Biosci Biotechnol Biochem, 1998 Sep, 62(9), 1809 - 11 Expression and purification of cytokine receptor homology domain of human granulocyte-colony stimulating factor receptor in Escherichia coli; Tanaka R et al.; In an attempt to generate a stable non-glycosylated cytokine receptor homology (CRH) domain (Tyr97-Ala309) of human granulocyte-colony stimulating factor (G-CSF) receptor, two free cysteines in the CRH domain were converted to serine by site-directed mutagenesis . Taking advantage of the tight regulation for the expression of T7 RNA polymerase, the mutated CRH domain was successfully expressed in Escherichia coli (E . coli) with a pelB signal sequence at its NH2-terminus and with a His tag at its COOH-terminus . The processed and secreted CRH domain after solubilization and in vitro refolding retained G-CSF binding activity, and its yield (approximately 40 micrograms/30 ml culture) was more than 100-fold higher than that of the mouse CRH domain expressed by the MalE fusion system in E . coli. Biosci Biotechnol Biochem, 1998 Sep, 62(9), 1795 - 8 Cloning, sequencing, and expression of an endoglucanase gene from the rumen anaerobic fungus Neocallimastix frontalis MCH3; Fujino Y et al.; A cDNA clone encoding an endo-1,4-beta-glucanase from a rumen fungus, Neocallimastix frontalis MCH3, was isolated . The nucleotide sequence showed that the gene, celA, encoded a multidomain enzyme containing a family 5 catalytic domain and a reiterated sequence that is involved in the association of a multienzyme complex, the cellulosome . The enzyme expressed in Escherichia coli showed the highest activity against carboxymethylcellulose at 40 degrees C and pH 8.5. Biosci Biotechnol Biochem, 1998 Sep, 62(9), 1731 - 8 Molecular cloning and sequence analysis of two endoinulinase genes from Aspergillus niger; Ohta K et al.; Two genomic DNAs encoding endoinulinase from Aspergillus niger 12 were cloned and sequenced . Open reading frames (ORFs) of the endoinulinase genes, inuA and inuB, both consisted of 1,548 nucleotides encoding 516 amino acids . It was suggested that the coding regions were not interrupted by introns . The ORFs differed from each other by 23 nucleotide substitutions or by eight amino acid replacements, indicating that the inuA and inuB genes arose by gene duplication . Each mature enzyme of 493 amino acids was preceded by a hydrophobic signal peptide of 23 amino acids . The enzymes contained two Cys residues and five potential sites for N-linked glycosylation . Partial amino acid sequences of the secreted enzyme suggested that it originated from the inuB gene product . The deduced amino acid sequences of the mature A . niger enzymes showed 73% identity with that of the Penicillium purpurogenum endoinulinase. Biochim Biophys Acta, 1998 Nov 10, 1376(3), 455 - 66 Genetic analysis of lipid-protein interactions in Escherichia coli membranes; Dowhan W; Phospholipids play essential roles in defining the membrane permeability barrier, in regulating cellular processes, in providing a support for organization of many membrane-associated processes, and in providing precursors for the synthesis of macromolecules . Although in vitro experiments have provided important information on the role of protein-lipid interactions in cell function, such approaches are limited by the lack of a direct measure for phospholipid function . Genetic approaches can provide direct evidence for a specific role for phospholipids in cell function provided cell viability or membrane structure is not compromised . This review will summarize recent genetic approaches that when coupled with biochemical studies have led to a better understanding of specific functions for phospholipids at the molecular level. Biochim Biophys Acta, 1998 Nov 8, 1442(2-3), 409 - 14 A gene cluster for 6-deoxy-L-talan synthesis in Actinobacillus actinomycetemcomitans; Nakano Y et al.; The serotype c antigen of Actinobacillus actinomycetemcomitans consists of 6-deoxy-l-talose . A gene cluster involved in the synthesis of serotype-specific polysaccharide antigen was cloned from the chromosomal DNA of A . actinomycetemcomitans NCTC 9710 (serotype c) . This cluster consisted of 17 open reading frames . Escherichia coli produced the polysaccharide that reacts with the serotype c-specific antibody when transformed with a plasmid containing the cluster . Comparing the structure of the gene cluster with a similar cluster from A . actinomycetemcomitans Y4 (serotype b), which produces a polysaccharide consisting of l-rhamnose and d-fucose, revealed that a 5.7 kb region containing seven genes in the cluster from strain Y4 was replaced by a 3.8 kb region containing three genes in strain NCTC 9710 . The results suggest that these region, as well as dTDP-6-deoxy-l-talose-forming dTDP-4-keto-l-rhamnose reductase, is essential to the production of extracellular polysaccharide specific to serotype c. Biochim Biophys Acta, 1998 Nov 8, 1442(2-3), 347 - 52 Characterization of a GTP-binding protein in the ADP-ribosylation factor subfamily from Leishmania tarentolae; Sturm NR et al.; We report the cloning and characterization of a gene, LtARL, which encodes a small GTP-binding protein, from the protozoan Leishmania tarentolae . Hybridization analysis of genomic DNA under high stringency conditions indicates the single-copy nature of LtARL . LtARL is transcribed and yields a approximately 0.9 kb mRNA that is processed at the 5' end by trans-splicing . When expressed in Escherichia coli, LtArl binds GTP with a low stoichiometry and in a phospholipid-independent manner . Based on the greatest sequence identity with Homo sapiens Arl3 and lipid-independent binding of guanine nucleotides we designate this gene LtARL and the encoded protein LtArl. Biochim Biophys Acta, 1998 Nov 8, 1442(2-3), 296 - 303 Specific recognition of the cyanobacterial psbA promoter by RNA polymerases containing principal sigma factors; Shibato J et al.; The psbA2 gene of a unicellular cyanobacterium, Microcystis aeruginosa K-81, encodes a D1 protein homolog in the reaction center of photosynthetic Photosystem II . To clarify the promoter recognition by a sigma factor of RNA polymerase, in vivo and in vitro analyses were performed for the photosynthetic gene . Although the specific transcript from the psbA2 promoter, whose sequence is of Escherichia coli consensus type, was observed in both cyanobacterium K-81 and E . coli cells, the expression was light-dependent in K-81 whereas it was constitutive in E . coli under the conditions of light and darkness (L/D) . The specific psbA2-dependent transcripts were also detected in vitro by RNA polymerases containing the principal sigma factors, E . coli sigma70 and K-81 sigmaA1 (constitutively exists in K-81 grown under L/D cycles) . Furthermore, a series of promoter fragments were constructed to confirm minimal cis elements for the in vitro psbA2 transcription . A -80 to +6 or -38 to +46 region, the sequences of which consisted of a core promoter (-38 to +6), was identified as the potential minimal cis element using the RNA polymerase fraction (*EsigmaA1) containing sigmaA1 partially purified from K-81 . These results suggest that the psbA2 transcription with the minimal sequence was induced by the RNA polymerase (EsigmaA1) containing the principal sigma factor, sigmaA1, under both light and dark conditions in K-81. Biochim Biophys Acta, 1998 Nov 8, 1442(2-3), 245 - 51 Down-regulation of CCAAT/enhancer binding proteins alpha, beta and delta in adipose tissue by intracerebroventricular leptin in rats; Qian H et al.; In our previous report, intracerebroventricular (i.c.v.) administration of leptin caused fat depletion by an induced adipocyte apoptosis in addition to influencing lipid metabolism . To uncover the biochemical mechanisms that mediate this response, the present study was designed to determine whether CCAAT/enhancer binding proteins (C/EBP)alpha, -beta and -delta play a role in the leptin-induced fat depletion . Expressions of C/EBPalpha, -beta and -delta in epididymal fat tissues were examined by Western immunoblot and in situ immunocytochemical analysis after 5 days of i.c.v . treatment . Young and old rats (3 and 8 months old) were treated with or without 5 micrograms/day leptin . The expression of C/EBPalpha, -beta and -delta was decreased by i.c.v . leptin treatment in young rats as compared with controls (P<0.05) . However, leptin did not influence the expression of C/EBPalpha, -beta and -delta in adipose tissues of 8-month-old rats . The basal level of expression of C/EBPbeta was greater in 8-month-old rats than in 3-month-old rats, (P<0.05) whereas the basal expression of C/EBPalpha and -delta was not different between age groups . These results were confirmed by in situ immunocytochemical analysis . The present study suggests that leptin-induced down-regulation of C/EBPalpha, -beta and -delta might influence adipocyte differentiation and growth in a number of ways. Brain Res, 1998 Nov 16, 811(1-2), 156 - 60 Ambiguus nucleus neurons innervating the abdominal esophagus are malpositioned in the reeler mouse; Fujimoto Y et al.; To examine whether the migration of ambiguus nucleus (NA) neurons is affected in the reeler mouse, recombinant replication-deficient adenoviral vector carrying E . coli-galactosidase gene (lacZ) was injected into the abdominal esophagus of the reeler mouse and normal control at two months of age prior to 5 days of sacrifice of the animals . In the normal control, lacZ-positive neurons were found in the compact formation of the NA, whereas, in the reeler, they were scattered from the base of the fourth ventricle to the ventro-lateral margin of the medulla . The present study confirmed that NA neurons are malpositioned in the reeler mouse, suggesting that the migration of NA neurons is guided by the reelin-related protein (Reelin) . J Biol Chem, 1998 Nov 13, 273(46), 30750 - 6 Regulation of the cyanide-resistant alternative oxidase of plant mitochondria . Identification of the cysteine residue involved in alpha-keto acid stimulation and intersubunit disulfide bond formation; Rhoads DM et al.; The cyanide-resistant alternative oxidase of plant mitochondria is a homodimeric protein whose activity can be regulated by a redox-sensitive intersubunit sulfhydryl/disulfide system and by alpha-keto acids . After determining that the Arabidopsis alternative oxidase possesses the redox-sensitive sulfhydryl/disulfide system, site-directed mutagenesis of an Arabidopsis cDNA clone was used to individually change the two conserved Cys residues, Cys-128 and Cys-78, to Ala . Using diamide oxidation and chemical cross-linking of the protein expressed in Escherichia coli, Cys-78 was shown to be: 1) the Cys residue involved in the sulfhydryl/disulfide system; and 2) not required for subunit dimerization . The C128A mutant was stimulated by pyruvate, while the C78A mutant protein had little activity and displayed no stimulation by pyruvate . Mutating Cys-78 to Glu produced an active enzyme which was insensitive to pyruvate, consistent with alpha-keto acid activation occurring through a thiohemiacetal . These results indicate that Cys-78 serves as both the regulatory sulfhydryl/disulfide and the site of activation by alpha-keto acids . In light of these results, the previously observed effects of sulfhydryl reagents on the alternative oxidase of isolated soybean mitochondria were re-examined and were found to be in agreement with a single sulfhydryl residue being the site both of alpha-keto acid activation and of the regulatory sulfhydryl/disulfide system. J Biol Chem, 1998 Nov 13, 273(46), 30568 - 75 Molecular cloning and functional expression of a water-soluble chlorophyll protein, a putative carrier of chlorophyll molecules in cauliflower; Satoh H et al.; A cDNA for a water-soluble chlorophyll (Chl) protein (WSCP) from cauliflower (Brassica oleracea L . var botrys) was cloned and sequenced . The cDNA contained an open reading frame encoding 19 residues for a signal peptide and 199 residues for the mature form of WSCP . The sequence showed extensive homology to drought-stress-related, 22-kDa proteins in some Brassicaceae plants . Functional WSCP was expressed in Escherichia coli as a fusion protein with a maltose-binding protein (MBP) . When the recombinant MBP-WSCP was incubated with thylakoid membranes, the MBP-WSCP removed Chls from these membranes . During this process, the monomer of the apo-MBP-WSCP successfully bound Chls and was converted into tetrameric holo-MBP-WSCP . The reconstituted MBP-WSCP exhibited absorption and fluorescent spectra identical to those of the native WSCP purified from cauliflower leaves . The Chl a/b ratio in native WSCP indicates a high content of Chl a, which was mainly due to the higher affinity of MBP-WSCP for Chl a . WSCP is the first example of a hydrophilic protein that can transfer Chls from thylakoid hydrophobic proteins . Possible functions of WSCP are discussed. J Biol Chem, 1998 Nov 13, 273(46), 30415 - 8 Membrane topology of the 60-kDa Oxa1p homologue from Escherichia coli; Saaf A et al.; We have characterized the membrane topology of a 60-kDa inner membrane protein from Escherichia coli that is homologous to the recently identified Oxa1p protein in Saccharomyces cerevisiae mitochondria implicated in the assembly of mitochondrial inner membrane proteins . Hydrophobicity and alkaline phosphatase fusion analyses suggest a membrane topology with six transmembrane segments, including an N-terminal signal-anchor sequence not present in mitochondrial Oxa1p . In contrast to partial N-terminal fusion protein constructs, the full-length protein folds into a protease-resistant conformation, suggesting that important folding determinants are present in the C-terminal part of the molecule. J Biol Chem, 1998 Nov 13, 273(46), 30279 - 86 Lipid products of phosphoinositide 3-kinase interact with Rac1 GTPase and stimulate GDP dissociation; Missy K et al.; A number of reports suggest that under different conditions leading to cytoskeleton reorganization the GTPase Rac1 and possibly RhoA are downstream targets of phosphoinositide 3-kinase (PI 3-kinase) . In order to gain more insight into this particular signaling pathway, we have addressed the question of a possible direct interaction of PI 3-kinase products with the Rho family GTPases RhoA, Rac1, and Cdc42 . Using recombinant proteins, we found that Rac1 and, to a lesser extent, RhoA but not Cdc42 were capable to selectively bind to phosphatidylinositol 3,4,5-trisphosphate (PtdIns(3,4,5)P3) in a mixture of crude brain phosphoinositides . Nucleotide-depleted Rac1 was the most efficient, but the GDP- and GTP-bound forms retained significant PtdIns(3,4,5)P3 binding activity . This protein-lipid association involved electrostatic as well as hydrophobic interactions, since both phosphate groups located at specific positions of the inositol ring and fatty-acyl chains were absolutely required . Based on the sequence of Rac1, two potential binding sites were identified, one at the C terminus and one in the extra alpha-helical domain . Deletion of these two domains resulted in a complete loss of binding to PI 3-kinase products . Finally, PtdIns(3, 4,5)P3 strongly stimulated GDP dissociation from Rac1 in a dose-dependent manner . In agreement, data obtained in intact cells suggest that PtdIns(3,4,5)P3 might target Rac1 to peculiar membrane domains, allowing formation of specific clusters containing not only small GTPases but other partners bearing pleckstrin homology domains such as specific exchange factors required for Rac1 and RhoA activation. J Biol Chem, 1998 Nov 13, 273(46), 30232 - 8 Reversible induction of ATP synthesis by DNA damage and repair in Escherichia coli . In vivo NMR studies; Dahan-Grobgeld E et al.; Early metabolic events in Escherichia coli exposed to nalidixic acid, a topoisomerase II inhibitor and an inducer of the SOS system, were investigated by in vivo NMR spectroscopy, a technique that permits monitoring of bacteria under controlled physiological conditions . The energetics of AB1157 (wild type) and of its isogenic, SOS-defective mutants, recBC, lexA, and DeltarecA, were studied by 31P and 19F NMR before, during, and after exposure to nalidixic acid . The content of the NTP in E . coli embedded in agarose beads and perfused at 36 degreesC was found to be 4.3 +/- 1.1 x 10(-18) mol/cell, yielding a concentration of approximately 2.7 +/- 0.7 mM . Nalidixic acid induced in the wild type and mutants a rapid 2-fold increase in the content of the NTP, predominantly ATP . This induction did not involve synthesis of uracil derivatives or breakdown of RNA and caused cell proliferation to stop . Removal of nalidixic acid after 40 min of treatment rescued the cells and resulted in a decrease of ATP to control levels and resumption of proliferation . However, in DeltarecA cells, which were more sensitive to the activity of the drug, ATP elevation could not be reversed, and ATP content continued to increase faster than in control cells . The results ruled out association between the elevation of ATP and the induction of the SOS system and suggested involvement of a process reminiscent of apoptosis in the stimulation of ATP synthesis . Thus, the presence of the RecA protein was found to be essential for reversing the ATP increase and cell rescue, possibly by its function in repair of DNA damage. J Med Chem, 1998 Nov 5, 41(23), 4550 - 5 Synthesis and enzymic evaluation of 4-mercapto-6-oxo-1, 4-azaphosphinane-2-carboxylic acid 4-oxide as an inhibitor of mammalian dihydroorotase; Manthey MK et al.; The design, synthesis, and enzymic evaluation of cis- and trans-4-mercapto-6-oxo-1,4-azaphosphinane-2-carboxylic acid 4-oxide 5 against mammalian dihydroorotase is presented . The design strategy for 5 was based on the strong affinity of phosphinothioic acids for zinc and that 5 also resembles the postulated tetrahedral transition state for the enzyme-catalyzed reaction . The synthesis of 5 utilized a novel protection/deprotection sequence upon 4-hydroxy-6-oxo-1, 4-azaphosphinane-2-carboxylic acid 4-oxide 4, followed by incorporation of alpha-phenyl benzenemethanethiol and exhaustive deprotection to afford 5 in 40% overall yield from 4 . The activities of both isomers of 5 as inhibitors of mammalian dihydroorotase were marginally greater than that of the parent phosphinic acid 4, indicating a weak binding enhancement due to the phosphinothioic acid moiety. Metab Brain Dis, 1998 Sep, 13(3), 211 - 23 DNA fragmentation and HSP72 gene expression by adenovirus-mediated gene transfer in postischemic gerbil hippocampus and ventricle; Kitagawa H et al.; A replication defective adenoviral vector containing the E . coli lacZ gene (AdCMVnLacZ) was directly injected into right hippocampus and lateral ventricle immediately after 5 min of transient global ischemia in gerbils . The relations between the lacZ gene expression and DNA fragmentation or heat shock protein 72 (HSP72) immunoreactivity were examined up to 21 days post ischemia . The lacZ gene was transiently expressed at 1 day in the hippocampus except around the CA1 region, while a large number of the periventricular cells strongly expressed the lacZ gene from 8 h to 7 days . In CA1 layer, terminal deoxynucleotidyl dUTP nick end labeling (TUNEL) positive cells, which were present only adjacent to the needle track at 8 h to 1 day, became more extensive in the whole CA1 layer at 3 to 7 days . TUNEL-positive cells were also detected around the DG at 1 day, around the needle track at 8 h to 3 days, and in the choroid plexus cells at 7 days . HSP72 staining was detected in the subiculum at 1 to 3 days, the dentate granule cells at 8 h to 1 day, and in the CA3 or CA4 pyramidal cells at 1 to 3 days . Some lacZ expressing cells were double-positive with HSP72 in DG, while the majority of those were distinguished from the TUNEL-positive cells . Pyramidal neurons were almost completely lost in the CA1 sector at 7 days after the ischemia . The present study demonstrates the successful LacZ gene transfer into the hippocampus and ventricle of postischemic gerbil brain except in the vulnerable CA1 layer by adenoviral vector injection . However, adenovirus-mediated gene transfer may induce indirect apoptotic cell death in the DG and ventricle, in addition to direct traumatic injury around the needle track. FEBS Lett, 1998 Oct 16, 437(1-2), 75 - 80 Isolation and characterization of single-chain Fv genes encoding antibodies specific for Drosophila Poxn protein; Hassanzadeh GH G et al.; The usefulness of intrabodies as specific inhibitors of gene function has been extensively demonstrated in cell culture assays . However, very few experiments have been conducted with intrabodies expressed in whole organisms . To evaluate the intrabody technology in Drosophila, we focused on poxn protein, since its effects can be easily studied . We purified the recombinant poxn protein . We next isolated three single-chain variable fragments (scFv) which specifically recognize poxn protein . Two scFvs, designated alpha-Poxn2 and alpha-Poxn4, react with both denatured and native Poxn with half maximal inhibition values of 100 nM and 40 nM, respectively . The alpha-Poxn5 scFv also recognizes denatured Poxn but either does not recognize native Poxn or its half maximal inhibition value for native Poxn is high. J Biotechnol, 1998 Aug 27, 63(3), 179 - 86 Elimination of an HuIFN alpha 2b readthrough species, produced in Escherichia coli, by replacing its natural translational stop signal; Cesar Sanchez J et al.; When human interferon-alpha 2b (HuIFN alpha 2b) was expressed intracellularly in Escherichia coli as insoluble aggregates, a HuIFN alpha 2b molecular species of high molecular weight was detected, even after immunoaffinity chromatography and characterized by mass spectrometry and automatic sequencing . This HuIFN alpha 2b species was synthesized by an inefficient reading of the UGA natural stop codon, stopping the translation at another UGA in frame placed 10 codons downstream of the HuIFN alpha 2b stop signal . To avoid this translational readthrough process the UGA termination codon was replaced by UAA, which is frequently used in highly expressed E . coli genes . Simultaneously, almost all the HuIFN alpha 2b gene 3' noncoding region was removed . Analysis by SDS-PAGE and enzyme-linked immunosorbent assay revealed the elimination of the undesired HuIFN alpha 2b molecular species and an almost twofold increase in the expression level . These results indicate that both factors, the stop codon used and the length of the transcription unit should be taken into account when the expression in E . coli of heterologous proteins is desired. Intensive Care Med, 1998 Sep, 24(9), 967 - 72 Influence of N-acetylcysteine treatment on endotoxin-induced microcirculatory disturbances; Schmidt W et al.; OBJECTIVES: To determine the influence of N-acetylcysteine (NAC) in a treatment model, its effects on endotoxin-induced leukocyte-endothelial cell adhesion, vascular leakage, and venular microhemodynamics in postcapillary venules of rat mesentery . DESIGN: Prospective, randomized, controlled, experimental study . SETTING: Animal research laboratory . SUBJECTS: 40 male Wistar rats . INTERVENTIONS: The rats randomly received one of four treatments: infusion of saline (SAL) or Escherichia coli lipopolysaccharides (LPS) followed by treatment with saline (SAL) or NAC (150 mg.kg-1 body weight) 30 min after induction of endotoxemia . MEASUREMENTS AND MAIN RESULTS: Leukocyte adherence, red blood cell velocity, and vessel diameters in postcapillary venules of rat mesentery were evaluated every 30 min over a period of 120 min using in vivo videomicroscopy . Vascular permeability was determined by measuring the extravasation of fluorescence-labeled albumin . Venular wall shear rate was calculated from red cell velocity, and vessel diameter . NAC in rats without endotoxemia (SAL + NAC group) compared to the control group (SAL + SAL) did not change microcirculatory parameters in postcapillary venules of rat mesentery . In both LPS-treated groups (LPS + SAL and LPS + NAC), leukocyte adherence increased after just 30 min . NAC treatment prevented a further increase in leukocyte adherence and attenuated the extravasation of fluorescence-labeled albumin during endotoxemia . Venular diameters remained unchanged, while erythrocyte velocity decreased in the LPS + SAL group . This led to a lower venular wall shear rate in this group . CONCLUSIONS: Treatment with NAC attenuates endotoxin-induced leukocyte adherence and macromolecular leakage in postcapillary venules of rat mesentery, showing that NAC is also effective after the onset of endotoxemia. J R Coll Surg Edinb . 1998 Oct;43(5):357. Necrotizing fasciitis following gall-bladder perforation; Rehman A et al.; Necrotizing fasciitis continues to carry a very high mortality and prolonged morbidity . Gallstones have previously not been reported as a cause of this condition . We report a patient who presented with gallbladder perforation leading to necrotizing fasciitis of the anterior abdominal wall . The only organism isolated was Escherichia Coli, cultured from necrotic issue. Microbiology, 1998 Oct, 144 ( Pt 10), 2697 - 704 The mabA gene from the inhA operon of Mycobacterium tuberculosis encodes a 3-ketoacyl reductase that fails to confer isoniazid resistance; Banerjee A et al.; A target of the anti-tuberculosis drugs isoniazid (INH) and ethionamide (ETH) has been shown to be an enoyl reductase, encoded by the inhA gene . The mabA (mycolic acid biosynthesis A) gene is located immediately upstream of inhA in Mycobacterium tuberculosis, Mycobacterium bovis and Mycobacterium smegmatis . The MabA protein from M . tuberculosis was expressed in Escherichia coli and shown to have 3-ketoacyl reductase activity, consistent with a role in mycolic acid biosynthesis . In M . smegmatis, inhA and mabA are independently transcribed, but in M . tuberculosis and M . bovis BCG, mabA and inhA constitute a single operon . Several INH-ETH-resistant M . tuberculosis clinical isolates contain point mutations in the ribosome-binding site of mabA in the mabA-inhA operon . However, genetic dissection of this operon reveals that the INH-ETH-resistance phenotype is encoded only by inhA, and not by mabA. Regul Pept, 1998 Sep 25, 75-76, 215 - 20 Endogenous neuropeptide Y mediates vasoconstriction during endotoxic and hemorrhagic shock; Qureshi NU et al.; Neuropeptide Y (1-36), NPY, is a sympathetic vasoconstrictor whose activities in blood vessels is determined by the presence of vasoconstrictive Y1 receptors and the enzyme dipeptidyl peptidase IV (DPPIV), which converts NPY to non-vasoconstrictive peptides . While the role of the NPY system has been established during cold water stress, its role in hypotensive conditions has not; yet, exogenous NPY improves hemodynamics and survival in rats with endotoxic shock . We used a new selective non-peptidergic Y1 receptor antagonist, BIBP-3226, to determine the role of the endogenous NPY/Y1 system in endotoxic shock (induced by i.v . injection of 10 mg/kg of Escherichia coli lipopolysaccharide 0127:B8, LPS) and hemorrhagic shock (bleeding of 15 ml/kg over 1.5 min) . Conscious rats received a bolus of BIBP-3226 or the vehicle 5 min before endotoxin challenge or induction of hemorrhage, followed by continuous infusion . Mean arterial pressure (MAP) at 5 min after LPS administration dropped in the control group by 15%, compared to 36% in the BIBP-3226-treated group (p < 0.01) . Similarly, the hemorrhage-induced drop in MAP in the control group was 32% at 5 min, compared to 53% in the BIBP-treated rats (p < 0.01) . Plasma NPY levels were unchanged in the endotoxic shock group, but were significantly elevated in the hemorrhagic shock group . BIBP-3226 pretreatment abrogated the increased plasma NPY levels after hemorrhagic shock . Endogenous NPY contributes to blood pressure recovery during endotoxic and hemorrhagic shock. J Allergy Clin Immunol, 1998 Oct, 102(4 Pt 1), 579 - 91 Recombinant birch pollen allergens (rBet v 1 and rBet v 2) contain most of the IgE epitopes present in birch, alder, hornbeam, hazel, and oak pollen: a quantitative IgE inhibition study with sera from different populations; Niederberger V et al.; BACKGROUND: Pollen from trees of the order Fagales are important allergen sources in most parts of the world . Clinical, immunochemical, and molecular biology studies indicate that they contain cross-reactive allergens . The major birch pollen allergen, Bet v 1, and birch profilin, Bet v 2, a highly cross-reactive allergen, have been cloned and expressed in Escherichia coli . OBJECTIVE: The purpose of this study was to demonstrate the presence of allergens in Fagales pollens that share IgE epitopes with recombinant Bet v 1 and Bet v 2 and to determine the percentage of birch, alder, hornbeam, hazel, and oak pollen-specific IgE that can be preabsorbed with rBet v 1 and rBet v 2 from 102 sera of different populations of subjects allergic to Fagales tree pollen . METHODS: The presence of rBet v 1- and rBet v 2-homologous allergens in tree pollen extracts was investigated by IgE immunoblot inhibition experiments, and the percentage of tree (birch, alder, hornbeam, hazel, and oak) pollen-specific IgE that was bound by a mixture of rBet v 1 and rBet v 2 was determined by RAST-based quantitative IgE inhibition experiments . The clinical significance of IgE antibody cross-reactivity was studied by skin prick testing with rBet v 1, rBet v 2, and Fagales pollen extracts . RESULTS: Natural birch, alder, hornbeam, hazel, and oak pollen contain allergens that share IgE epitopes with rBet v 1 and rBet v 2 . A combination of rBet v 1 and rBet v 2 accounted for 82% of tree pollen-specific IgE on average . Most of the tree pollen-specific IgE was directed against rBet v 1 . CONCLUSION: rBet v 1 and rBet v 2 contain most of the Fagales pollen-specific IgE epitopes and may therefore substitute natural tree pollen extracts not only for diagnosis but also for patient-tailored immunotherapy of tree pollen allergy. Microbiology, 1998 Oct, 144 ( Pt 10), 2905 - 14 The localization of KpsC, S and T, and KfiA, C and D proteins involved in the biosynthesis of the Escherichia coli K5 capsular polysaccharide: evidence for a membrane-bound complex; Rigg GP et al.; Biosynthesis of the Escherichia coli K5 polysaccharide requires the KfiA, KfiB, KfiC and KfiD proteins . The subsequent transport of the polysaccharide onto the cell surface requires the KpsC, KpsD, KpsE, KpsM, KpsS and KpsT proteins, which are conserved between different group II capsular polysaccharides . The KfiA and KfiC, together with the KpsC, KpsS and KpsT proteins, were purified and polyclonal antisera to each protein generated . These antisera, together with one previously generated (by others) against the purified KfiD protein, were used in Western blot analysis to locate the corresponding proteins within the cell . Analysis of membrane fractions revealed that KfiA (involved in initiation of polysaccharide synthesis), KfiC (K5 glycosyl transferase) and the KfiD protein (UDP-glucose dehydrogenase) were associated with the inner membrane . The KpsC, KpsS, and KpsT proteins involved in polysaccharide transport were associated with the inner membrane and this membrane association occurred in the absence of any other capsule-related proteins . The effect of mutations in individual kps genes on the localization of each protein was determined . Mutations in the kpsC, kpsM, kpsS and kpsT genes resulted in a loss of membrane targeting for KfiA and KfiC, suggesting some form of hetero-oligomeric membrane-bound biosynthetic complex . Osmotic shock caused the release of KfiA, KfiC, KpsC and KpsS from the inner membrane into the periplasm, suggesting that the polysaccharide biosynthetic complex may be associated with sites of adhesion between the inner and outer membrane. Microbiology, 1998 Oct, 144 ( Pt 10), 2865 - 72 Roles for GcvA-binding sites 3 and 2 and the Lrp-binding region in gcvT::lacZ expression in Escherichia coli; Stauffer LT et al.; GcvA and Lrp are both necessary for activation of the gcv operon . The upstream GcvA-binding sites 3 and 2 were separated from the Lrp-binding region and the rest of the gcv control region . Moving these sites by 1 or 2 helical turns of DNA further from the gcv promoter reduces, but does not eliminate, either GcvA-mediated activation or repression of a gcvT::lacZ gene fusion . However, moving these sites by 1.5 or 2.5 helical turns of DNA results in a GcvA-mediated super-repression of the operon . This repression is dependent on Lrp and is partially dependent on GcvR . Lrp bound to the gcv control region induces a bend in the DNA . Based on these results, a model for gcv regulation is presented in which Lrp plays a primarily structural role, by bending the DNA and GcvA functions as the activator protein. Microbiology, 1998 Oct, 144 ( Pt 10), 2855 - 64 Selection and characterization of mercury-independent activation mutants of the Tn501 transcriptional regulator, MerR; Parkhill J et al.; MerR is the transcriptional regulator of the mercury-resistance (mer) operon of transposon Tn501, acting at the mer promoter as both an activator in the presence of mercuric salts and a repressor in their absence . This paper reports a method for selection of constitutive activator mutants, which activate transcription in the absence of HgII, and the characterization of these MerRAC proteins . At least two mutations in the MerR protein were found necessary for strong mercury-independent activation, and these mutations lie in the C-terminal two-thirds of the MerR protein near the HgII-binding cysteines . A triple mutation was shown to increase activation over the corresponding double mutations . All mutant proteins caused further activation in the presence of HgII . The data support a mechanism in which a conformational change of one or both MerR subunits in the homodimer drives a distortion of DNA bound to a helix-turn-helix structure in the N-terminal region . A mutation in this putative helix-turn-helix region severely reduced both the repressor and activator functions of MerR. Bioorg Med Chem, 1998 Sep, 6(9), 1563 - 75 Solid-phase extraction on C18 silica as a purification strategy in the solution synthesis of a 1-thio-beta-D-galactopyranoside library; Nilsson UJ et al.; A novel strategy for the purification of carbohydrate-based chemical libraries synthesized in solution was developed . Purification of reaction products was accomplished by means of solid-phase extraction enabled by protecting the 2-, 3-, 4-, and 6-hydroxyl groups of a galactose derivative as their hydrophobic O-laurates . The presence of multiple O-laurates allowed adsorption of reaction products onto C18 silica while reagents and by-products were washed away with MeOH . Products were quantitatively eluted with pentane . Purification of products using solid-phase extraction offers the combined advantages of solution synthesis (normal solution reactivity and ease of reaction monitoring) with those of solid-phase synthesis (facile product isolation permitting the use of large excesses of reagents) . To demonstrate the utility of the hydrophobic recovery-procedure, tetra-O-lauroyl-beta-D-galactopyranose-1-thiol was subjected to high-yielding reactions with a panel of Michael-acceptors and an alpha-chloro ketone . The resulting ketone adducts were then either reduced to the alcohols or reductively aminated with a selection of amino acids to give 30 different 1-thio-beta-D-galactosides as mixtures of four diastereomers after removal of protecting groups . At each step, the product was separated from the reagents and their by-products by simple adsorption onto C18 silica, washing with MeOH and elution of product with pentane . After completion of the combinatorial chemistry sequence, the O-laurates were cleaved by methanolysis and the product methyl laurate in turn removed from the desired water-soluble products by C18 adsorption . Individual library members were thus conveniently produced on 10-30 mg scales at purity levels of > 90% . One of the 1-thio-beta-D-galactosides thus produced was found to be a competitive inhibitor of the beta-galactosidase from E . coli with Ki value of 1.7 microM. Biochem Mol Biol Int, 1998 Oct, 46(2), 307 - 19 The expressed alpha domain of mouse metallothionein-I from Escherichia coli displays independent structure and function; Xiong Y et al.; The alpha domain of mouse metallothionein-I (mMT-I) was expressed in E . coli as a C-terminus of the 26 KD glutathione-S-transferase and purified from the cell lysates . The amino acid composition and molecular weight of the expressed protein are as expected . The metal-binding stoichimetry was determined to show that divalent metals bind to the expressed alpha domain at the desired ratio of 4:1 . The ultraviolet absorbance, circular dichroism spectra and the atomic force microscopy indicate that it can form the proper metal-thiolate structure as in the whole MT molecule . The apparent affinity of the expressed alpha domain to bind cadmium is 1.8-fold stronger than the recombinant mMT-I when detected by the reaction with DTNB . The ability to scavenge hydroxyl free radicals remains higher than the whole MT molecule . All the results demonstrate that the expressed alpha-domain from E . coli exhibits independent biochemical and physiological structure/function without the assistance of beta domain. Antonie Van Leeuwenhoek, 1998 Apr, 73(3), 245 - 54 Metal resistance and plasmid DNA in Thiobacillus ferrooxidans; Chisholm IA et al.; The minimal inhibitory concentrations of copper and nickel were determined for each of fifteen isolates of T . ferrooxidans native to a Cu/Ni tailings environment . Ten isolates were inhibited by 160 mM Cu2+ or less, and ten were inhibited by 160 mM Ni2+ or less . The isolates were screened for plasmid DNA using an alkaline lysis method and CCC plasmid forms were confirmed using the Hintermann technique . Two isolates were found to be devoid of plasmid DNA, and only one isolate contained more than two plasmids . Variability existed in plasmid size, although the majority were larger than the standard pBR322 (4.3 kbp) . One plasmid was selected for further analysis using restriction endonucleases . EcoRI, HindIII and KpnI all cleaved the plasmid in two locations, and PstI cleaved the plasmid in six locations . PstI-digested fragments of the plasmid were ligated into pBR322, and the recombinant plasmids were transformed into Escherichia coli ATCC 8739 . Four genetically-different transformants resulted, and each was grown in media containing 2.0 mM Cu2+ and compared to the growth of a control under similar conditions . There was no conferred copper resistance in E . coli, although one recombinant plasmid appeared to decrease the tolerance for E . coli ATCC 8739 to Cu2+. Clin Diagn Lab Immunol, 1998 Nov, 5(6), 766 - 72 Use of the cell division protein FtsZ as a means of differentiating among Bartonella species; Kelly TM et al.; Genes coding for homologs of the highly conserved cell division protein FtsZ were isolated from Bartonella henselae and Bartonella quintana, the causative agents of cat scratch disease and trench fever, respectively . DNA fragments coding for the ftsZ open reading frames (ORFs) were cloned into Escherichia coli following PCR amplification with primers based on the ftsZ sequence of the closely related species Bartonella bacilliformis . The amino acid sequences predicted from the cloned B . henselae and B . quintana ftsZ ORFs are 81 to 83% identical to the corresponding protein in B . bacilliformis . Like the FtsZ protein of B . bacilliformis, the B . henselae and B . quintana homologs are about twice as large as the FtsZ proteins reported in most other organisms . Localized sequence differences within the C-terminal coding regions of the Bartonella ftsZ genes were used as the basis for species-specific identification of these organisms at both the DNA and protein levels . Oligonucleotide primers which permit the amplification of an ftsZ fragment from each of the Bartonella species without amplifying DNA from the other two species were designed . Anti-FtsZ antisera raised in rabbits against synthetic peptides corresponding to the relatively divergent C-terminal regions were shown via Western blot analysis to react only with the FtsZ protein from the cognate Bartonella species . These observations raise the possibility that the differences in ftsZ sequences can be used as the basis for diagnostic tests to differentiate among these closely related pathogens. Biochemistry, 1998 Nov 3, 37(44), 15575 - 82 Redox potentials for yeast, Escherichia coli and human glutathione reductase relative to the NAD+/NADH redox couple: enzyme forms active in catalysis; Veine DM et al.; The flavoenzyme glutathione reductase catalyzes the NADPH-dependent reduction of glutathione disulfide, yielding two molecules of glutathione . The oxidation-reduction potentials, Eox/EH2 (two-electron reduced enzyme), for yeast, Escherichia coli, and human glutathione reductase have been determined between pH 6.0 and 9.8 relative to the nonphysiological substrate couple NAD+/NADH and were found to be -237, -243, and -227 mV (+/-5 mV) at pH 7.0 and 20 degreesC, respectively . The potential as a function of pH demonstrated slopes of -51, -45, and -42 mV/pH unit, respectively, at low pH and -37, -31, and -34 mV/pH unit, respectively, at high pH . The change in slope indicated pKa values of 7.4, 8.5, and 7.6, respectively . The slopes indicate that two protons are associated with the two-electron reduction of Eox at low pH and that only one proton is involved with the two-electron reduction of Eox at high pH, provided that the effects of nearby titratable residues are considered in the data analysis . The influence of four such groups, Cys50, Cys45, His456', and either Tyr107 or the flavin-(N3), has been included (residue numbering refers to the yeast sequence) . The enzyme loses activity upon deprotonation of the acid-base catalyst at high pH . Since the pKa ascribed to the EH2-to-EH- ionization is lower than the pKa of the acid-base catalyst, both the EH2 and EH- forms of glutathione reductase must be catalytically active, in contrast to the closely related enzyme lipoamide dehydrogenase, for which only EH2 is active. Nucleic Acids Res, 1998 Nov 15, 26(22), 5199 - 202 Rat 7,8-dihydro-8-oxoguanine DNA glycosylase: substrate specificity, kinetics and cleavagemechanism at an apurinic site; Prieto Alamo MJ et al.; Reactive oxygen species produce different lesions in DNA . Among them, 7,8-dihydro-8-oxoguanine (8-oxoG) is one of the major oxidative products implicated in mutagenesis . This lesion is removed from damaged DNA by base excision repair, and genes coding for 8-oxoG-DNA glycosylases have been isolated from bacteria, yeast and human cells . We have isolated and characterized the cDNA encoding the rat 8-oxoG-DNA glycosylase (rOGG1) . Expression of the cDNA in the fgp mutY Escherichia coli double mutant allowed the purification of the untagged rOGG1 protein . It excises 8-oxoG from DNA with a strong preference for duplex DNA containing 8-oxoG:C base pairs . rOGG1 also acts on formamidopyrimidine (FaPy) residues, and the K m values on 8-oxoG and FaPy residues are 18.8 and 9.7 nM, respectively . When acting on an oligonucleotide containing an 8-oxoG residue, rOGG1 shows a beta-lyase activity that nicks DNA 3' to the lesion . However, rOGG1 acts on a substrate containing an apurinic site by a beta-delta elimination reaction and proceeds through a Schiff base intermediate . Expression of rOGG1 in E.coli fpg mutY suppresses its spontaneous mutator phenotype. Nucleic Acids Res, 1998 Nov 15, 26(22), 5170 - 5 The FIS protein fails to block the binding of DnaA protein to oriC, the Escherichia coli chromosomal origin; Margulies C et al.; The Escherichia coli chromosomal origin contains several bindings sites for factor for inversion stimulation (FIS), a protein originally identified to be required for DNA inversion by the Hin and Gin recombinases . The primary FIS binding site is close to two central DnaA boxes that are bound by DnaA protein to initiate chromosomal replication . Because of the close proximity of this FIS site to the two DnaA boxes, we performed in situ footprinting with 1, 10-phenanthroline-copper of complexes formed with FIS and DnaA protein that were separated by native gel electrophoresis . These studies show that the binding of FIS to the primary FIS site did not block the binding of DnaA protein to DnaA boxes R2 and R3 . Also, FIS appeared to be bound more stably to oriC than DnaA protein, as deduced by its reduced rate of dissociation from a restriction fragment containing oriC . Under conditions in which FIS was stably bound to the primary FIS site, it did not inhibit oriC plasmid replication in reconstituted replication systems . Inhibition, observed only at high levels of FIS, was due to absorption by FIS binding of the negative superhelicity of the oriC plasmid that is essential for the initiation process. Nucleic Acids Res, 1998 Nov 15, 26(22), 5123 - 33 Evidence that MutY is a monofunctional glycosylase capable of forming a covalent Schiff base intermediate with substrate DNA; Williams SD et al.; The Escherichia coli adenine glycosylase MutY is involved in the repair of 7,8-dihydro-8-oxo-2'-deoxyguanosine (OG):A and G:A mispairs in DNA . DNA strand cleavage via beta-elimination (beta-lyase) activity coupled with MutY's removal of misincorporated adenine bases was sought using both qualitative and quantitative methods . The qualitative assays demonstrate formation of a Schiff base intermediate which is characteristic of DNA glycosylases catalyzing a concomitant beta-lyase reaction . Borohydride reduction of the Schiff base results in the formation of a covalent DNA-MutY adduct which is easily detected in SDS-PAGE experiments . However, quantitative activity assays which monitor DNA strand scission accompanying base release suggest MutY behaves as a simple monofunctional glycosylase . Treatment with base effects DNA strand cleavage at apurinic/apyrimidinic (AP) sites arising via simple glycosylase activity . The amount of cleaved DNA in MutY reactions treated with base is much greater than that in non-base treated reactions, indicating that AP site generation by MutY is not associated with a concomitant beta-lyase step . As standards, identical assays were performed with a known monofunctional enzyme (uracil DNA glycosylase) and a known bifunctional glycosylase/lyase (FPG), the results of which were used in comparison with those of the MutY experiments . The apparent inconsistency between the data obtained for MutY by the qualitative and quantitative methods underscores the current debate surrounding the catalytic activity of this enzyme, and a detailed explanation of this controversy is proposed . The work presented here lays ground for the identification of specific active site residues responsible for the chemical mechanism of MutY enzyme catalysis. Nucleic Acids Res, 1998 Nov 15, 26(22), 5109 - 15 Utilizing the C-terminal cleavage activity of a protein splicing element to purify recombinant proteins in a single chromatographic step; Chong S et al.; A conventional affinity protein purification system often requires a separate protease to separate the target protein from the affinity tag . This paper describes a unique protein purification system in which the target protein is fused to the C-terminus of a modified protein splicing element (intein) . A small affinity tag is inserted in a loop region of the endonuclease domain of the intein to allow affinity purification . Specific mutations at the C-terminal splice junction of the intein allow controllable C-terminal peptide bond cleavage . The cleavage is triggered by addition of thiols such as dithiothreitol or free cysteine, resulting in elution of the target protein while the affinity-tagged intein remains immobilized on the affinity column . This system eliminates the need for a separate protease and allows purification of a target protein without the N-terminal methionine . We have constructed general cloning vectors and demonstrated single-column purification of several proteins . In addition, we discuss several factors that may affect the C-terminal peptide bond cleavage activity. Nucleic Acids Res, 1998 Nov 15, 26(22), 5102 - 8 Point and deletion mutations eliminate one or both methyl group transfers catalysed by the yeast TRM1 encoded tRNA (m22G26)dimethyltransferase; Liu J et al.; Guanosine at position 26 in eukaryotic tRNAs is usually modified to N2 , N2 -dimethylguanosine (m22G26) . In Saccharomyces cerevisiae , this reaction is catalysed by the TRM1 encoded tRNA (m22G26)dimethyltransferase . As a prerequisite for future studies, the yeast TRM1 gene was expressed in Escherichia coli and the His-tagged Trm1 protein (rTrm1p) was extensively purified . rTrm1p catalysed both the mono- and dimethylation of G26 in vivo in Escherichia coli tRNA and in vitro in yeast trm1 mutant tRNA . The TRM1 gene from two independent wild-type yeast strains differed at 14 base positions causing two amino acid exchanges . Exchange of the original Ser467 for Leu caused a complete loss of enzyme activity in vitro against trm1 yeast tRNA . Comparatively short N- or C-terminal deletions from the 570 amino acid long Trm1 polypeptide decreased or eliminated the enzyme activity, as did some point mutations within these regions . This indicated that the protein is not a two domain peptide with the enzyme activity localised to one of the domains, but rather that both ends of the polypeptide seem to interact to influence the conformation of those parts that make up the RNA-binding site and/or the active site of the enzyme. Mutagenesis, 1998 Sep, 13(5), 507 - 14 Role of DNA repair by (A)BC excinuclease and Ogt alkyltransferase in the final distribution of LacI-d mutations induced by N-butyl-N-nitrosourea in Escherichia coli; Ferrezuelo F et al.; In the absence of nucleotide excision repair, the additional deficiency of the DNA alkyltransferase (ATase) encoded by the constitutive ogt gene of Escherichia coli caused a marked increase in mutation induction by N-butyl-N-nitrosourea (BNU) . Irrespective of the presence or absence of the Ogt ATase, little mutagenic response was detected in Uvr+ bacteria in the concentration range 0-8 mM BNU, indicating that most premutagenic DNA lesions induced at these concentrations are efficiently recognized and repaired by the nucleotide excision repair system . Increased susceptibility to mutagenesis by BNU was detected in Uvr- Ogt+ bacteria, but the Uvr- Ogt- double mutant exhibited much higher sensitivity . These data suggest that the Ogt ATase can replace to a great extent the repair capacity of the (A)BC excinuclease . Forward mutations induced by 6 mM BNU within the initial part of the lacI gene of E.coli were recovered from Uvr+ Ogt-, Uvr- Ogt+ and Uvr- Ogt- bacteria . A total of 454 independent mutations were characterized by DNA sequence analysis . The BNU-induced spectra were dominated by G:C-->A:T transitions, consistent with the major role of the O6-alkylguanine miscoding lesion in mutagenesis by alkylating agents . Specific sites for G:C-->A:T transitions were recovered more or less frequently in one genetic background versus the others, giving statistically significant differences among the spectra (P < 10(-6)) . We examined the influence of DNA repair by (A)BC excinuclease and Ogt ATase on the 5'-flanking base associated with the BNU-induced G:C-->A:T transitions; preferences different from those previously reported for other alkylnitrosoureas were detected . We discuss how these differences might be caused by BNU producing branched chain derivatives, in addition to the expected linear chain adducts, and by possible preferences with respect to both the initial distribution of O6-butylguanine lesions and their repairability. Mutagenesis, 1998 Sep, 13(5), 487 - 97 Spontaneous and ENU-induced mutation spectra at the cII locus in Big Blue Rat2 embryonic fibroblasts; Watson DE et al.; Big Blue Rat2 embryonic fibroblasts carry the lambda-Liz shuttle vector which is also present in the Big Blue mouse and rat . Mutations in the Big Blue systems have most often been measured at the lacI locus . However, a method for positive selection of mutations at the lambda cII locus was recently described . This assay appears to have many advantages over the use of lacI as a mutational target, but it has yet to be well characterized in mammalian mutagenesis studies . The objective of these studies was to determine the spontaneous and ethylnitrosourea (ENU)-induced mutant frequencies (MFs) and mutational spectra at cII using Big Blue Rat2 embryonic fibroblasts . The average spontaneous MF was 13 +/- 1.4 x 10(-5) . The average induced MF was 60 +/- 10 x 10(-5) 10 days following a 30 min treatment with 0.1 mg/ml ENU . Eighty four independent spontaneous mutants were sequenced: 23 (27.4%) were frameshift mutations and 61 (72.6%) were base substitutions . Two spontaneous frameshift hotspots were detected, both in mononucleotide runs . G:C-->A:T transitions were the most common type of base substitution in cII; of these 71% occurred at CpG sites . The ENU-induced mutational spectrum at cII (44 mutants) consisted of 42 base substitutions (95.5%) and two -1 frameshift mutations (4.5%) . Compared with the spontaneous spectrum, the ENU-induced spectrum had significantly fewer frameshift mutations (4.5 versus 27%) and base substitutions occurred predominantly at A:T base pairs (71 versus 34%) . Overall, the spontaneous cII mutational spectrum reported here differs slightly from spontaneous spectra reported at the Big Blue lacI locus, but the mutational spectra and base substitution MFs following treatment with ENU were comparable at both loci . These data support the continued use of cII as a selectable marker in mutagenesis studies involving cells or tissues that carry a lambda transgene. Curr Biol, 1998 Oct 22, 8(21), R768 - 70 Protein translocation: delivering virulence into the host cell; Gauthier A et al.; A wide variety of plant and human bacterial pathogens use a specialized 'type III' protein secretion system to deliver virulence factors into host cells . Appendage-like surface structures have recently been identified on several bacterial pathogens and there are indications that these may be conduits for virulence factor delivery. Biotechnol Appl Biochem, 1998 Dec, 28 ( Pt 3), 207 - 13 A new derivatizing agent, trimethylammoniopropyl methanethiosulphonate, is efficient for preparation of recombinant brain-derived neurotrophic factor from inclusion bodies; Inoue M et al.; Derivatization with trimethylammoniopropyl methanethiosulphonate (TAPS-sulphonate) enabled brain-derived neurotrophic factor (BDNF) to be prepared efficiently from Escherichia coli inclusion bodies . Reduced BDNF obtained from inclusion bodies solubilized by urea and reduced by dithiothreitol was suggested to form a complex with itself or with other compounds such as lipids . It could hardly be adsorbed on to cation-exchange resin for partial purification prior to a refolding reaction . Reversible derivatization of cysteine residues was tested as a method of dissociating BDNF from such complexes . However, even if a methyl or aminoethyl group was introduced, BDNF could not be dissociated readily . Derivatization with TAPS-sulphonate brought about good dissociation of BDNF, and more than 50% adsorbed on to the cation-exchange resin . BDNF derivatized with TAPS-sulphonate refolded well, and the refolded samples showed the same biological activity as purified BDNF . Derivatization with TAPS-sulphonate would increase the intermolecular repulsion of BDNF, due to the positively charged character of the quaternized amine, and inhibit complex formation . Thus, TAPS-sulphonate is effective for the preparation of BDNF under denatured conditions. J Mol Biol, 1998 Nov 13, 283(5), 985 - 92 Identification of a defined epitope on the surface of the active RecA-DNA filament using a monoclonal antibody and three-dimensional reconstruction; Yu X et al.; Studies of the Escherichia coli RecA protein are expected to illuminate mechanisms of DNA recombination and repair in bacteria, and in all higher organisms as well, due to the functional and structural homology with the eukaryotic Rad51 protein . The active form of the RecA protein is a helical filament formed on DNA in the presence of ATP or ATP analogs, and this has been studied at low-resolution by electron microscopy . An atomic model of the protein comes from an X-ray crystallographic study of a filament formed in the absence of DNA and ATP . This filament is believed to be an inactive, storage form of the protein . A key step in generating an atomic model of the active filament, and a detailed model for function, is to understand the large conformational changes that occur between these two states . Towards this end, we have decorated active RecA-DNA filaments with monoclonal antibodies (ARM191) against a known epitope (residues 285 to 320) to determine the position of this epitope in the low-resolution structure . Electron microscopy and three-dimensional reconstruction of the RecA-antibody complex reveal that the lobe containing the epitope is very disordered on the surface of the filament, but in a position similar to that in the inactive crystal filament . The antibody binding also induces a significant conformational change in the RecA filament . This study shows that the basic orientation of the subunit is likely to be similar within the inactive and active filaments, and that the large movement of mass that occurs between these two states must involve other residues than the 285-320 region . J Mol Biol, 1998 Nov 13, 283(5), 947 - 61 The DNA binding characteristics of the trimeric EcoKI methyltransferase and its partially assembled dimeric form determined by fluorescence polarisation and DNA footprinting; Powell LM et al.; The type I DNA restriction and modification systems of enteric bacteria display several enzymatic activities due to their oligomeric structure . Partially assembled forms of the EcoKI enzyme from E . coli K12 can display specific DNA binding properties and modification methyltransferase activity . The heterodimer of one specificity (S) subunit and one modification (M) subunit can only bind DNA whereas the addition of a second modification subunit to form M2S1 also confers methyltransferase activity . We have examined the DNA binding specificity of M1S1 and M2S1 using the change in fluorescence anisotropy which occurs on binding of a DNA probe labelled with a hexachlorofluorescein fluorophore . The dimer has much weaker affinity for the EcoKI target sequence than the trimer and slightly less ability to discriminate against other DNA sequences . Binding of both proteins is strongly dependent on salt concentration . The fluorescence results compare favourably with those obtained with the gel retardation method . DNA footprinting using exonucleaseIII and DNaseI, and methylation interference show no asymmetry, with both DNA strands being protected by the dimer and the trimer . This indicates that the dimer is a mixture of the two possible forms, M1S1 and S1M1 . The dimer has a footprint on the DNA substrate of the same length as the trimer implying that the modification subunits are located on either side of the DNA helical axis rather than lying along the helical axis . Arch Biochem Biophys, 1998 Nov 1, 359(1), 99 - 106 Thermosensitive phenotype of yeast mutant lacking thioredoxin peroxidase; Lee SM et al.; A soluble protein from Saccharomyces cerevisiae specifically provides protection against a thiol-containing oxidation system but not against an oxidation system without thiol . This 25-kDa protein acts as a peroxidase but requires a NADPH-dependent thioredoxin system or a thiol-containing intermediate, and was thus named thioredoxin peroxidase (TPx) . The protective role of TPx in the cellular defense against heat shock (42 or 48 degreesC), which may increase oxidative stress in cells sufficiently to form reactive oxygen species harmful to cellular function, was investigated in a wild-type and a mutant yeast strain in which the tsa gene that encodes TPx was disrupted by homologous recombination . Upon exposure under aerobic conditions to heat shock there was a distinct difference between these two strains in growth kinetics and viability . The wild-type strain was more resistant to killing by heat than the mutant strain . In addition, the expression of the tsa gene in Escherichia coli caused an increase in thermotolerance . The expression of the tsa gene increased under heat shock; however, modulation of activities of other antioxidant enzymes, such as catalase, superoxide dismutase, glucose 6-phosphate dehydrogenase, and glutathione reductase as well as the total glutathione level, remained unaltered in both strains under heat shock . The induction of heat shock protein HSP104 was not significantly different in the two strains under heat shock . The results indicate that the lack of TPx expression may be solely responsible for the thermosensitive phenotype of tsa mutant cells . When the oxidation of 2', 7'-dichlorofluorescin was used to examine hydroperoxide production in yeast cells, tsa mutant cells showed a 2.5- to 3.5-fold increase in fluorescence upon exposure to heat stress compared to wild-type cells . The antioxidant, N-acetylcysteine, prevented intracellular peroxide formation in response to heat shock . The carbonyl content of extract, the indicative marker of oxidative damage to protein, from tsa mutant cells was higher than that from wild-type cells . These results suggest that TPx may play a direct role in cellular defense against heat shock, presumably functioning as an antioxidant protein . Arch Biochem Biophys, 1998 Nov 1, 359(1), 82 - 8 Membrane topology of cytochrome P450 2B4 in Langmuir-Blodgett monolayers; Shank-Retzlaff ML et al.; Using Langmuir-Blodgett monolayers of both phosphatidylethanolamines and phosphatidylcholines as membrane mimics, we have examined the topology of cytochrome P450 2B4 anchoring . The interaction of wild-type P450 2B4 with phosphatidylethanolamine monolayers can be characterized as a biphasic reaction, with the initial fast phase explained by the specific insertion of membrane-spanning segments of the protein into the monolayer . Injection of cytochrome b5 (b5) beneath dipalmitoyl-phosphatidylcholine monolayers also resulted in biphasic kinetics . Regardless of the nature of the lipid employed, neither a truncated cytochrome P450 2B4 (P450 2B4 Delta2-27) lacking the amino-terminal hydrophobic residues widely believed to be the major transmembrane segment nor a soluble b5 fragment (Deltab5) lacking its carboxy terminus anchor exhibit the fast-phase behavior characteristic of specific insertion . To further characterize the membrane topology of P450 2B4, its insertion area in DPPE monolayers was measured and analyzed with use of the Gibbs equation for adsorption at an interface . The mean molecular insertion area derived from isotherms of P450 2B4 in a DPPE monolayer at a pressure of 19 mN/m, 680 +/- 95 A2 is large enough to accommodate two to four transmembrane helices . The large insertion area and the fact that the truncated cytochrome retains as much as 30% of its membrane localization when expressed in Escherichia coli (Pernecky, S . J., Larson, J . R., Philpot, R . M., and Coon, M . J . (1993) Proc . Natl . Acad . Sci . USA 90, 2651-2655) suggest that this cytochrome is not deeply embedded but that other regions, in addition to the amino-terminal 26 residues, may be involved in the interaction of cytochrome P450 with the membrane . Arch Biochem Biophys, 1998 Nov 1, 359(1), 31 - 41 Impact of the presequence of a mitochondrium-targeted precursor, preadrenodoxin, on folding, catalytic activity, and stability of the protein in vitro; Goder V et al.; Bovine preadrenodoxin, an adrenocortical precursor protein destined for mitochondrial import, was expressed in Escherichia coli as an {2Fe-2S} cluster-containing protein . It was found in inclusion bodies, purified from there, and finally reconstituted to obtain soluble holo-protein . The impact of the presequence on folding of the protein using biochemical and biophysical approaches has been investigated . Upon unfolding the preprotein reveals a decrease in the denaturational enthalpy and heat capacity compared with mature adrenodoxin, indicating an incomplete unfolding of the preprotein with remaining residual structure . Moreover, the data obtained show that the presequence is solvent exposed in aqueous solution with no preference for secondary structure elements and that it does not disturb the accurate folding of the mature part of the protein . The latter conclusion is also based on the finding that the precursor in vitro exhibits electron transfer function comparable to the mature protein, adrenodoxin . While the reduction of cytochrome c, reflecting the interaction between adrenodoxin and its reductase, and the interaction with CYP11B1 have not been significantly affected by the presence of the presequence, the binding affinity of preadrenodoxin to CYP11A1 is 5.5-fold lower than that of the mature form . Biochemistry, 1998 Nov 3, 37(44), 15542 - 7 Excess nucleoside triphosphates (or zinc) allow recovery of alkaline phosphatase activity following refolding under reducing conditions; Ghosh N et al.; The contribution of ATP and other nucleotides to the stabilization of non-native structures has been described for some proteins . We report here the effect of GTP, ATP, and their nonhydrolyzable analogues on the denaturation and renaturation of the enzyme Escherichia coli alkaline phosphatase . We show that GTP, ATP, and their nonhydrolyzable analogues considerably stimulate renaturation of AP in the presence of 2-mercaptoethanol where spontaneous renaturation is completely arrested due to reduction of S-S bonds . GTP is the most efficient inducer of reconstitution of the active site and appears to play a specific role besides being a substrate . The reconstituted protein was found to be in the reduced form despite having near-normal activity . The self-refolded oxidized form and the GTP-refolded reduced form had the same KM/kcat values and showed similar structural properties . We conclude that GTP can not only induce reconstitution of dimerization-competent monomers because of its substrate nature but also act as a modulator of the activity of AP . We also report here on the Zn2+-assisted reconstitution of E . coli AP under reducing condition . The prior formation of a disulfide bond for positioning the active site residues in the proper geometry is unnecessary under this condition. Biochemistry, 1998 Nov 3, 37(44), 15503 - 12 Reactions catalyzed by tetrahydrobiopterin-free nitric oxide synthase; Rusche KM et al.; Murine macrophage nitric oxide synthase (NOS) was expressed in E . coli and purified in the presence (holoNOS) or absence (H4B-free NOS) of (6R)-tetrahydro-L-biopterin (H4B) . Isolation of active enzyme required the coexpression of calmodulin . Recombinant holoNOS displayed similar spectral characteristics and activity as the enzyme isolated from murine macrophages . H4B-free NOS exhibited a Soret band at approximately 420 nm and, by analytical gel filtration, consisted of a mixture of monomers and dimers . H4B-free NOS catalyzed the oxidation of NG-hydroxy-L-arginine (NHA) with either hydrogen peroxide (H2O2) or NADPH and O2 as substrates . No product formation from arginine was observed under either condition . The amino acid products of NHA oxidation in both the H2O2 and NADPH/O2 reactions were determined to be citrulline and Ndelta-cyanoornithine (CN-orn) . Nitrite and nitrate were also formed . Chemiluminescent analysis did not detect the formation of nitric oxide (*NO) in the NADPH/O2 reaction . The initial inorganic product of the NADPH/O2 reaction is proposed to be the nitroxyl anion (NO-) based on the formation of a ferrous nitrosyl complex using the heme domain of soluble guanylate cyclase as a trap, and the formation of a ferrous nitrosyl complex of H4B-free NOS during turnover of NHA and NADPH . NO- is unstable and, under the conditions of the reaction, is oxidized to nitrite and nitrate . At 25 degreesC, the H2O2-supported reaction had a specific activity of 120 +/- 14 nmol min-1 mg-1 and the NADPH-supported reaction had a specific activity of 31 +/- 6 nmol min-1 mg-1 with a KM,app for NHA of 129 +/- 9 microM . HoloNOS catalyzed the H2O2-supported reaction with a specific activity of 815 +/- 30 nmol min-1 mg-1 and the NADPH-dependent reaction to produce *NO and citrulline at 171 +/- 20 nmol min-1 mg-1 with a KM, app for NHA in the NADPH reaction of 36.9 +/- 0.3 microM. Biochemistry, 1998 Nov 3, 37(44), 15442 - 8 Dependence of the 16S rRNA decoding region structure on Mg2+, subunit association, and temperature; Noah JW et al.; The effects of Mg2+ concentration, subunit association, and temperature on the structure of 16S rRNA in the Escherichia coli ribosome were investigated using UV cross-linking and gel electrophoresis analysis . Mg2+ concentrations between 1 and 20 mM and temperatures between 5 and 55 degreesC had little effect on the frequency of 12 of the 14 cross-links in 30S subunits and modest effects on the same cross-links in 70S ribosomes . In contrast, two cross-links, C967 x C1400 and C1402 x C1501, involving rRNA in the decoding region are present in 30S subunits only above 3 mM Mg2+, increase in frequency at higher Mg2+ concentration, and are both more frequent when 50S subunits are included in the reactions . In 70S ribosomes, the cross-link C1402 x C1501 increases but the cross-link C967 x C1400 decreases at higher Mg2+ concentrations . One cross-link, C1397 x U1495, is detected only in 70S ribosomes and decreases in frequency as Mg2+ concentration is increased . An additional cross-link, A1093 x C1182, decreases upon subunit association . The cross-link frequency differences indicate that the arrangement of the decoding region of the 16S rRNA, but not in the rest of the subunit, is readily altered by Mg2+ ions and subunit association. Biochemistry, 1998 Nov 3, 37(44), 15408 - 13 Interaction of guanine nucleotides with the signal recognition particle from Escherichia coli; Jagath JR et al.; The bacterial signal recognition particle (SRP) is an RNA-protein complex . In Escherichia coli, the particle consists of a 114 nt RNA, a 4.5S RNA, and a 48 kDa GTP-binding protein, Ffh . GDP-GTP exchange on, and GTP hydrolysis by, Ffh are thought to regulate SRP function in membrane targeting of translating ribosomes . In the present paper, we report the equilibrium and kinetic constants of guanine nucleotide binding to Ffh in different functional complexes . The association and dissociation rate constants of GTP/GDP binding to Ffh were measured using a fluorescent analogue of GTP/GDP, mant-GTP/GDP . For both nucleotides, association and dissociation rate constants were about 10(6) M-1 s-1 and 10 s-1, respectively . The equilibrium constants of nonmodified GTP and GDP binding to Ffh alone and in SRP, and in the complex with the ribosomes were measured by competition with mant-GDP . In all cases, the same 1-2 microM affinity for GTP and GDP was observed . Binding of both GTP and GDP to Ffh was independent of Mg2+ ions . The data suggest that, at conditions in vivo, (i) there will be rapid spontaneous GDP-GTP exchange, and (ii) the GTP-bound form of Ffh, or of SRP, will be predominant. Biochemistry, 1998 Nov 3, 37(44), 15363 - 75 Fourier transform infrared analysis of purified lactose permease: a monodisperse lactose permease preparation is stably folded, alpha-helical, and highly accessible to deuterium exchange; Patzlaff JS et al.; The lactose permease, encoded by the lacY gene of Escherichia coli, is an integral membrane protein that functions as a proton and lactose symporter . In this study, we have characterized a novel monodisperse, purified preparation of lactose permease, as well as functionally reconstituted lactose permease, using spectroscopic techniques . The purification of monodisperse lactose permease has been aided by the development of a lacY gene product containing an amino-terminal six histidine affinity tag . In the novel purification method described here, lactose permease is purified from beta-dodecyl maltoside-solubilized membrane vesicles using three sequential column steps: hydroxyapatite, nickel-nitriloacetic acid (Ni-NTA) affinity, and cation-exchange chromatography . The hydroxyapatite step was shown to be essential in reducing aggregation of the final purified protein . Amino acid composition analysis and sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) analysis support the conclusion that the protein has been purified to greater than 90% homogeneity . The protein has been successfully reconstituted and has been shown to be active for lactose transport . Fourier transform infrared (FT-IR) spectroscopy has been performed on monodisperse lactose permease and on proteoliposomes containing functional lactose permease . FT-IR spectroscopy supports the conclusion that the monodisperse lactose permease preparation is 80% alpha-helical and stably folded at 20 degreesC; thermal denaturation is first detected at 70 degreesC . Because the purified protein is also readily susceptible to 2H exchange, these results suggest that the protein is conformationally flexible and that 2H exchange is facilitated as the result of conformational fluctuations from the folded state. Biochemistry, 1998 Nov 3, 37(44), 15277 - 88 Structural determinants of the bifunctional corn Hageman factor inhibitor: x-ray crystal structure at 1.95 A resolution; Behnke CA et al.; Corn Hageman factor inhibitor (CHFI) is a bifunctional 127 residue, 13.6 kDa protein isolated from corn seeds . It inhibits mammalian trypsin and Factor XIIa (Hageman Factor) of the contact pathway of coagulation as well as alpha-amylases from several insect species . Among the plasma proteinases, CHFI specifically inhibits Factor XIIa without affecting the activity of other coagulation proteinases . We have isolated CHFI from corn and determined the crystallographic structure at 1.95 A resolution . Additionally, we have solved the structure of the recombinant protein produced in Escherichia coli at 2.2 A resolution . The two proteins are essentially identical . The proteinase binding loop is in the canonical conformation for proteinase inhibitors . In an effort to understand alpha-amylase inhibition by members of the family of 25 cereal trypsin/alpha-amylase inhibitors, we have made three-dimensional models of several proteins in the family based on the CHFI coordinates and the coordinates determined for wheat alpha-amylase inhibitor 0.19 {Oda, Y., Matsunaga, T., Fukuyama, K., Miyazaki, T., and Morimoto, T . (1997) Biochemistry 36, 13503-13511} . From an analysis of the models and a structure-based sequence analysis, we propose a testable hypothesis for the regions of these proteins which bind alpha-amylase . In the course of the investigations, we have found that the cereal trypsin/alpha-amylase inhibitor family is evolutionarily related to the family of nonspecific lipid-transfer proteins of plants . This is a new addition to the group which now consists of the trypsin/alpha-amylase inhibitors, 2S seed storage albumins, and the lipid-transfer family . Apparently, the four-helix conformation has been a successful vehicle in plant evolution for providing protection from predators, food for the embryo, and lipid transfer. Biochemistry, 1998 Nov 3, 37(44), 15266 - 76 Allostery in rabbit pyruvate kinase: development of a strategy to elucidate the mechanism; Friesen RH et al.; Isozymes of pyruvate kinase (PK) expressed in rabbit muscle and kidney show different allosteric kinetics . The only amino acid changes in the two isozymes, originating from alternative RNA splicing, occur at a stretch of 55 amino acids in the C domain near the subunit interface . The self-correcting distance geometry (SECODG) program DIAMOD was used to calculate a homology model of these interfacial contacts in the four helix bundle of the kidney PK dimer, based on the X-ray structure of the tetrameric rabbit muscle PK {Larsen et al . (1994) Biochemistry 33, 6301-6309} . Energy refinement with the program FANTOM, using the ECEPP/2 force field to assess packing and electrostatic interactions between the two subunits, yielded two groups of energetically favorable conformations . The primary difference in the two groups is the loop conformation of residue Pro 402, which is serine in muscle PK . In one loop conformation, the conserved Lys 421 can form an intersubunit salt bridge as observed in the muscle PK crystal structure . The other loop conformation favors an alternative intrasubunit salt bridge, similar to that found in the Escherichia coli PK structure, which was not used for generating the model . The intersubunit salt bridge leads to an intersubunit hydrogen bonding between Lys 421 of one subunit and Tyr 443 of the other . To provide direct evidence on the roles of these residues, site-directed mutagenesis of the muscle PK gene was conducted . Converting Ser 402 to a proline and Tyr 443 to a phenylalanine changed neither the secondary nor the tetrameric structure, as measured by far UV-CD and sedimentation velocity, respectively . However, the S402P mutant exhibits steady-state kinetics, indicating that the mutant is more reponsive to regulation by effectors, while the mutant Y443F was essentially equivalent to wild-type muscle PK protein except for a lower affinity to phosphoenolpyruvate . These findings suggest a pivotal role for a few key residues in the allosteric regulation in PK. Arch Microbiol, 1998 Nov, 170(6), 464 - 8 The biotin protein MadF of the malonate decarboxylase from Malonomonas rubra; Berg M et al.; The gene for the biotin protein MadF of the Na+-pumping malonate decarboxylase from Malonomonas rubra was expressed in Escherichia coli together with the gene for the biotin ligase birA . MadF was partially purified from cell lysates by ammonium sulfate precipitation . Almost pure biotin protein was obtained by subsequent gel chromatography . With recombinant MadF, malonate decarboxylase activity of M . rubra cell extracts previously inactivated by avidin was recovered . Thus, the biological activity of recombinant MadF was proven . Despite the coexpression of birA, MadF was poorly biotinylated . This effect was not caused by an insufficient cofactor supply due to elevated protein levels at constant biotin uptake rates . Attempts to improve the cofactor incorporation were made by site-directed mutagenesis, by coexpression of madK, and by N-terminal elongation of MadF . These measures improved the fraction of MadF containing biotin to maximally 5% . These results might indicate the existence of a biotin ligase in M . rubra with an altered substrate specificity different from that of BirA. Arch Microbiol, 1998 Nov, 170(6), 418 - 26 The vhuU gene encoding a small subunit of a selenium-containing {NiFe}-hydrogenase in Methanococcus voltae appears to be essential for the cell; Pfeiffer M et al.; We developed a general method for the site-specific deletion of gene sequences to obtain new selectable markers in the archaeon Methanococcus voltae . Using a deletion in the hisA gene, a vector was integrated into the chromosome by homologous recombination, thereby reconstituting histidine prototrophy . The vector contained the beta-glucuronidase gene uidA of Escherichia coli as a reporter under the control of an M . voltae promoter that normally drives the expression of a selenium-free {NiFe}-hydrogenase after selenium deprivation . This construct has allowed us to check whether the selenium supply was sufficiently low to induce the transcription of the genes encoding the selenium-free hydrogenases . We tried to introduce a chromosomal deletion of the vhuU gene of the archaeon M . voltae by gene replacement and by keeping the cells under selenium deprivation . The gene vhuU encodes the very small, selenocysteine-containing subunit that is part of the primary reaction center of the Vhu hydrogenase . All transformants bearing the deletion also contained the vhuU wild-type gene . Therefore, the vhuU gene appears to be essential for the cell even under conditions that lead to the induction of the selenium-free homologue Vhc of the Vhu hydrogenase. Eur J Biochem, 1998 Oct 1, 257(1), 263 - 73 Structural information on a cecropin-like synthetic peptide, Shiva-3 toxic to the sporogonic development of Plasmodium berghei; Boisbouvier J et al.; This study is a contribution towards the understanding of the mode of action of Shiva-3 and more generally that of cecropin-like peptides . Structural information on Shiva-3 (a cecropin-like synthetic peptide) in water and in a membrane-mimicking environment (trifluoroethanol alcohol, SDS) were obtained using analytical centrifugation, CD and NMR spectroscopies . A total of 20 converged structures were retained on the basis of 197 non-redundant experimental constraints, including 166 distance constraints from the nuclear Overhauser effect measurements and 31 dihedral angle restraints derived from the purged COSY experiments . Some results obtained in presence of SDS are also presented . The toxic effects of the peptides obtained by cleavage (trypsin and lysine-C hydrolysis) of Shiva-3 on Escherichia coli and on Plasmodium berghei sporogonic stages are reported . Biological effects are discussed in relation to the calculated structure . The antiparasite activity and the low mosquito toxicity of Shiva-3 make this peptide a good candidate for genetic transformation of mosquito vectors which warrants further studies aimed at the improvement of the molecule. Eur J Biochem, 1998 Oct 1, 257(1), 242 - 8 Caspase 3 specifically cleaves p21WAF1/CIP1 in the earlier stage of apoptosis in SK-HEP-1 human hepatoma cells; Park JA et al.; We report here that p21WAF1/CIP1, an inhibitor of cyclin kinases, underwent proteolytic processing into a smaller fragment, p14, in the early stage of apoptosis in SK-HEP-1 cells . Apoptosis was induced by either staurosporine or ginsenoside Rh2, a ginseng saponin with a dammarane skeleton . Proteolytic processing was the result of caspase-3 activity, which accompanied the early changes in cell morphology and DNA fragmentation . p21WAF1/CIP1 translated in vitro was cleaved into a p14 fragment when incubated with cell extracts obtained from either ginsenoside Rh2-treated or staurosporine-treated cells . Cleavage was equally inhibited in both cases by adding Ac-DEVD-CHO, a specific caspase-3 inhibitor, but not by Ac-YVAD-CHO, a specific caspase-1 inhibitor . Similarly, p21WAF1/CIP1 was efficiently cleaved by recombinant caspase-3, overexpressed in Escherichia coli . Moreover, the endogenous p21WAF1/CIP1 of untreated cell extracts was also cleaved by recombinant caspase 3, as measured by immunoblotting . Mutation analysis allowed identification of two caspase-3 cleavage sites, DHVD112/L and SMTD149/F, which are located within or near the interaction domains for cyclins, Cdks, and proliferating cell nuclear antigen (PCNA) . Taken together, these results show that ginsenoside Rh2 and staurosporine increase caspase-3 activity, which in turn directly cleaves p21WAF1/CIP1 during the early stages of apoptosis . We propose that proteolytic cleavage of p21WAF1/CIP1 is a functionally relevant event that allows release of the cyclin/Cdk complex from the p21WAF1/CIP1 inhibitor, resulting in the elevated levels of cyclin/Cdk kinase activity seen in the earlier stage of apoptosis. Eur J Biochem, 1998 Oct 1, 257(1), 236 - 41 Nonenzymatic palmitoylation at Cys 3 causes extra-activation of the alpha-subunit of the stimulatory GTP-binding protein Gs; Mollner S et al.; Treatment of crude stimulatory GTP-binding protein of adenylyl cyclase (Gs) from turkey erythrocyte membranes with hydroxylamine results in twofold enhancement of adenylyl cyclase activity following reconstitution with adenylyl cyclase type V expressed in Spodoptera frugiperda cells (Sf9) cells . Enhancement by hydroxylamine of immunoaffinity purified Gs was still 1.5-fold, while that of Gs purified according to the multiple-step procedure by Northup, J . K., Sternweis, P . C., Smigel, M . D., Schleifer, L . S., Ross, E . M . & Gilman, A . G . (Proc . Natl Acad . Sci . USA 78, 6516-6520, 1980) was close to unity . The alpha-subunit of the stimulatory GTP-binding protein expressed in Escherichia coli (r(alpha)s), likewise, failed to show an effect by hydroxylamine . Surprisingly, guanosine 5'-O-(3-thiotriphosphate) (GTP{S})-liganded r(alpha)s, treated with palmitoyl-CoA for 14 h at 4 degrees C resulted in sixfold enhancement of reconstitutive activity . In contrast, that of the GDP-liganded r(alpha)s(beta)gamma heterotrimer was not improved by palmitoylation and consecutive activation with GTP{S}, although incorporation of {3H}palmitate into momomer and heterotrimer was identical . While adenylyl cyclase type-V activity reconstituted by r(alpha)s x GTP{S} was not influenced by betagamma-subunits, that activated by palmitoylated r(alpha)s x GTP{S} was considerably inhibited, suggesting a higher affinity of palmitoylated r(alpha)s for betagamma-subunits . On treatment of either form with the proteinase Lys-C, less than 25% of the label was found in a stable M(r) 38000 fragment with intact C terminus, but lacking the N-terminal portion . Absence of the latter did not affect activation by r(alpha)s, but caused a >90% loss of extra-activation by palmitoylated r(alpha)s . The results also indicate that nonenzymatic, much in the same way as physiological enzymatic, palmitoylation of alpha(s) occurs predominantly on Cys 3. Eur J Biochem, 1998 Oct 1, 257(1), 55 - 61 Characterisation of two putative protein Ser/Thr kinases from actinomycete Streptomyces granaticolor both endowed with different properties; Vomastek T et al.; The structural genes, pkg4 and pkg3, encoding two putative protein serine/threonine kinases in Streptomyces granaticolor, have been cloned and sequenced . The genes were isolated after screening genomic sublibraries with specific probes obtained by PCR amplification of chromosomal DNA using degenerate primers which correspond to amino acid sequences highly conserved in eukaryotic protein Ser/Thr kinases . The sequences of these genes predict polypeptide chains of 761 and 780 amino acids for Pkg4 and Pkg3, respectively . The genes are separated by only 2 bp and therefore probably constitute an operon . pkg4, which is positioned upstream of pkg3, contains a UUALeu codon suggesting a developmental-dependent mode of expression . The amino-terminal half of both proteins clearly shares similarities with the family of protein Ser/Thr kinases . Both proteins studied also possess a region rich in Pro and Ala residues and a repeating motif of 11 amino acid residues, the function of which is unknown, in the carboxy-terminal domain . Expression of pkg4 in Escherichia coli gave rise to two different forms: a soluble protein autophosphorylated at threonine residues and an insoluble form phosphorylated at threonine and serine residues . In contrast, when pkg3 was expressed in E . coli, no autophosphorylation was detected either in vivo or in vitro. Eur J Biochem, 1998 Oct 1, 257(1), 21 - 30 Alliinase {S-alk(en)yl-L-cysteine sulfoxide lyase} from Allium tuberosum (Chinese chive)--purification, localization, cDNA cloning and heterologous functional expression; Manabe T et al.; Alliinase {S-alk(en)yl-L-cysteine sulfoxide lyase}, a pyridoxal-phosphate-(Pxy-P)-dependent enzyme, is responsible for the degradative conversion of S-alk(en)yl-L-cysteine sulfoxide to volatile odorous sulfur-containing metabolites in Allium plants . We have purified alliinase from shoots of Allium tuberosum (Chinese chive) to apparent homogeneity by SDS/polyacrylamide gel electrophoresis . A cDNA clone encoding alliinase was isolated from a cDNA library constructed from whole plants of A . tuberosum by hybridization screening with a synthetic 50-residue oligonucleotide encoding a conserved region of the alliinases from onion and garlic . The isolated cDNA encoded a protein of 476 amino acid residues with a molecular mass of 54083 Da . The deduced amino acid sequence exhibited 66-69% identities with those of reported alliinases from onion, garlic and shallot . The partial amino acid sequence, which was determined for a V8 protease-digested peptide fragment of the purified alliinase, was perfectly matched with the sequence deduced from the cDNA . An expression vector of recombinant alliinase cDNA was constructed in yeast . The catalytically active protein was in the soluble fraction of transformed yeast . Site-directed mutagenesis experiments indicated that Lys280 was essential for the catalytic activity and, thus, a possible Pxy-P-binding residue . The mRNA expression of the alliinase gene comprising a multigene family in the shoots of green plants was twofold higher than that in the roots of green plants; however, the expression in the shoots of etiolated plants was only 13% that in green shoots, although the expression in the roots was not remarkably different between in green and etiolated plants . Immunohistochemical investigation indicated that the alliinase protein is predominantly accumulated in the bundle sheath cells of shoots of A . tuberosum. Eur J Biochem, 1998 Oct 1, 257(1), 1 - 10 Partial characterization and three-dimensional-structural localization of eight mutations in exon 7 of the human phenylalanine hydroxylase gene associated with phenylketonuria; Bjorgo E et al.; The molecular basis for the metabolic defect in patients with phenylketonuria has been characterized for seven missense point mutations (R252G/Q, L255V/S, A259V/T and R270S) and a termination mutation (G272X) in an evolutionarily conserved motif of exon 7 in the catalytic domain of the human phenylalanine hydroxylase (hPAH) gene . The mutations were expressed in three heterologous in vitro systems . When expressed as fusion proteins with maltose-binding protein in Escherichia coli five of the mutant proteins demonstrated a defect in the normal ability of hPAH to fold and assemble as homotetramer/dimer, and they were mostly recovered as inactive aggregated forms . Only for the R252Q and L255V mutants were catalytically active tetramer and dimer recovered and for R252G some dimer, i.e . 20% (R252Q, tetramer), 44% (L255V, tetramer) and 4.4% (R252G, dimer) of the activity for the respective wild-type (wt) forms . When expressed by a coupled in vitro transcription-translation system, all the mutant enzymes were recovered as a mixture of non-phosphorylated and phosphorylated forms with a low homospecific activity (i.e . maximum 11% of wt-hPAH for the L255V mutant) . When transiently expressed in human embryonic kidney (A293) cells a very low level of immunoreactive PAH protein was recovered in spite of normal PAH mRNA levels . All these mutations resulted in variant hPAH proteins which revealed a defect in oligomerization, an increased sensitivity to limited proteolysis in vitro, reduced cellular stability and a variable reduction in their catalytic activity . All these effects seem to result from structural perturbations of the monomer, and based on the crystal structure of the catalytic domain of hPAH, an explanation is provided for the impact of the mutations on the folding and oligomerization of the monomers. Int J Radiat Biol, 1998 Oct, 74(4), 511 - 9 Characterization of DNA damage induced by gamma-radiation-derived water radicals, using DNA repair enzymes; Kuipers GK et al.; PURPOSE: To characterize the DNA damage profiles due to gamma-radiation induced water radicals . MATERIALS AND METHODS: Double stranded (ds) phiX174 DNA was irradiated in aqueous solution with gamma-rays under different gassing conditions (O2, N2O or N2) and the damage profiles were determined using DNA repair enzymes . RESULTS: The DNA damage profile under O2 is characterized by about equal numbers of direct single-strand breaks (ssb) and Fpg sensitive sites, whereas endonuclease III and exonuclease III sites are formed in lower amounts . The DNA damage profiles under N2O and N2 in phosphate buffer consist predominantly of direct single-strand breaks . Fpg sensitive sites dominate the DNA damage profile under N2 in phosphate buffer in the presence of the radical scavenger 2-methyl propan-2-ol, where (almost) only .H atoms are present . CONCLUSIONS: Both .OH radicals and .H atoms induce direct single-strand breaks, but .OH radicals are the most effective ones . Fpg sensitive sites are induced in high amounts by both .OH radicals and H atoms, but when both types of radicals are present, the formation of Fpg sensitive sites is prevented . Hydrated electrons (e(aq)-) contribute to inactivation of DNA, although only a very small fraction of the e(aq)- is involved in this process. Brain, 1998 Oct, 121 ( Pt 10), 1895 - 902 Induction of experimental autoimmune neuritis with peripheral myelin protein-22; Gabriel CM et al.; Two myelin proteins, P2 basic protein and P0 glycoprotein, can induce experimental autoimmune neuritis (EAN), a model of human inflammatory neuropathy . We investigated whether peripheral nerve myelin protein-22 (PMP22), the gene for which is duplicated in hereditary motor sensory neuropathy type la, can also induce EAN . PMP22 cDNA produced by the reverse transcriptase-polymerase chain reaction from rat sciatic nerve was expressed in Escherichia coli as a fusion protein with glutathione-S-transferase (GST) . Ten Lewis rats were immunized with purified PMP22 fusion protein (50-100 microg) and eight controls with the same amount of GST . Two additional animals were immunized with each of two peptides (250 microg) of the human PMP22 extracellular sequences . Animals were examined daily until 20 days following immunization, when they underwent neurophysiological examination . A serum sample was then taken, prior to perfusion with glutaraldehyde and removal of the sciatic nerves and cauda equina . PMP22-immunized animals developed antibodies to the fusion protein and five out of 10 developed limp tails . No changes were observed in controls immunized with GST or in animals immunized with peptide . The mean compound motor action potentials elicited in the foot muscles by stimulation of the sciatic nerve at the sciatic notch and of the tibial nerve at the ankle were significantly reduced in the PMP22-immunized group (P < 0.05) . Spinal roots from the group of animals immunized with PMP22 showed sparse infiltration of mononuclear cells, oedema and demyelination . PMP22 now deserves consideration as an autoantigen in human acute inflammatory demyelinating polyradiculoneuropathy. Oncogene, 1998 Oct 22, 17(16), 2143 - 8 Conservation of function and primary structure in the BRCA1-associated RING domain (BARD1) protein; Ayi TC et al.; The BRCA1 gene encodes a tumor suppressor that has been implicated in hereditary forms of breast and ovarian cancer . During S phase of the cell cycle, BRCA1 polypeptides are found in discrete nuclear bodies ('BRCA1 nuclear dots') together with HsRad51, a human homolog of the E . coli recA protein, and BARD1, a protein that interacts with BRCA1 to form a stable heterodimer . BARD1 is structurally similar to BRCA1 in that both molecules harbor an amino-terminal RING domain and two carboxy-terminal BRCT domains . Here we describe the amino acid sequence and expression pattern of murine Bard1 . A comparison of the mouse and human sequences reveals that the recognizable protein motifs of BARD1 are well conserved, including the RING domain, the three tandem ankyrin repeats, and, to a lesser extent, the two BRCT domains . However, the remaining sequences of BARD1 display a markedly lower degree of phylogenetic conservation, comparable to those reported for BRCA1 and BRCA2 . Moreover, murine Bard1 retains the ability to associate in vivo with BRCA1, and its expression pattern in adult mice mirrors that of Brca1, with elevated levels of RNA transcripts found in the testes and spleen . These data suggest that BRCA1 and BARD1 have co-evolved to participate in a common pathway of tumor suppression. Mol Immunol, 1998 Jun, 35(8), 469 - 78 Cloning and immunological characterization of the allergen Hel a 2 (profilin) from sunflower pollen; Asturias JA et al.; Sunflower (Helianthus annuus) sensitization is not always related with occupational allergy . We have isolated the allergen profilin (Hel a 2) from this Compositae plant, cloned and sequenced five cDNAs encoding for full-length or partial Hel a 2 . Natural sunflower profilin reacted with specific IgE in the 121 sera tested, at a frequency of 30.5% . Expression of the cDNA encoding Hel a 2 in Escherichia coli and a simple purification procedure by poly-L-proline chromatography allowed immunological characterization of the recombinant allergen . Binding of monoclonal antibodies against sunflower profilin revealed that some epitopes responsible for antigen-specific IgG production were not present in the recombinant allergen . High cross-reactivity has been found between recombinant Hel a 2 and profilins from other Compositae plants and also from botanically distant plants. J Food Prot, 1998 Oct, 61(10), 1269 - 74 The incidence of Escherichia coli on beef carcasses and its association with aerobic mesophilic plate count categories during the slaughter process; Siragusa GR et al.; An analysis of 535 prefabricated beef carcass samples taken in three processing plants demonstrated an association between the mesophilic aerobic plate count (APC) class and the incidence of obtaining an Escherichia coli-positive sample . Beef carcasses were sampled from three separate plants; one was a fed-beef processing plant and the other two were cow/bull plants . Samples were obtained by sponging and were analyzed for APC and E . coli . When samples were classified into four APC levels or classes (class 1: < 2, class 2: > or = 2 and < 3, class 3: > or = 3 and < 4, and class 4: > or = 4 log CFU/cm2), a trend indicating that samples from higher APC classes were more likely to be positive for E . coli biotype 1 was observed . Of the APC class 4 samples (> or = 4 log CFU/cm2), 88% were positive for the presence of E . coli, as opposed to 21% in APC class 1 (< 2 log CFU/cm2) . Univariate chi-square analysis of the resulting contingency tables from reclassified data (class 1: < 2, class 2: > or = 2 and < 3, and class 3: > or = 3 log CFU/cm2) indicated a strong association between APC class and the incidence (presence or absence) of an E . coli-positive sample . Using multivariate analysis to account for influences of plant and within plant processing site, the data indicated a strong positive linear trend between the presence of E . coli and the APC class. Genes Cells, 1998 Aug, 3(8), 511 - 20 Escherichia coli HU protein suppresses DNA-gyrase-mediated illegitimate recombination and SOS induction; Shanado Y et al.; BACKGROUND: The HU protein is an abundant DNA binding protein of bacteria and is a major constituent of the bacterial nucleoid . HU protein is known to be involved in several fundamental biological functions, including DNA supercoiling, DNA replication, site-specific DNA inversion, and transposition . It is generally thought that a functional relationship exists between HU protein and DNA gyrase . RESULTS: We found that an hupA hupB double mutant displays enhanced spontaneous illegitimate recombination during the formation of lambdabio transducing phage in Escherichia coli . Nucleotide sequence analysis of the resulting transducing phages showed that the E . coli bio and lambda recombination sites did not have any homologous sequence . This mutation also enhanced the spontaneous expression of SOS functions . Furthermore, either overproduced GyrA protein or a temperature-sensitive gyrB mutation suppressed the illegitimate recombination enhanced by the defect of HU protein . CONCLUSION: These results show that the defect of HU induces illegitimate recombination and SOS response, which are probably mediated by DNA gyrase, implying that HU protein plays roles in suppression of illegitimate recombination and SOS response through interaction with DNA gyrase. Nature, 1998 Oct 22, 395(6704), 808 - 13 The G protein G alpha12 stimulates Bruton's tyrosine kinase and a rasGAP through a conserved PH/BM domain; Jiang Y et al.; Heterotrimeric guanine-nucleotide-binding proteins (G proteins) are signal transducers that relay messages from many receptors on the cell surface to modulate various cellular processes . The direct downstream effectors of G proteins consist of the signalling molecules that are activated by their physical interactions with a G alpha or Gbetagamma subunit . Effectors that interact directly with G alpha12 G proteins have yet to be identified . Here we show that G alpha12 binds directly to, and stimulates the activity of, Bruton's tyrosine kinase (Btk) and a Ras GTPase-activating protein, Gap1m, in vitro and in vivo . G alpha12 interacts with a conserved domain, composed of the pleckstrin-homology domain and the adjacent Btk motif, that is present in both Btk and Gap1m . Our results are, to our knowledge, the first to identify direct effectors for G alpha12 and to show that there is a direct link between heterotrimeric and monomeric G proteins. Gastroenterology, 1998 Nov, 115(5), 1186 - 96 Guinea pig gastric mucosal cells produce abundant superoxide anion through an NADPH oxidase-like system; Teshima S et al.; BACKGROUND & AIMS: Superoxide anion (O2-) plays an important role in gastric pathophysiology . The aims of this study were to identify O2--producing activity in gastric mucosal cells and to elucidate its possible roles in inflammatory responses of the cells . METHODS: The amount of O2- was measured by the reduction of cytochrome c, and O2--producing cells were visualized by nitroblue tetrazolium reaction . Cytosolic components of the phagocyte reduced nicotinamide adenine dinucleotide phosphate (NADPH) oxidase were detected by immunoblotting and immunocytochemical analyses with antibodies against p47-phox and p67-phox . RESULTS: Gastric pit cells, but not parietal cells, spontaneously released O2- at 50 nmol . mg protein-1 . h-1 . NADPH or guanosine 5'-O-(3-thiotriphosphate) increased the release more than threefold, whereas diphenylene iodonium inhibited it . A reconstituted cell-free system showed that both membrane fraction and neutrophil-related cytosolic components were required for the activity . p47-phox and p67-phox were expressed in the cells . Live Helicobacter pylori organisms and their culture supernatants significantly increased the O2- release . Furthermore, H . pylori lipopolysaccharide could enhance the release more effectively than Escherichia coli lipopolysaccharide . The O2--dependent activation of nuclear factor kappaB occurred in these primed cells . CONCLUSIONS: Gastric pit cells may actively regulate inflammatory responses of gastric mucosa through a phagocyte NADPH oxidase-like activity. Appl Environ Microbiol, 1998 Nov, 64(11), 4217 - 25 Partial characterization of the Streptomyces lividans xlnB promoter and its use for expression of a thermostable xylanase from Thermotoga maritima; Chen CC et al.; Xylanase activity assays were used to screen a Streptomyces coelicolor genomic library in Escherichia coli, and a xylanase gene that is 99% identical to the xylanase B gene (xlnB) of S . lividans (GenBank accession no . M64552) was identified . The promoter region of this gene was identified by using a transcriptional fusion between the upstream region of the S . coelicolor xlnB gene and the xylE reporter gene . Transcription from the xlnB promoter was found to be induced by xylan and repressed by glucose . A single apparent transcription start site was identified by both primer extension analysis and in vitro run off transcription assays . Analysis of deletions of the promoter identified a region required for glucose repression . By using the transcriptional and protein localization signals of the Streptomyces xlnB gene, an in-frame translational fusion between the end of the xlnB signal sequence and the ATG of the Thermotoga maritima xynA gene was constructed . The xynA gene encodes a thermostable xylanase that has been demonstrated to be useful in the bleaching of Kraft pulp . The xlnB-xynA gene fusion was expressed in Streptomyces, and the activity of the protein produced was thermostable and was localized to the supernatant fraction of harvested cells. Appl Environ Microbiol, 1998 Nov, 64(11), 4210 - 6 An automated fluorescent PCR method for detection of shiga toxin-producing Escherichia coli in foods; Chen S et al.; An automated fluorescence-based PCR system (a model AG-9600 AmpliSensor analyzer) was investigated to determine whether it could detect Shiga toxin-producing Escherichia coli (STEC) . The AmpliSensor PCR assay involves amplification-mediated disruption of a fluorogenic DNA signal duplex (AmpliSensor) that is homologous to conserved target sequences in a 323-bp amplified fragment of Shiga toxin genes stx1, stx2, and stxe . Using the Amplisensor assay, we detected 113 strains of STEC belonging to 50 different serotypes, while 18 strains of non-Shiga-toxin-producing E . coli and 68 strains of other bacteria were not detected . The detection limits of the assay were less than 1 to 5 CFU per PCR mixture when pure cultures of five reference strains were used and 3 CFU per 25 g of food when spiked ground beef samples that were preenriched overnight were used . The performance of the assay was also evaluated by using 53 naturally contaminated meat samples and 48 raw milk samples . Thirty-two STEC-positive samples that were confirmed to be positive by the culture assay were found to be positive when the AmpliSensor assay was used . Nine samples that were found to be positive when the PCR assay was used were culture negative . The system described here is an automated PCR-based system that can be used for detection of all serotypes of STEC in food or clinical samples. Protein Eng, 1998 Sep, 11(9), 797 - 802 The spontaneous gating activity of OmpC porin is affected by mutations of a putative hydrogen bond network or of a salt bridge between the L3 loop and the barrel; Liu N et al.; Porins are trimeric channel-forming proteins of the outer membrane of Escherichia coli . Each subunit contains 16 beta-strands forming a transmembrane beta-barrel whose pore is constricted by the third extracellular loop (L3) . We investigated the effects of site-directed mutations at two critical regions of the OmpC porin: (i) the D315A mutation targets a key component of a putative hydrogen bond network linking the L3 loop to the adjacent barrel wall and (ii) the D118Q, R174Q and R92Q mutations target putative salt bridges at the root of the L3 loop . We purified the outer membrane fractions obtained from each mutant and reconstituted them in liposomes suitable for electrophysiology . Patch clamp experiments showed that the frequency of spontaneous transitions between open and closed states is increased in the D315A, D118Q and R92Q mutants but unchanged in the R174Q mutant . These transitions are not driven by transmembrane voltage changes and represent the thermal oscillations between functionally distinct conformations . The asymmetric voltage-dependent inactivation of the channels is not affected by the mutations, however, suggesting different molecular mechanisms for the spontaneous and voltage-dependent gating processes . We propose that the positioning or flexibility of the L3 loop across the pore, as governed by the putative hydrogen-bond network and a salt bridge, play a role in determining the frequency of spontaneous channel gating. Protein Eng, 1998 Sep, 11(9), 775 - 82 Comparative study of binase and barnase: experience in chimeric ribonucleases; Schulga A et al.; Chimeric enzymes were constructed to elucidate the differences in physicochemical properties of two related bacterial RNases, barnase and binase . Chimeras (Ba26Bi, Ba73Bi, Ba26Bi73Ba and Bi73Ba) contain six to thirteen residue substitutions relative to barnase, which are beyond the active site . The catalytic activity of RNases toward GpU, GpC and poly(I), as well as conformational distinctions and heat denaturation parameters, were studied . Thermal denaturation of binase, barnase and chimeric RNases is a two-state transition . The mutation-induced changes in the free energy of unfolding of barnase deduced from thermal and urea denaturation nearly coincide . The kinetic parameters for GpU and GpC demonstrate that the chimeras fall into two groups: barnase-like and binase-like . This division is determined by the origin of their C-terminal part (residues 73-110) which is also responsible for their thermostability at pH 2.4 . An inverse linear dependence was found between kcat for poly(I) and denaturation temperature of RNases at pH 5.5, which points out that certain lability of the protein molecule appears to be necessary for efficient polynucleotide cleavage. Photochem Photobiol, 1998 Oct, 68(4), 527 - 31 Assessment of the effects of various UV sources on inactivation and photoproduct induction in phage T7 dosimeter; Fekete A et al.; The correlation between the biologically effective dose (BED) of a phage T7 biological dosimeter and the induction of cyclobutane pyrimidine dimers (CPD) and (6-4) photoproducts ((6-4)PD) in the phage DNA was determined using seven various UV sources . The BED is the inactivation rate of phage T7 expressed in HT7 units . The CPD and (6-4)PD were determined by lesion-specific monoclonal antibodies in an immunodot-blot assay . The various lamps induced these lesions at different rates; the relative induction ratios of CPD to (6-4)PD increased with increasing effective wavelength of irradiation source . The amount of total adducts per phage was compared to the BED of phage T7 dosimeter, representing the average number of UV lesions in phage . For UVC (200-280 nm radiation) and unfiltered TL01 the number of total adducts approximates the reading; however, UV sources having longer effective wavelengths produced fewer CPD and (6-4)PD . A possible explanation is that although the most relevant lesions by UVC are the CPD and (6-4)PD, at longer wavelengths other photoproducts can contribute to the lethal damage of phages . The results emphasize the need to study the biological effects of solar radiation because the lesions responsible for the lethal effect may be different from those produced by various UV sources. Anal Chem, 1998 Oct 15, 70(20), 4433 - 40 Coupling 2D SDS-PAGE with CNBr cleavage and MALDI-TOFMS: a strategy applied to the identification of proteins induced by a hypochlorous acid stress in Escherichia coli; Dukan S et al.; A protocol including 2D SDS-PAGE, electroblotting proteins onto nitrocellulose membranes, and CNBr cleavage, followed by MALDI-MS analysis of intact proteins and peptide fragments and a database search, has been optimized and applied to the rapid identification of the Escherichia coli response to hypochlorous acid . The methodology has proved to be efficient from the point of view of sensitivity (picomole range) and selectivity . In particular, MALDI analysis of proteins and CNBr fragments by directly dissolving the membrane in an acetone solution of matrix, without previous elution, is reliable and reproducible . The accuracy of the MW determination is somewhat reduced compared to that of methods involving elution and purification of proteins and digests; nevertheless, the utilization of large MW windows combined with the pI entry in database searches had allowed, for most of the spots, the selection of only one protein candidate . Finally, 19 proteins exhibiting a response to hypochlorous acid stress have been confirmed or identified on the basis of this protocol. Fukuoka Igaku Zasshi, 1998 Sep, 89(9), 277 - 81 Obstructive jaundice due to multiple hepatic abscesses; Dohmen K et al.; A 63-year-old Japanese male with diabetes mellitus developed obstructive jaundice following the onset of multiple hepatic abscesses . Percutaneous transhepatic cholangiography showed intrahepatic bile duct irregularity and dilatations accompanied by a complete obstruction of the right branch of the intrahepatic bile duct . Three kinds of organisms were cultured from the blood and the drained bile . The cholangiographic changes returned to the normal after the liver abscesses subsided following biliary drainage and the administration of intravenous antibiotics. Vaccine, 1998 Dec, 16(20), 2069 - 76 Mucosal immunoadjuvant activity of recombinant Escherichia coli heat-labile enterotoxin and its B subunit: induction of systemic IgG and secretory IgA responses in mice by intranasal immunization with influenza virus surface antigen; Verweij WR et al.; The Escherichia coli heat-labile enterotoxin (LT) is a very potent mucosal immunogen . LT also has strong adjuvant activity towards coadministered unrelated antigens and is therefore of potential interest for development of mucosal vaccines . However, despite the great demand for such mucosal vaccines, the use of LT holotoxin as an adjuvant is essentially precluded by its toxicity . LT is composed of an A subunit, carrying t