 contains both negatively and positively charged amino acid residues . The amino acid sequences of the 2B10 VH region and a region of MSA-1 from the Wellcome strain of P . falciparum (amino acids 1002 to 1115) share 43% similarity, and the amino acid sequences between the 2B10 VL region and another segment of the same MSA-1 (amino acids 1247 to 1394) share 48% similarity . We conclude that the interactions between erythrocyte receptors and their ligands, 2B10 and MSA-1, are related and that the C-terminal region of MSA-1 is the erythrocyte binding domain.

Infect Immun, 1996 Jan, 64(1), 305 - 9
Augmented immunological activities of recombinant lipopolysaccharide possessing the mannose homopolymer as the O-specific polysaccharide; Paeng N et al.; Recombinant lipopolysaccharide possessing the mannose homopolymer as the O-specific polysaccharide was manufactured genetically by transforming Escherichia coli K-12 with various rfb genes capable of synthesizing the mannose homopolymer . Recombinant lipopolysaccharide exhibited levels of anticomplement activity, adjuvant activity, and regional lymph node-enlarging activity much higher than those exhibited by the original rough-type lipopolysaccharide from E . coli K-12 or lipopolysaccharide possessing the heteropolysaccharide from E . coli O111 . Immunological activities of recombinant lipopolysaccharide were as strong as those of wild-type lipopolysaccharide possessing the mannose homopolymer . Characteristic activities of wild-type lipolysaccharide possessing the mannose homopolymer were exhibited by recombinant lipopolysaccharide . The abilities of lipopolysaccharide to activate B cells polyclonally and to produce cytokines did not seem to be related to the presence of the mannose homopolymer . Therefore, it was suggested that the mannose homopolymer in the O-specific polysaccharide might exclusively enhance anticomplement activity, adjuvant activity, and regional lymph node-enlarging activity among various lipid A activities.

Infect Immun, 1996 Jan, 64(1), 154 - 60
Endothelial cell GlcNAc beta 1-4GlcNAc epitopes for outer membrane protein A enhance traversal of Escherichia coli across the blood-brain barrier; Prasadarao NV et al.; Inadequate knowledge of pathogenesis and pathophysiology has contributed to the high mortality and morbidity associated with neonatal Escherichia coli meningitis . We have shown previously that outer membrane protein A (OmpA) contributes to E . coli K1 membrane invasion of brain microvascular endothelial cells . In this study we report that this OmpA+ K1 E . coli invasion of brain microvascular endothelial cells was inhibited by wheat germ agglutinin and chitooligomers prepared from the polymer of 1,4-linked GlcNAc, chitin . The specificity of the interaction between OmpA and GlcNAc beta 1-4GlcNAc epitopes was verified by the demonstration that chitotriose-bound OmpA and wheat germ agglutinin-bound brain microvascular endothelial cell membrane proteins inhibit E . coli K1 invasion . Of interest, OmpA+ E . coli invasion into systemic endothelial cells did not occur, but invasion similar to that of brain microvascular endothelial cells was observed when systemic cells were treated with alpha-fucosidase, suggesting that the GlcNAc beta 1-4GlcNAc moieties might be substituted with L-fucose on these cells . More importantly, the chitooligomers prevented entry of E . coli K1 into the cerebrospinal fluid of newborn rats with experimental hematogenous E . coli meningitis, suggesting that the GlcNAc beta 1-4GlcNAc epitope of brain microvascular endothelial cells indeed mediates the traversal of E . coli K1 across the blood-brain barrier . A novel strategy with the use of soluble receptor analog(s) may be feasible in the prevention of devastating neonatal E . coli meningitis.

Infect Immun, 1996 Jan, 64(1), 100 - 7
Molecular characterization, occurrence, and immunogenicity in infected sheep and cattle of two minor outer membrane proteins of Brucella abortus; Tibor A et al.; Screening of a Brucella abortus genomic library with two sets of monoclonal antibodies allowed the isolation of the genes corresponding to two minor outer membrane proteins (OMP10 and OMP19) found in this bacterial species . Sequence analysis of the omp10 gene revealed an open reading frame capable of encoding a protein of 126 amino acids . The nucleotide sequence of the insert producing the OMP19 protein contains two overlapping open reading frames, the largest of which (177 codons) was shown to encode the protein of interest . Analysis of the N-terminal sequences of both putative proteins revealed features of a bacterial signal peptide, and homology to the bacterial lipoprotein processing sequence was also observed . Immunoblotting with monoclonal antibodies specific for OMP10 or OMP19 showed that both proteins are present in the 34 Brucella strains tested, representing all six Brucella species and all their biovars . The OMP19 detected in the five Brucella ovis strains examined migrated at an apparent molecular weight that is slightly higher than those of the other Brucella species, confirming the divergence of B . ovis from these species . OMP10 and OMP19 were produced in recombinant Escherichia coli and purified to homogeneity for serological analysis . A large fraction of sera from sheep naturally infected with Brucella melitensis were reactive with these proteins in an enzyme-linked immunosorbent assay, whereas sera from B . abortus-infected cattle were almost completely unreactive in this assay.

Genes Dev, 1996 Jan 1, 10(1), 93 - 102
trachealess encodes a bHLH-PAS protein that is an inducer of tracheal cell fates in Drosophila; Wilk R et al.; The embryonic tracheal system in Drosophila develops from placodes of precursor cells on the ectoderm . A transcription factor of the basic helix-loop-helix (bHLH)-PAS family, which is expressed in the nuclei of the tracheal cells throughout development, was identified . The protein shows the highest degree of homology to the Single-minded (Sim) protein . The transcript represents the previously identified trachealess (trh) locus, essential for tracheal development . Ectopic expression of trh leads to generation of extra tracheal pits and branches and to the expression of tracheal markers by patches of ectodermal cells . The expression of trh is consistent with a biphasic mode of transcriptional regulation . Expression is first induced by exogenous cues and is subsequently autoregulated . trh is also expressed and required in the posterior spiracles and the salivary gland ducts . The role of Trachealess in the formation of several tubular tissues in the embryo suggests that it may induce a general fate of branched tubular structures of epithelial origin.

Genes Dev, 1996 Jan 1, 10(1), 16 - 26
DNA-binding determinants of the alpha subunit of RNA polymerase: novel DNA-binding domain architecture; Gaal T et al.; The Escherichia coli RNA polymerase alpha-subunit binds through its carboxy-terminal domain (alpha CTD) to a recognition element, the upstream (UP) element, in certain promoters . We used genetic and biochemical techniques to identify the residues in alpha CTD important for UP-element-dependent transcription and DNA binding . These residues occur in two regions of alpha CTD, close to but distinct from, residues important for interactions with certain transcription activators . We used NMR spectroscopy to determine the secondary structure of alpha CTD, alpha CTD contains a nonstandard helix followed by four alpha-helices . The two regions of alpha CTD important for DNA binding correspond to the first alpha-helix and the loop between the third and fourth alpha-helices . The alpha CTD DNA-binding domain architecture is unlike any DNA-binding architecture identified to date, and we propose that alpha CTD has a novel mode of interaction with DNA . Our results suggest models for alpha CTD-DNA and alpha CTD-DNA-activator interactions during transcription initiation.

Arch Biochem Biophys, 1996 Jan 1, 325(1), 8 - 19
Expression of biologically active human SPARC in Escherichia coli; Bassuk JA et al.; Human SPARC has been cloned by the polymerase chain reaction from an endothelial cell cDNA library and expressed in Escherichia coli as a biologically active protein . Transcriptional expression of the insert cDNA was dependent on the activation of the T7 RNA polymerase promoter by isopropylgalactopyranoside . Two forms of recombinant SPARC (rSPARC) protein were recovered from BL21 (DE3) E . coli after transformation with the plasmid pSPARCwt: a soluble, monomeric form of rSPARC and an insoluble, aggregated form sequestered in inclusion bodies . The isolation of the soluble form of rSPARC was accomplished by anion-exchange, nickel-chelate affinity, and gel filtration chromatographies . The isolated protein was an intact, full-length polypeptide of 293 amino acids by the following criteria: N-terminal amino acid sequence, reaction with anti-SPARC immunoglobulins specific for N-terminal and C-terminal sequences, and interaction of the C-terminal histidine tag of rSPARC with a nickel-chelate affinity resin . Circular dichroism and intrinsic fluorescence spectroscopy indicated that the conformation of rSPARC was dependent on interaction with Ca2- ions . The recombinant protein inhibited cell spreading and bound specifically to bovine aortic endothelial cells . Levels of bacterial endotoxin (< 18 pg/microgram rSPARC) present in rSPARC preparations were below the threshold that affects the behavior of these endothelial cells . These conformational and biological properties of rSPARC are consistent with previously described characteristics of the native protein . The purification of biologically active rSPARC, as well as mutated forms of the protein, will provide sufficient quantities of protein for the determination of structure/function relationships.

Arch Biochem Biophys, 1996 Jan 1, 325(1), 129 - 38
Alterations of heparan sulfate moieties in cultured endothelial cells exposed to endotoxin; Colburn P et al.; In previous studies, we observed that exposure to endotoxin markedly reduces the level of heparan sulfate proteoglycans in the extracellular matrix of cultured endothelial cells and at the same time causes the accumulation of proteoglycans bearing glycosaminoglycan chains of reduced size in the conditioned medium (P . Colburn, E . Kobayashi, and V . Buonassisi, 1994, J . Cell . Physiol . 159, 121-130) . We have now investigated the structural and ligand-binding features which distinguish the matrix glycosaminoglycan moiety and the nature of the alterations of the truncated glycosaminoglycans . The matrix glycosaminoglycans are less sulfated than those of other cellular compartments and are more extensively degraded by heparitinase I, yielding a larger proportion of smaller oligosaccharides . In the binding assays, matrix glycosaminoglycans had greater specificity than those of the cell surface for a synthetic peptide patterned on the carboxyl-terminal sequence of an N-glycan sulfated protein synthesized by the endothelial cell . The nature of the alteration caused by exposure to endotoxin consists in the loss of a region rich in sulfate, located at the nonreducing end of the glycosaminoglycan chain . We also determined that only proteoglycans with intact chains are found in the extracellular matrix of endotoxin-treated cells.

J Bacteriol, 1996 Jan, 178(2), 470 - 6
Regulation of Escherichia coli starvation sigma factor (sigma s) by ClpXP protease; Schweder T et al.; In Escherichia coli, starvation (stationary-phase)-mediated differentiation involves 50 or more genes and is triggered by an increase in cellular sigma s levels . Western immunoblot analysis showed that in mutants lacking the protease ClpP or its cognate ATPase-containing subunit ClpX, sigma s levels of exponential-phase cells increased to those of stationary-phase wild-type cells . Lack of other potential partners of ClpP, i.e., ClpA or ClpB, or of Lon protease had no effect . In ClpXP-proficient cells, the stability of sigma s increased markedly in stationary-phase compared with exponential-phase cells, but in ClpP-deficient cells, sigma s became virtually completely stable in both phases . There was no decrease in ClpXP levels in stationary-phase wild-type cells . Thus, sigma s probably becomes more resistant to this protease in stationary phase . The reported sigma s-stabilizing effect of the hns mutation also was not due to decreased protease levels . Studies with translational fusions containing different lengths of sigma s coding region suggest that amino acid residues 173 to 188 of this sigma factor may directly or indirectly serve as at least part of the target for ClpXP protease.

J Bacteriol, 1996 Jan, 178(1), 321 - 4
Autoregulation of iclR, the gene encoding the repressor of the glyoxylate bypass operon; Gui L et al.; The aceBAK operon was partially induced by a multicopy plasmid which carried the promoter region of the gene which encodes its repressor, iclR . Gel shift and DNase I analyses demonstrated that IclR binds to its own promoter . Disruption of iclR increased the expression of an iclR::lacZ operon fusion . Although aceBAK and iclR are both regulated by IclR, aceBAK expression responds to the carbon source, while expression of iclR does not.

J Bacteriol, 1996 Jan, 178(1), 301 - 5
Cloning and overexpression in Escherichia coli of the genes encoding NAD-dependent alcohol dehydrogenase from two Sulfolobus species; Cannio R et al.; The gene adh encoding a NAD-dependent alcohol dehydrogenase from the novel strain RC3 of Sulfolobus sp . was cloned and sequenced . Both the adh gene from Sulfolobus sp . strain RC3 and the alcohol dehydrogenase gene from Sulfolobus solfataricus (DSM 1617) were expressed at a high level in Escherichia coli, and the recombinant enzymes were purified, characterized, and compared . Only a few amino acid replacements were responsible for the different kinetic and physicochemical features investigated.

J Bacteriol, 1996 Jan, 178(1), 289 - 92
Purification and in vitro phosphorylation of Myxococcus xanthus AsgA protein; Li Y et al.; The deduced amino acid sequence of the Myxococcus xanthus AsgA protein contains an N-terminal domain that is homologous to the receiver of response regulators and a C-terminal domain that is homologous to the transmitter of histidine protein kinases . We overexpressed affinity-tagged AsgA in Escherichia coli, purified the recombinant protein, and showed that AsgA has autokinase activity in vitro . The results of chemical-stability assays suggest that AsgA is phosphorylated on a histidine and provide no evidence for transfer of the phosphoryl group to the conserved aspartate of the receiver domain.

J Bacteriol, 1996 Jan, 178(1), 280 - 3
Frur mediates catabolite activation of pyruvate kinase (pykF) gene expression in Escherichia coli; Bledig SA et al.; Expression of a pykF-lacZ fusion was studied as a function of the carbon source in wild-type strains and strains lacking or overproducing the FruR protein of Escherichia coli . FruR controls the response to the carbon source by repressing pykF expression more strongly under gluconeogenic than under glycolytic conditions, a phenomenon we term catabolite activation.

J Bacteriol, 1996 Jan, 178(1), 273 - 9
Allelic exchange in Mycobacterium tuberculosis with long linear recombination substrates; Balasubramanian V et al.; Genetic studies of Mycobacterium tuberculosis have been greatly hampered by the inability to introduce specific chromosomal mutations . Whereas the ability to perform allelic exchanges has provided a useful method of gene disruption in other organisms, in the clinically important species of mycobacteria, such as M . tuberculosis and Mycobacterium bovis, similar approaches have thus far been unsuccessful . In this communication, we report the development of a shuttle mutagenesis strategy that involves the use of long linear recombination substrates to reproducibly obtain recombinants by allelic exchange in M . tuberculosis . Long linear recombination substrates, approximately 40 to 50 kb in length, were generated by constructing libraries in the excisable cosmid vector pYUB328 . The cosmid vector could be readily excised from the recombinant cosmids by digestion with PacI, a restriction endonuclease for which there exist few, if any, sites in mycobacterial genomes . A cosmid containing the mycobacterial leuD gene was isolated, and a selectable marker conferring resistance to kanamycin was inserted into the leuD gene in the recombinant cosmid by interplasmid recombination in Escherichia coli . A long linear recombination substrate containing the insertionally mutated leuD gene was generated by PacI digestion . Electroporation of this recombination substrate containing the insertionally mutated leuD allele resulted in the generation of leucine auxotrophic mutants by homologous recombination in 6% of the kanamycin-resistant transformants for both the Erdman and H37Rv strains of M . tuberculosis . The ability to perform allelic exchanges provides an important approach for investigating the biology of this pathogen as well as developing new live-cell M . tuberculosis-based vaccines.

J Bacteriol, 1996 Jan, 178(1), 240 - 7
Identification of the O antigen polymerase (rfc) gene in Escherichia coli O4 by insertional mutagenesis using a nonpolar chloramphenicol resistance cassette; Lukomski S et al.; Computer analysis of the O4 polysaccharide gene cluster of Escherichia coli revealed the presence of two open reading frames (ORFs) encoding strongly hydrophobic polypeptides . O antigen polymerase, which is encoded by the rfc gene, is a potential membrane protein and therefore should be hydrophobic . To identify the rfc gene, these two ORFs were subjected to insertional mutagenesis . A chloramphenicol resistance cassette was designed which, when properly inserted, does not cause a polar effect in downstream genes . Each of two ORFs, cloned into a plasmid vector, was inactivated with this cassette . Two types of mutants bearing chromosomal insertions of the cassettes in each ORF were constructed by homologous recombination . These mutants were characterized by PCR, Southern blotting, and transverse-alternating-field electrophoresis . Only one class of mutants exhibited the expected O polymerase-deficient phenotype; they produced O4-specific, semirough lipopolysaccharide . Therefore, this ORF was identified as the rfc gene . The chromosomal rfc mutation was complemented in trans by the rfc gene expressed from a plasmid vector.

J Bacteriol, 1996 Jan, 178(1), 24 - 34
Escherichia coli fliAZY operon; Mytelka DS et al.; We have cloned the Escherichia coli fliAZY operon, which contains the fliA gene (the alternative sigma factor sigma F) and two novel genes, fliZ and fliY . Transcriptional mapping of this operon shows two start sites, one of which is preceded by a canonical E sigma F-dependent consensus and is dependent on sigma F for expression in vivo and in vitro . We have overexpressed and purified sigma F and demonstrated that it can direct core polymerase to E sigma F-dependent promoters . FliZ and FliY are not required for motility but may regulate sigma F activity, perhaps in response to a putative cell density signal that may be detected by FliY, a member of the bacterial extracellular solute-binding protein family 3.

J Bacteriol, 1996 Jan, 178(1), 232 - 9
A novel alpha-ketoglutarate reductase activity of the serA-encoded 3-phosphoglycerate dehydrogenase of Escherichia coli K-12 and its possible implications for human 2-hydroxyglutaric aciduria; Zhao G et al.; Escherichia coli serA-encoded 3-phosphoglycerate (3PG) dehydrogenase catalyzes the first step of the major phosphorylated pathway of L-serine (Ser) biosynthesis . The SerA enzyme is evolutionarily related to the pdxB gene product, 4-phosphoerythronate dehydrogenase, which catalyzes the second step in one branch of pyridoxal 5'-phosphate coenzyme biosynthesis . Both the Ser and pyridoxal 5'-phosphate biosynthetic pathways use the serC(pdxF)-encoded transaminase in their next steps . In an analysis of these parallel pathways, we attempted to couple the transaminase and dehydrogenase reactions in the reverse direction . Unexpectedly, we found that the SerA enzyme catalyzes a previously undetected reduction of alpha-ketoglutarate (alpha KG) to 2-hydroxyglutaric acid (HGA) . Numerous criteria ruled out the possibility that this SerA alpha KG reductase activity was caused by contamination in the substrate or purified enzyme preparations . HGA was confirmed as the product of the SerA alpha KG reductase reaction by thin-layer chromatography and by enzyme assays showing that both the D- and L-isomers of HGA were substrates for the reverse (dehydrogenase) reaction . Detailed steady-state kinetic analyses showed that alpha KG reduction (apparent Michaelis-Menten constant {Km(app)} = 88 microM; apparent catalytic constant {kcat(app)} = 33.3 s-1) and 3-phosphohydroxypyruvate reduction (Km(app) = 3.2 microM; kcatapp = 27.8 s-1), which is the reverse reaction of 3PG oxidation, were the major in vitro activities of the SerA enzyme . The SerA alpha KG reductase was inhibited by Ser, D-HGA, 3PG, and glycine (Gly), whereas the D-HGA dehydrogenase was inhibited by Ser, alpha KG, 3-phosphohydroxypyruvate, and Gly . The implications of these findings for the regulation of Ser biosynthesis, the recycling of NADH, and the enzymology of 2-hydroxyacid dehydrogenases are discussed . Since the same pathway of Ser biosynthesis seems to be present in all organisms, these results suggest that a mutation in the human SerA homolog may contribute to the neurometabolic diseases D- and L-2-hydroxyglutaric aciduria, which lead to the accumulation of D-HGA and L-HGA, respectively.

J Bacteriol, 1996 Jan, 178(1), 223 - 31
Torque generation in the flagellar motor of Escherichia coli: evidence of a direct role for FliG but not for FliM or FliN; Lloyd SA et al.; Among the many proteins needed for assembly and function of bacterial flagella, FliG, FliM, and FliN have attracted special attention because mutant phenotypes suggest that they are needed not only for flagellar assembly but also for torque generation and for controlling the direction of motor rotation . A role for these proteins in torque generation is suggested by the existence of mutations in each of them that produce the Mot- (or paralyzed) phenotype, in which flagella are assembled and appear normal but do not rotate . The presumption is that Mot- defects cause paralysis by specifically disrupting functions essential for torque generation, while preserving the features of a protein needed for flagellar assembly . Here, we present evidence that the reported mot mutations in fliM and fliN do not disrupt torque-generating functions specifically but, instead, affect the incorporation of proteins into the flagellum . The fliM and fliN mutants are immotile at normal expression levels but become motile when the mutant proteins and/or other, evidently interacting flagellar proteins are overexpressed . In contrast, many of the reported fliG mot mutations abolish motility at all expression levels, while permitting flagellar assembly, and thus appear to disrupt torque generation specifically . These mutations are clustered in a segment of about 100 residues at the carboxyl terminus of FliG . A slightly larger carboxyl-terminal segment of 126 residues accumulates in the cells when expressed alone and thus probably constitutes a stable, independently folded domain . We suggest that the carboxyl-terminal domain of FliG functions specifically in torque generation, forming the rotor portion of the site of energy transduction in the flagellar motor.

J Bacteriol, 1996 Jan, 178(1), 199 - 203
The nucleotide concentration determines the specificity of in vitro transcription activation by the sigma 54-dependent activator FhlA; Hopper S et al.; An in vitro transcription system has been set up for formate- and FhlA-dependent transcription activation at the -12/-24 promoter of the fdhF gene from Escherichia coli by sigma 54-RNA polymerase . It requires the presence of the upstream activation sequence on supercoiled DNA . Transcription is independent from the effector formate at nucleoside triphosphate concentrations of 400 microM and above and completely dependent on the presence of the effector when the concentration is lowered to 300 microM . Inclusion of nucleoside diphosphates in the system raises the nucleoside triphosphate level at which specific induction by formate can take place . The threshold level of FhlA relative to that of template DNA required for transcription activation in the absence of formate was lowered at a high nucleoside triphosphate concentration . On the other hand, transcription activation at the fdhF promoter lacking the upstream activation sequence requires an increased ratio of FhlA to promoter plus the presence of formate; high ATP concentrations cannot bypass the effect of formate . These results are interpreted in terms of a model which implies that FhlA must undergo a change in its oligomeric state for transcription activation and that this oligomerization is favored by high nucleoside triphosphate concentrations, by the effector formate, and by the target DNA . In the absence of the target DNA, FhlA can line up at unspecific DNA and activate transcription; in this case, however, presence of formate and a higher FhlA concentration are required to stabilize and increase the amount of active oligomer.

J Bacteriol, 1996 Jan, 178(1), 184 - 90
Preferential binding of Escherichia coli RecF protein to gapped DNA in the presence of adenosine (gamma-thio) triphosphate; Hegde SP et al.; Escherichia coli RecF protein binds, but does not hydrolyze, ATP . To determine the role that ATP binding to RecF plays in RecF protein-mediated DNA binding, we have determined the interaction between RecF protein and single-stranded (ss)DNA, double-stranded (ds)DNA, and dsDNA containing ssDNA regions (gapped {g}DNA) either alone or in various combinations both in the presence and in the absence of adenosine (gamma-thio) triphosphate, gamma-S-ATP, a nonhydrolyzable ATP analog . Protein-DNA complexes were analyzed by electrophoresis on agarose gels and visualized by autoradiography . The type of protein-DNA complexes formed in the presence of gamma-S-ATP was different with each of the DNA substrates and from those formed in the absence of gamma-S-ATP . Competition experiments with various combinations of DNA substrates indicated that RecF protein preferentially bound gDNA in the presence of gamma-S-ATP, and the order of preference of binding was gDNA > dsDNA > ssDNA . Since gDNA has both ds- and ssDNA components, we suggest that the role for ATP in RecF protein-DNA interactions in vivo is to confer specificity of binding to dsDNA-ssDNA junctions, which is necessary for catalyzing DNA repair and recombination.

J Bacteriol, 1996 Jan, 178(1), 149 - 55
Characterization and phylogeny of the pfp gene of Amycolatopsis methanolica encoding PPi-dependent phosphofructokinase; Alves AM et al.; The actinomycete Amycolatopsis methanolica employs a PPi-dependent phosphofructokinase (PPi-PFK) (EC 2.7.1.90) with biochemical characteristics similar to those of both ATP- and PPi-dependent enzymes during growth on glucose . A 2.3-kb PvuII fragment hybridizing to two oligonucleotides based on the amino-terminal amino acid sequence of PPi-PFK was isolated from a genomic library of A . methanolica . Nucleotide sequence analysis of this fragment revealed the presence of an open reading frame encoding a protein of 340 amino acids with a high degree of similarity to PFK proteins . Heterologous expression of this open reading frame in Escherichia coli gave rise to a unique 45-kDa protein displaying a high level of PPi-PFK activity . The open reading frame was therefore designated pfp, encoding the PPi-PFK of A . methanolica . Upstream and transcribed divergently from pfp, a partial open reading frame (aroA) similar to 3-deoxy-D-arabino-heptulosonate-7-phosphate synthase-encoding genes was identified . The partial open reading frame (chiA) downstream from pfp was similar to chitinase genes from Streptomyces species . A phylogenetic analysis of the ATP- and PPi-dependent proteins showed that PPi-PFK enzymes are monophyletic, suggesting that the two types of PFK evolved from a common ancestor.

Hepatology, 1996 Jan, 23(1), 115 - 22
Identification of the 37-kd rat liver protein that forms an acetaldehyde adduct in vivo as delta 4-3-ketosteroid 5 beta-reductase; Zhu Y et al.; Acetaldehyde, the first product of alcohol metabolism, is highly reactive . Several proteins have been shown to be covalently modified by acetaldehyde in vivo . We have previously reported the detection of a cytosolic 37-kd protein-acetaldehyde adduct (-AA) in the liver of alcohol-fed rats . The liver extract from an alcohol-fed rat was subjected to 2-dimensional (2D) sodium dodecylsulfate (SDS)-polyacrylamide gel electrophoresis (PAGE), transferred to polyvinylidene difluoride (PVDF) membrane, and the 37-kd protein-AA spot was digested with trypsin and sequenced for amino acids . Degenerate oligonucleotides corresponding to a peptide sequence of the protein-AA were used as the probe to screen a lambda gt11 rat liver complementary DNA (cDNA) library . A clone that extended to a potential ATG start codon was identified . The open reading frame was 978 nucleotides long, encoding 326 amino acid residues . The sequence matched that of rat liver delta 4-3-ketosteroid 5 beta-reductase . The cloned cDNA was expressed in Escherichia coli using pGEX-KG as the vector . The expressed protein was found to be of correct molecular weight . It reacted with an antibody that recognized the unmodified liver 37-kd protein by Western blotting . Peptide profiles of tryptic-digested recombinant protein and the purified rat liver 37-kd protein were similar and yielded the same peptide sequence . delta 4-3-ketosteroid 5 beta-reductase catalyzes the reduction of key intermediates during bile acid biosynthesis . Whether modification of the 5 beta-reductase by acetaldehyde affects the enzyme activity and bile acid synthesis remains to be studied.

Nat Struct Biol, 1996 Jan, 3(1), 74 - 86
The crystal structure of GMP synthetase reveals a novel catalytic triad and is a structural paradigm for two enzyme families; Tesmer JJ et al.; The crystal structure of GMP synthetase serves as a prototype for two families of metabolic enzymes . The Class I glutamine amidotransferase domain of GMP synthetase is found in related enzymes of the purine, pyrimidine, tryptophan, arginine, histidine and folic acid biosynthetic pathways . This domain includes a conserved Cys-His-Glu triad and is representative of a new family of enzymes that use a catalytic triad for enzymatic hydrolysis . The structure and conserved sequence fingerprint of the nucleotide-binding site in a second domain of GMP synthetase are common to a family of ATP pyrophosphatases, including NAD synthetase, asparagine synthetase and argininosuccinate synthetase.

Arterioscler Thromb Vasc Biol, 1996 Jan, 16(1), 64 - 71
Adhesion of blood platelets is inhibited by VCL, a recombinant fragment (leucine504 to lysine728) of von Willebrand factor; Sixma JJ et al.; VCL, fragment Leu504 to Lys728 of von Willebrand factor (vWF) expressed in Escherichia coli, contains the glycoprotein (GP) Ib-binding domain of vWF . This fragment inhibited ristocetin-induced platelet aggregation with an IC50 of 0.2 mumol/L and botrocetin-induced platelet aggregation with an IC50 of 0.08 mumol/L . We studied the antiadhesive profile of VCL by adding it to blood that was circulated over various adhesive surfaces . VCL inhibited adhesion to endothelial cell matrix, which served as a model of the vessel wall . Maximal inhibition at a high shear rate of 1600 s-1 was stronger (60%) than at a low shear rate of 300 s-1 (40%) . Half maximal inhibition was found to be 1.5 mumol/L at both shear rates . The role of various adhesive molecules was investigated in more detail by coating glass coverslips with collagen type I, laminin, fibronectin, or vWF . Fibrinogen was studied as well . Platelet adhesion to laminin and vWF was not inhibited by VCL . Adhesion to collagen, fibronectin, and fibrinogen was particularly inhibited at a high shear rate . VCL coated to a coverslip caused a concentration-dependent adhesion that was blocked by antibodies against GPIb, which block interaction with vWF . Binding studies showed a nonsaturable ristocetin binding of VCL to platelets that was blocked by vWF or inhibitory antibodies against GPIb . Binding to collagen was weak, and VCL did not inhibit binding of vWF at a 5000-fold excess . From these data, we conclude that VCL inhibits adhesion in all cases in which adhesion is vWF dependent by competing for vWF binding to activated GPIb . The lack of inhibition of adhesion to vWF as a single molecule may be explained by assuming that this adhesion is determined by interaction of nonactivated GPIb with vWF that has been changed in conformation by adsorption . Studies investigating thrombus formation on the connective tissue of an atherosclerotic plaque in a human coronary artery showed that VCL was able to partially prevent this thrombus formation . VCL may be of value in preventing adhesion and thrombus formation under conditions in which these processes are dependent on vWF.

Arch Surg, 1996 Jan, 131(1), 44 - 50
Calcium and calmodulin regulate lipopolysaccharide-induced alveolar macrophage production of tumor necrosis factor and procoagulant activity; Lo CJ et al.; BACKGROUND: Alterations in macrophage (M phi) function are responsible, in part, for adult respiratory distress syndrome and multiple organ failure developing in patients with sepsis . Elucidation and control of these M phi mechanisms during sepsis are crucial to our understanding of this disease and, ultimately, to improving survival of these patients . OBJECTIVE: To investigate the involvement of calcium flux in endotoxin-induced alveolar M phi production of tumor necrosis factor (TNF) and procoagulant (PC) activity . DESIGN: Rabbit alveolar M phi obtained by bronchoalveolar lavage were exposed to endotoxin in the form of lipopolysaccharide (LPS) extracted from Escherichia coli 0111:B4 in the presence of different specific calcium agonists and antagonists . The TNF expression was measured in the supernatant by L929 bioassays . The PC activity was determined in cell lysates by a one-step coagulation assay . RESULTS: Macrophages activated by LPS produce enormous levels of TNF and PC . Either W7 (20 mumol/L), a calmodulin antagonist, or TMB-8 (50 mumol/L), which prevents calcium release from the endoplasmic reticulum, inhibited production of both TNF and PC activity . Verapamil (50 mumol/L) alone or combined with TMB-8 significantly inhibited both TNF and PC production by LPS-stimulated M phi . Elevating intracellular calcium ({Ca2+}i), using the calcium ionophore, A23187, or thapsigargin alone, did not induce M phi production of TNF but significantly augmented LPS-stimulated TNF production . CONCLUSION: Our results indicate that increased intracellular calcium causing signal transduction activation through the calmodulin pathway is a necessary, but insufficient, component of the LPS signaling in M phi.

J Surg Oncol, 1996 Jan, 61(1), 42 - 8
Cytosine deaminase gene as a potential tool for the genetic therapy of colorectal cancer; Rowley S et al.; The bacterial enzyme cytosine deaminase (CD) catalyzes the conversion of 5-fluorocytosine (5-FC) to the lethal 5-fluorouracil (5-FU) and so provides a useful system for selective killing of gene-modified mammalian tumor cells . Cloning of the CD gene from Escherichia coli and expression in human tumor cell lines enabled these cells to convert 3H-labeled 5-FC into 3H-5-FU . Two CD-expressing human tumor cell lines (adenocarcinoma cell line KM12 and glioblastoma cell line T1115) became 200-fold more sensitive to 5-FC than the nonexpressing parental cell lines . At least 90% of the cells are killed within 7 days . CD-expressing cells are able to kill nonexpressing cells when grown in the same culture flask (bystander effect) . The CD gene may be used as a suicide system for in situ chemotherapy or as a safety mechanism abrogating the expression of other genes.

J Nucl Med, 1996 Jan, 37(1), 87 - 94
Monitoring gene therapy with cytosine deaminase: in vitro studies using tritiated-5-fluorocytosine; Haberkorn U et al.; Genetically modified mammalian cells that express the cytosine deaminase (CD) gene are able to convert the nontoxic prodrug 5-fluorocytosine (5-FC) to the toxic metabolite 5-fluorouracil (5-FU) . PET with 18F-5-FC may be used for in vivo measurement of CD activity in genetically modified tumors . METHODS: A human glioblastoma cell line was stably transfected with the Escherichia coli CD gene . After incubation of lysates of CD-expressing cells and control cells with 3H-5-FC high-performance liquid chromatography (HPLC) was performed . The uptake of 5-FC was measured after various incubation times using therapeutic amounts of 5-FC . In addition, saturation and competition experiments with 5-FC and 5-FU were performed . Finally, the efflux was measured . RESULTS: We found that 3H-5-FU was produced in CD-expressing cells, whereas in the control cells only 3H-5-FC was detected . Moreover, significant amounts of 5-FU were found in the medium of cultured cells, which may account for the bystander effect observed in previous experiments . However, uptake studies revealed a moderate and nonsaturable accumulation of radioactivity in the tumor cells, suggesting that 5-FC enters the cells only through diffusion . Although a significant difference in 5-FC uptake was seen between CD-positive and control cells after 48 hr of incubation, no difference was observed after 2 hr of incubation . Furthermore, a rapid efflux could be demonstrated . CONCLUSION: 5-Fluorocytosine transport may be a limiting factor for this therapeutic procedure . Quantitation with PET has to rely more on dynamic studies and modeling, including HPLC analysis of the plasma, than on nonmodeling approaches.

Am J Respir Crit Care Med, 1996 Jan, 153(1), 391 - 7
Effect of a specific neutrophil elastase inhibitor, ONO-5046, on endotoxin-induced acute lung injury; Sakamaki F et al.; Because excessive neutrophil elastase (NE) activity is involved in the pathogenesis of acute lung injury, we speculated that administering anti-NE might prevent lung injury . In a guinea pig model of acute lung injury induced by Escherichia coli endotoxin (lipopolysaccharide {LPS}), we investigated the effect of ONO-5046, a low-molecular-weight and specific inhibitor of NE . ONO-5046 produced concentration-dependent inhibition of guinea pig NE, whereas there were no inhibitory effects on neutrophil chemotaxis or the expression of adhesion molecules in endothelial cells . Detectable NE activity in bronchoalveolar lavage (BAL) fluid was present in the LPS-alone group . No NE activity in BAL fluid was detected in the LPS+ONO-5046 groups . Neutrophil counts in BAL fluid, the lung tissue wet to dry weight ratio, and the lung tissue or BAL fluid to plasma ratio of 125I-albumin were increased in the LPS-alone group as compared with the saline group (p < 0.05) . In the LPS+ONO-5046 group, neutrophil counts in BAL fluid, the lung tissue wet to dry weight ratio and BAL fluid to plasma ratio of 125I-albumin were decreased as compared with the LPS-alone group (p < 0.05) . These data suggest that ONO-5046 can attenuate LPS-induced acute lung injury.

Radiat Res, 1996 Jan, 145(1), 24 - 30
Rejoining of gamma-radiation-induced single-strand breaks in plasmid DNA by human cell extracts: dependence on the concentration of the hydroxyl radical scavenger, Tris; Hodgkins PS et al.; The rejoining of single-strand breaks induced by gamma irradiation in plasmid DNA under different scavenging conditions is described using human cell extracts . As the scavenging capacity of the irradiated solution increases from 1.5 x 10(7) to 3 x 10(8) s-1 using Tris-HCl as a scavenger, the ratio of single- to double-strand breaks is reduced from approximately 70:1 to 40:1 . After irradiation, a proportion of DNA molecules have no initial strand breaks but contain damage that is converted to strand breaks when incubated either at 37 degrees C or in the presence of cellular extract . Repair of damage by the extracts is dependent upon the scavenging capacity of the irradiated solution . Optimal rejoining is observed when the scavenging capacity is < 1.5 x 10(7) s-1, and results in the repair of some initial strand breaks . As the scavenging capacity increases to 3 x 10(8) s-1 the proportion of breaks repaired is significantly reduced . The relative increase in the yield of double-strand breaks and reduced repairability of single-strand breaks at a scavenging capacity of 3 x 10(8) s-1 is consistent with the concept that the severity of damage increases upon increasing the scavenger concentration.

J Acquir Immune Defic Syndr Hum Retrovirol, 1996 Jan 1, 11(1), 20 - 30
Recombinant human immunodeficiency virus type 1 reverse transcriptase is heterogeneous; Wilson JE et al.; Recombinant wild type (wt) and T215Y HIV-1 reverse transcriptase (RT) were isolated using three methods designated A, B, and C . The three samples of wt RT were kinetically indistinguishable with respect to dTTP turnover on poly(rA).p(dT)10 . However, whereas the kinetic constants for dTTP and AZTTP for both T215Y B and T215Y C were similar to those of wt protein, T215Y A exhibited a twofold increase in Km value for dTTP and a 13-fold increase in Ki value for AZTTP with respect to wt protein purified in the same manner . We further investigated this observation by studying the denaturation of wt RT by urea . The urea denaturation curves monitored by fluorescence and circular dichroism spectroscopy were not coincident with the denaturation curve monitored by enzyme activity and yielded Cm values (the concentration of urea at which 50% of the protein is denatured) of 4.1 and 2.0 M urea, respectively . The noncoincidence of the transition curves reflects two separable, sequential, noncooperative conformational changes in the molecule: (a) from a catalytically active to an inactive conformation, and (b) from a catalytically inactive to a denatured, unfolded conformation . We therefore used denaturation as detected by changes in enzyme activity to compare the conformational stability of the three samples of wt and T215Y RT A, B, and C . The Cm values for T215Y RT did not differ from those of the respective wt; however, differences in Cm values were noted depending on how the protein was isolated . This suggested that the heterogeneity of the recombinant RT was due to small differences in conformation at or near the active site.

J Virol, 1996 Jan, 70(1), 658 - 62
Phosphorylation and nuclear localization of the varicella-zoster virus gene 63 protein; Stevenson D et al.; The protein encoded by varicella-zoster virus open reading frame 63 and carboxy-terminal deletions of the same were expressed either as fusion proteins at the carboxy terminus of the maltose-binding protein in Escherichia coli or independently in transfected mammalian cells . The truncations contained amino acids 1 to 142 (63 delta N) or 1 to 210 (63 delta K) of the complete 278-amino-acid primary sequence . Recombinant casein kinase II phosphorylated the 63F and 63 delta KF fusion proteins in vitro but did not phosphorylate the 63 delta NF fusion protein, implying that phosphorylation occurred between amino acids 142 and 210 . Immunoprecipitation of 35S- or 32P-labelled extracts of cells transfected with plasmids expressing 63, 63 delta N, or 63 delta K also indicated that in situ phosphorylation most likely occurred between amino acids 142 and 210 . These combined results suggest that casein kinase II plays a significant role in the phosphorylation of the varicella-zoster virus 63 protein . Indirect immunofluorescence of transfected cells indicated nuclear localization of the 63 protein and cytoplasmic localization of 63 delta K and 63 delta N, implying a requirement for sequences between amino acids 210 and 278 for efficient nuclear localization.

J Virol, 1996 Jan, 70(1), 533 - 40
Identification of a minimal hydrophobic domain in the herpes simplex virus type 1 scaffolding protein which is required for interaction with the major capsid protein; Hong Z et al.; Recent biochemical and genetic studies have demonstrated that an essential step of the herpes simplex virus type 1 capsid assembly pathway involves the interaction of the major capsid protein (VP5) with either the C terminus of the scaffolding protein (VP22a, ICP35) or that of the protease (Pra, product of UL26) . To better understand the nature of the interaction and to further map the sequence motif, we expressed the C-terminal 30-amino-acid peptide of ICP35 in Escherichia coli as a glutathione S-transferase fusion protein (GST/CT) . Purified GST/CT fusion proteins were then incubated with 35S-labeled herpes simplex virus type 1-infected cell lysates containing VP5 . The interaction between GST/CT and VP5 was determined by coprecipitation of the two proteins with glutathione Sepharose beads . Our results revealed that the GST/CT fusion protein specifically interacts with VP5, suggesting that the C-terminal domain alone is sufficient for interaction with VP5 . Deletion analysis of the GST/CT binding domain mapped the interaction to a minimal 12-amino-acid motif . Substitution mutations further revealed that the replacement of hydrophobic residues with charged residues in the core region of the motif abolished the interaction, suggesting that the interaction is a hydrophobic one . A chaotropic detergent, 0.1% Nonidet P-40, also abolished the interaction, further supporting the hydrophobic nature of the interaction . Computer analysis predicted that the minimal binding motif could form a strong alpha-helix structure . Most interestingly, the alpha-helix model maximizes the hydropathicity of the minimal domain so that all of the hydrophobic residues are centered around a Phe residue on one side of the alpha-helix . Mutation analysis revealed that the Phe residue is absolutely critical for the binding, since changes to Ala, Tyr, or Trp abrogated the interaction . Finally, in a peptide competition experiment, the C-terminal 25-amino-acid peptide, as well as a minimal peptide derived from the binding motif, competed with GST/CT for interaction with VP5 . In addition, a cyclic analog of the minimal peptide which is designed to stabilize an alpha-helical structure competed more efficiently than the minimal peptide . The evidence suggests that the C-terminal end of ICP35 forms an alpha-helical secondary structure, which may bind specifically to a hydrophobic pocket in VP5.

J Virol, 1996 Jan, 70(1), 37 - 46
Directed integration of viral DNA mediated by fusion proteins consisting of human immunodeficiency virus type 1 integrase and Escherichia coli LexA protein; Goulaouic H et al.; We tested whether the selection of target sites can be manipulated by fusing retroviral integrase with a sequence-specific DNA-binding protein . A hybrid protein that has the Escherichia coli LexA protein fused to the C terminus of the human immunodeficiency virus type 1 integrase was constructed . The fusion protein, IN1-288/LA, retained the catalytic activities in vitro of the wild-type human immunodeficiency virus type 1 integrase (WT IN) . Using an in vitro integration assay that included multiple DNA fragment as the target DNA, we found that IN1-288/LA preferentially integrated viral DNA into the fragment containing a DNA sequence specifically bound by LexA protein . No bias was observed when the LexA-binding sequence was absent, when the fusion protein was replaced by WT IN, or when LexA protein was added in the reaction containing IN1-288/LA . A majority of the integration events mediated by IN1-288/LA occurred within 30 bp of DNA flanking the LexA-binding sequence . The specificity toward the LexA-binding sequence and the distribution and frequency of target site usage were unchanged when the integrase component of the fusion protein was replaced with a variant containing a truncation at the N or C terminus or both, suggesting that the domain involved in target site selection resides in the central core region of integrase . The integration bias observed with the integrase-LexA hybrid shows that one effective means of altering the selection of DNA sites for integration is by fusing integrase to a sequence-specific DNA-binding protein.

J Virol, 1996 Jan, 70(1), 241 - 7
Mechanism for inhibition of influenza virus RNA polymerase activity by matrix protein; Watanabe K et al.; Influenza virus M1 protein has been shown to inhibit the transcription catalyzed by viral ribonucleoprotein complexes isolated from virions . Here, this inhibition mechanism was studied with the recombinant M1 protein purified from Escherichia coli expressing it from cDNA . RNA mobility shift assays indicated that both soluble and aggregate forms of the recombinant M1, which were separated by the glycerol density gradient, were bound to RNA . Once an M1-RNA complex was formed, free M1 was bound to the M1-RNA complex cooperatively rather than to free RNA . In addition, the recombinant M1 was capable of binding to preformed RNA-nucleocapsid protein complexes . The mechanism for inhibition of the viral RNA polymerase activity was analyzed by the in vitro RNA synthesis systems that depend on an exogenously added RNA template . These systems were more sensitive for evaluating the inhibition by M1 than the RNA synthesis system depending on an endogenous RNA template . The RNA synthesis inhibition was examined at four steps: cleavage of capped RNA; incorporation of the first nucleotide, GMP; limited elongation; and synthesis of full-size product . M1 inhibited RNA synthesis mainly at the early steps . The experiments with M1 mutant proteins containing amino acid deletions suggested that the M1 region between amino acid residues 91 and 111 was essential for anti-RNA synthesis activity, RNA binding, and oligomerization of M1 on RNA.

J Virol, 1996 Jan, 70(1), 127 - 32
Identification of the sequence on NS4A required for enhanced cleavage of the NS5A/5B site by hepatitis C virus NS3 protease; Shimizu Y et al.; In addition to NS3 protease, the NS4A protein is required for efficient cleavage of the nonstructural protein region of the hepatitis C virus polyprotein . To investigate the function and the sequence of NS4A required for the enhancement of NS3 protease activity, we developed an in vitro NS3 protease assay system consisting of three purified viral elements: (i) a recombinant NS3 protease which was expressed in Escherichia coli as a maltose-binding protein-NS3 fusion protein (MBP-NS3), (ii) synthetic NS4A fragments, and (iii) a synthetic peptide substrate which mimics the NS5A/5B junction . We showed that the NS3 protease activity of MBP-NS3 was enhanced in a dose-dependent manner by 4A18-40, which is a peptide composed of amino acid residues 18 to 40 of NS4A . The optimal activity was observed at a 10-fold molar excess of 4A18-40 over MBP-NS3 . The coefficient for proteolytic efficiency, kcat/Km, of NS3 protease was increased by about 40 times by the addition of a 10-fold molar excess of 4A18-40 . Using a series of truncations of 4A18-40, we estimated that amino acid residues 22 to 31 in NS4A (SVVIVGRIIL) constituted the core sequence for the effector activity . Single-substitution experiments with 4A21-34, a peptide composed of amino acid residues 21 to 34 of NS4A, suggested the importance of several residues (Val-23, Ile-25, Gly-27, Arg-28, Ile-29, and Leu-31) for its activity . In addition, we found that some single-amino-acid substitutions in 4A21-34 were able to inhibit the enhancement of NS3 protease activity by 4A18-40 . This approach has potential as a novel strategy for inhibiting the NS3 protease activity important for hepatitis C virus proliferation.

J Neurochem, 1996 Jan, 66(1), 20 - 5
3.6 kb of the 5' flanking DNA activates the mouse tyrosine hydroxylase gene promoter without catecholaminergic-specific expression; Morgan WW et al.; The tyrosine hydroxylase (TH) gene is expressed exclusively in cells and neurons that synthesize and release L-DOPA or catecholamines . To further understand the molecular genetic mechanisms that regulate this cell-type specific expression, a chimeric gene was prepared by linking 3.6 kb of the 5' flanking DNA of the mouse TH gene, including the +1 initiation site for transcription, to an E . coli beta-galactosidase reporter . This fusion gene (TH3.6LAC) was used to prepare transgenic mice, and the tissue distribution of expression of TH3.6LAC was determined by the measurement of beta-galactosidase enzymatic activity and/or by the detection of the transcription product of the chimeric gene by RNase protection assays . In two separate founder lines, TH3.6LAC expression was observed in every region of the brain that was examined, including the olfactory bulb, brainstem, cerebellum, diencephalon, hippocampus, striatum, and cerebral cortex . Expression of TH3.6LAC was observed in the adrenal gland of one founder line but not in the other . TH3.6LAC activation was undetectable in peripheral organs that were examined, including the liver, heart, salivary gland, kidney, lung, and spleen . Although 3.6 kb of the 5' regulatory DNA of the mouse TH gene is sufficient to activate the TH fusion gene in the mouse, it is not enough to restrict its expression to catecholaminergic cells.

DNA Res, 1995 Dec 31, 2(6), 239 - 46
Computer survey for likely genes in the one megabase contiguous genomic sequence data of Synechocystis sp . strain PCC6803; Hirosawa M et al.; Using the computer program GeneMark, the open reading frames (ORFs) previously assigned within the one megabase sequence data of the genome of the cyanobacterium, Synechocystis sp . strain PCC6803 (Kaneko et al., DNA Res . 2: 153-166, 1995), were re-examined . Matrices required by GeneMark for its statistical calculation were generated and modified by running a script termed GeneMark-Genesis that performed recursive application of GeneMark against the Synechocystis data and evaluated the probability scores for optimization . Based on the matrices thus generated, 752 of the 818 previously assigned ORFs (92%) were supported by GeneMark as likely coding sequences, of which 26 were predicted to start at more internal positions than previously assigned . In addition, 50 ORFs were newly identified as likely coding sequences, most of them being shorter than 300 bp . Thus, the procedure was proven to be very powerful to locate likely coding regions within the genomic sequence data of Synechocystis without having prior information concerning their similarity to the genes of other organisms . However, GeneMark did not predict 66 previously assigned ORFs as likely genes: 14 of them showed significant degrees of similarity to known genes and 10 others were found within IS-like elements . It seems that these genes, many of which appear to be exogenous origin, escaped detection by GeneMark as in the case of "class 3 (horizontally transferred) genes" of E . coli, which in turn suggests that genes of different phylogenetic origins might also be detected as such by modifying the matrices.

J Neuroimmunol, 1995 Dec 31, 63(2), 149 - 56
T cell antigenic and neuritogenic activity of recombinant human peripheral myelin P2 protein; Weishaupt A et al.; The major neuritogenic protein of peripheral nerve myelin is the P2 protein . Human P2 is a candidate autoantigen in inflammatory demyelinating diseases of the peripheral nervous system . Since human P2 is not readily available, we produced full-length recombinant human P2 protein (rhP2) in Escherichia coli . RhP2 was recognized by neuritogenic rat T cell lines and induced experimental autoimmune neuritis in Lewis rats . Production of rhP2 allowed the generation of human T cell lines reactive to the autologous protein . Studies of human T cell autoreactivity as well as efforts to use hP2 as a tolerogen will be facilitated by the large-scale expression of rhP2.

Neuroreport, 1995 Dec 29, 7(1), 57 - 60
Analysis of regulatory regions of the ciliary neutrophic factor gene in transgenic mice; Stefanuto G et al.; In order to study the regulatory regions of the human ciliary neurotrophic factor (CNTF) gene we made constructs containing sequences upstream and downstream of CNTF coding regions and the lacZ gene and analysed their expression in transgenic mice . We show that 240 bp upstream of the translation start codon are sufficient for the transcription of the lacZ gene . A further 4 kb upstream sequence is required for the expression of the transgene in Schwann cells . These two upstream regions together with a 2 kb downstream fragment drive high level of expression of the lacZ gene in the sciatic nerve . Our results indicate that these three fragments contain regulatory regions able to mimic the CNTF expression pattern in the mouse peripheral nervous system.

Ann N Y Acad Sci, 1995 Dec 29, 774, 59 - 72
Human dehydroepiandrosterone sulfotransferase . Purification, molecular cloning, and characterization; Falany CN et al.; Human tissues possess at least four distinct forms of cytosolic ST, three of which are involved in the sulfation of steroids . DHEA-ST is responsible for the majority of hydroxysteroid and bile acid sulfation in human tissues and abundant levels of the enzyme are present in human liver and adrenal tissues . In the adult human adrenal, DHEA-ST has been localized immunologically to the zona reticularis of the adrenal cortex . No age- or gender-related differences in the expression of DHEA-ST activity in adult human liver cytosols have been reported . The cDNA encoding DHEA-ST has been isolated from a human liver cDNA library and expressed in both mammalian COS cells and E . coli . Purification and molecular characterization studies suggest a single form of DHEA-ST in human tissues . The properties of DHEA-ST expressed in either mammalian or bacterial cells are very similar to those of the native enzyme . DHEA-ST can also bioactivate a number of procarcinogens to reactive electrophilic forms . Hydroxymethyl PAHs are sulfated and bioactivated at a relatively rapid rate by DHEA-ST, whereas 1'-hydroxysafrole and N-hydroxy-2-acetylaminofluorene are bioactivated to a lesser extent.

Gene, 1995 Dec 29, 167(1-2), 9 - 15
The argG gene of Streptomyces clavuligerus has low homology to unstable argG from other actinomycetes: effect of amplification on clavulanic acid biosynthesis; Rodriguez-Garcia A et al.; The argG gene of Streptomyces clavuligerus (Scl) has been cloned by complementation of argG mutants of Escherichia coli and S . lividans (Sl) . The argG nucleotide (nt) sequence showed that it corresponds to a new type of argG different from the corresponding genes of S . coelicolor (Sco) and Sl . It encodes a 43,250-Da protein that showed higher similarity to argininosuccinate synthetases (ASS) from Methanococcus vannielii and Methanosarcina barkeri than to ASS deduced from other Streptomyces argG . No hybridization of the Scl argG was found with the homologous genes of Sl or Sco . The argH gene was located downstream from argG in Scl . The genomic region around argG and argH in Scl was different from the homologous regions in other Streptomyces and is not genetically unstable, unlike in Sco and Sl . Amplification of argG in transformant Scl{pULAR113} results in a 2.3-fold increase in the production of clavulanic acid (CA) in relation to the control strain Scl{pIJ699}.

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