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