Escherichia coli

Trends Microbiol, 1996 Nov, 4(11), 444 - 52
Colonization factors of human enterotoxigenic Escherichia coli (ETEC); Gaastra W et al.; Enterotoxigenic Escherichia coli (ETEC) is the most common cause of childhood and travellers' diarrhoea . The ability of ETEC to adhere to the intestinal epithelium of the host is an important virulence determinant, and adhesion is mediated by proteinaceous surface appendages called colonization factors.

Thromb Haemost, 1996 Nov, 76(5), 710 - 4
O-glycosylation of fibrinogen from different mammalian species as revealed by the binding of Escherichia coli biotinylated lectins; L'Hote C et al.; After the demonstration that neither N-glycans nor neuraminic acid are involved in the binding of K88 lectins to the B beta and gamma chains of porcine fibrinogen and that their recognition was due to O-glycans (L'Hote C, Berger S, Bourgerie S, Duval-Ifiah Y, Julien R, Karamanos Y . Infect Immun 1995; 63: 1927-1932) it clearly appeared that these lectins could be used as probes to detect O-glycans on fibrinogens of other species . The conclusion of the present study is that many mammalian fibrinogens contain complex O-glycans on B beta and gamma chains . In addition, the combined use of the biotinylated K99 lectin and the Peanut agglutinin demonstrated the presence of sialylated T-antigens on the A alpha chains of all the fibrinogens examined . These lectins can now be used to determine differences on the glycosylation status of fibrinogens within one species and also to detect O-glycans on other glycoproteins.

Alcohol Clin Exp Res, 1996 Nov, 20(8), 1395 - 400
Cross-tolerance between acute alcohol intoxication and endotoxemia; Bautista AP et al.; This study tests two hypotheses: (1) prior exposure to LPS induces cross-tolerance for the hepatic effects of subsequent short-term alcohol intoxication; and (2) short-term alcohol intoxication renders the liver resistant to the effects of acute endotoxemia, resulting in reduced production of superoxide and tumor necrosis factor . In the first group of experiments, male Sprague-Dawley rats were treated intravenously with E . coli lipopolysaccharide (LPS) (0.5 mg/kg) 48 hr before they were given an intravenous bolus of ethanol (1.75 g/kg), followed by 250-300 mg/kg/hr) for 3-5 hr . Superoxide release in the perfused liver was measured after 3-hr ethanol infusion . Pretreatment with LPS attenuated ethanol-mediated superoxide anion release by the perfused liver . The stimulatory effect of phorbol myristate acetate on hepatic release of superoxide was also decreased . In the second group of experiments, rats previously treated with ethanol for 5 hr, received an intravenous injection of LPS (1 mg/kg) . At 90 min after LPS, sera were collected for tumor necrosis factor alpha assay . Hepatic release of superoxide anion was determined 3 hr after LPS . Acute ethanol intoxication for 5 hr significantly reduced LPS-induced serum tumor necrosis factor activity and free radical release by the perfused liver . LPS-induced mortality was also decreased . In both groups of experiments serum corticosteroid levels were reduced during cross-tolerance . These results demonstrate that cross-tolerance develops between acute alcohol intoxication and endotoxemia manifesting in reduced hepatic production of cytotoxic cytokines and superoxide anions.

Eur Respir J, 1996 Nov, 9(11), 2426 - 8
Vertebral osteomyelitis caused by thoracic empyema, or vice versa?
Liesker KR, Taconis WK, Plasmans CM, Schreurs AJ.
Thoracic empyema and vertebral osteomyelitis is a rare combination . We report a case of vertebral osteomyelitis possibly caused by a progressive thoracic empyema . The causative pathogen was Escherichia coli . Computed tomographic (CT) scan and magnetic resonance imaging (MRI) suggested the diagnosis of vertebral osteomyelitis, confirmed by transthoracic needle aspiration and operative findings . Aetiology and treatment are discussed.

Shock, 1996 Nov, 6(5), 357 - 64
SK&F 86002, a dual cytokine and eicosanoid inhibitor, attenuates endotoxin-induced cardiopulmonary dysfunction in the pig; Triplett EA et al.; Cytokines and eicosanoids are well documented important mediators of endotoxemia . Bicyclic imidazoles are a novel class of nonsteroidal anti-inflammatory compounds that display unique pharmacological profiles by reducing cytokine production and arachidonic acid metabolism . In this study, we evaluated the ability of the bicyclic imidazole, SK&F 86002, to attenuate endotoxin-induced cardiopulmonary dysfunction . Pigs were randomly assigned to one of four groups: LPS (n = 5), given .5 microgram/kg/h 055:B5 Escherichia coli lipopolysaccharide (LPS) intravenously (i.v.) for 6 h; saline (n = 5); SK&F 86002 (n = 3), given 50 mg/kg SK&F 86002 orally 30 min prior to anesthesia; and SK&F 86002 + LPS (n = 5) . Administration of LPS resulted in cardiopulmonary dysfunction characterized by decreased stroke volume and arterial oxygen tension, and increased room air alveolar-arterial oxygen gradient, pulmonary arterial pressure, pulmonary vascular resistance, and peak intratracheal pressure . Additionally, LPS administration was associated with leukopenia and increased pulmonary myeloperoxidase activity . Pretreatment with SK&F 86002 attenuated LPS induced hypotension, hypoxemia and bronchoconstriction and blocked the pulmonary hypertension . SK&F 86002 blocked the LPS-induced increase in myeloperoxidase activity, indicating a reduction in pulmonary neutrophil infiltration, but had no effect on systemic leukopenia . Pretreatment with SK&F 86002 significantly attenuated LPS-induced increases in plasma thromboxane B2 and tumor necrosis factor-alpha . We hypothesize that ameliorating effects of SK&F 86002 in this endotoxin model of cardiopulmonary dysfunction are related to inhibition of cytokine and eicosanoid biosynthesis.

Shock, 1996 Nov, 6(5), 345 - 50
Growth hormone and insulin-like growth factor I augment Escherichia coli-killing activity of murine peritoneal exudative cells; Inoue T et al.; Effects of growth hormone (GH) and insulin-like growth factor (IGF)-I on Escherichia coli-killing activity of murine peritoneal exudative cells (PECs) were investigated . Plasma from the mice, injected subcutaneously with saline, GH (4.8 mg/kg/day), or IGF-I(24 mg/kg/day) for 6 days, was mixed with E . coli and pooled murine PECs . Plasma from GH- and IGF-I-treated mice modestly but significantly augmented the E . coli-killing activity of PECs, as compared with that from saline controls . Plasma from IGF-I-treated mice also enhanced PEC interleukin 1 production . In the next experiment, PECs preincubated with medium, GH (10-1000 ng/mL), or IGF-I (50-5000 ng/mL) for 3 h were investigated for E . coli-killing activity . Preincubation of PECs with all concentrations of GH and IGF-I significantly enhanced the E . coli-killing activity of PECs, as compared with the medium control . These results indicate that GH and IGF-I enhance phagocytosis and the E . coli-killing activity of PECs, via a modestly increased plasma capacity to support these activities, as well as by a strong direct action.

Am J Physiol, 1996 Nov, 271(5 Pt 2), H1953 - 61
A time-dependent balance between endothelins and nitric oxide regulating portal resistance after endotoxin; Pannen BH et al.; To test whether endothelins are involved in the regulation of portal resistance after endotoxin pretreatment and whether their effects are modulated by nitric oxide (NO), rats received intraperitoneal injections of Escherichia coli lipopolysaccharide (LPS, 1 mg/kg body wt) or saline . Six and twenty-four hours later, livers were isolated and perfused . Analyses of portal pressure-flow (P-Q) relationships and epifluorescence microscopy were performed before and after administration of 1) the NO synthesis inhibitor N omega-nitro-L-arginine methyl ester (L-NAME, 10(-3) M), followed by L-arginine (2 x 10(-3) M), or 2) the endothelin ETA/ETB-receptor antagonist bosentan (2 x 10(-4) M), followed by L-NAME (10(-3) M) . LPS pretreatment increased all measures of resistance, which included total portal resistance, zero flow, incremental resistance (slopes of P-Q relationship), and sinusoid resistance . L-NAME had no effect in sham controls but increased all measures of resistance at 6 h after LPS and increased total and incremental resistance 24 h after LPS . L-Arginine reversed these changes . Bosentan reduced total and sinusoid resistance slightly in control livers and caused substantial reductions in all measures of resistance at 6 and 24 h after LPS; these were partially reversed after L-NAME at 6 but not at 24 h . Our data support the hypothesis that a critical balance between endothelin-mediated vasoconstrictor influences and NO-mediated vasodilator influences controls portal resistance after endotoxin pretreatment.

DNA Cell Biol, 1996 Nov, 15(11), 975 - 9
Interaction of rat Cdc37-related protein with retinoblastoma gene product; Ozaki T et al.; By using in vitro binding assays and the yeast two-hybrid system, we have found that a full-length rat Cdc37-related protein (RCdc37) could associate specifically with the retinoblastoma susceptibility gene product (pRB) . A series of GST-RCdc37 deletion mutants was constructed to define the amino acid sequence required for the interaction with pRB . A GST-RCdc37 (-20 approximately 229) possessed an activity to associate with pRB, whereas GST-RCdc37 (-20 approximately 165) lost its activity, indicating that the amino acid sequence between 166 and 229 of RCdc37 was essential for the association with pRB . Interestingly, there exists a highly conserved pRB-binding motif (LXCXE; X = any amino acid) that is essential for pRB binding of SV40 large T antigen, E1A, and E7 proteins . A similar experiment using a pRB deletion mutant revealed that the carboxy-terminal portion of pRB was required for binding to RCdc37.

Eur J Biochem, 1996 Nov 1, 241(3), 827 - 34
Expression and characterization of human perlecan domains I and II synthesized by baculovirus-infected insect cells; Groffen AJ et al.; We present the in vitro expression and purification of N-terminal fragments of human perlecan in insect cells . Three tailored fragments of human perlecan cDNA were introduced into the polyhedrin locus of baculovirus expression vectors (BEVs) encoding amino acids 1-196 (domain I), 1-404 (domain I + IIa) and 1-506 (domain I + IIab) . The integrity of the BEVs was checked by DNA sequencing, polymerase chain reaction, restriction enzyme analysis and Southern blotting . Northern hybridization and metabolic labeling with {35S}methionine showed that expression of the perlecan-(1-404)- and the -(1-506)- peptide was successful, but in the case of the perlecan-(1-196)-peptide no recombinant protein was produced . Immunoblotting showed that both the (1-404)-peptide and (1-506)-peptide are recognized by 95J10, a monoclonal antibody that was previously raised against perlecan-(24-404)-peptide expressed in Escherichia coli . Gel permeation and anion-exchange chromatography were applied to purify the recombinant proteins . Glycosaminoglycans were demonstrated to be present . Deglycosylation with chondroitinase ABC showed that the perlecan-(1-404)-peptide was glycosylated with chondroitin sulfate residues . Consistent with these results, glycosaminoglycans isolated from the perlecan-(1-404)-peptide were identified as chondroitin sulfate by agarose gel electrophoresis . Furthermore the perlecan-(1-404)-peptide showed affinity to immobilized basic fibroblast growth factor . The availability of baculovirus-derived recombinant perlecan fragments will facilitate domain-specific investigation of the structural and functional properties of perlecan in the future.

Eur J Biochem, 1996 Nov 1, 241(3), 750 - 5
Evidence for propeptide-assisted folding of the calcium-dependent protease of the cyanobacterium Anabaena; Baier K et al.; The Ca(2+)-dependent protease of the cyanobacterium Anabaena variabilis is a cytoplasmic enzyme with a substrate specificity like trypsin . Its previously published DNA sequence {Maldener, I., Lockau, W., Cai, Y . & Wolk, C . P . (1991) Mol . Gen . Genet . 225, 113-120} contained a sequencing error . Here we report the corrected sequence which shows, that the Ca(2+)-protease belongs to the family of subtilases (subtilisin-like serine proteases) . Consistent with its cytoplasmic localization, a pre-sequence is not found . The enzyme is produced as a precursor with a large amino-terminal propeptide . Expression of the pro-region and mature region (protease domain) in Escherichia coli cells in trans demonstrates that formation of the active enzyme requires the propeptide . The results demonstrate that propeptide-assisted protein folding also occurs with cytoplasmic enzymes, in support of the hypothesis that this mechanism is a widespread phenomenon.

J Chem Inf Comput Sci, 1996 Nov-Dec, 36(6), 1187 - 94
RASSE: a new method for structure-based drug design; Luo Z et al.; A novel method, RASSE, has been developed to suggest reasonable structures which can fit well to the binding sites of receptors . Molecules are generated by an iterative growing procedure in which atoms are added to existing fragments . Potential ligands are then picked out by special scoring rules . This atomgrowing based method is characterized by combinatorial searching of atom types and conformations . To some extent, it is the computer simulation of combinatorial chemistry . This method has been applied to the design of inhibitors for E . coli dihydrofolate reductase and human phospholipase A2 . The results demonstrate that this program is capable of generating reasonable structures, thus proving its power in drug design.

Nephrol Dial Transplant, 1996 Nov, 11(11), 2215 - 22
Sera from patients with anti-GBM nephritis including goodpasture syndrome show heterogenous reactivity to recombinant NC1 domain of type IV collagen alpha chains; Dehan P et al.; BACKGROUND: Goodpasture (GP) syndrome is defined by the clinical association of pulmonary haemorrhage with rapidly progressive glomerulonephritis . The disease is caused by pathogenic autoantibodies directed against type IV collagen, which is a major structural component of glomerular basement membranes (GBM) . METHODS: The non-collagenous domains (NC1) of all six human type IV collagen alpha chains was produced in E . coli as recombinant fusion proteins with glutathione-S transferase . Sera from 10 patients with different types of anti-GBM nephritis, including GP syndrome, were tested for reactivity with the six proteins using immunoblotting of denatured and reduced proteins and ELISA without reduction . RESULTS: All 10 sera reacted with the alpha 3 (IV) collagen chain by immunoblotting and ELISA . One serum also recognized the alpha 2(IV), alpha 4(IV), alpha 5(IV) and alpha 6(IV) chains by immunoblotting . ELISA measurements revealed reactivity of several other sera with alpha 2(IV), alpha 4(IV) or alpha 6(IV) but not with alpha 5(IV) collagen chains . No reactivity was observed with the alpha 1(IV) chain . CONCLUSION: Autoantibodies in anti-GBM nephritis may not be directed only against the alpha 3(IV) collagen chain and they frequently recognize conformational epitopes.

Am J Trop Med Hyg, 1996 Nov, 55(5), 570 - 6
Molecular cloning and antigenic mapping of heat-shock protein 70 from the malaria species Plasmodium bergheI; Fan JY et al.; We have isolated a 70-kD heat-shock protein (hsp-70) cDNA from Plasmodium berghei . A cDNA clone encoding the P . berghei hsp-70 was isolated and sequenced, demonstrating that it is highly homologous with other Plasmodium hsp-70s . One of the common features is a series of GGMP amino acid repeats at the carboxy terminus; there is also a long, AT-rich 5' untranslated region, a hallmark of other malarial RNAs . Hydropathy and antigenicity analyses suggest the presence of two hydrophilic domains . Recombinant peptides comprising different fragments of hsp-70 were expressed in Escherichia coli and assessed for antigenicity with antiserum from mice immunized with sonicated extracts of P . berghei . Antigenic sites map to regions that include the two hydrophilic domains.

Chem Biol, 1996 Nov, 3(11), 923 - 36
A new enzyme superfamily - the phosphopantetheinyl transferases; Lambalot RH et al.; BACKGROUND: All polyketide synthases, fatty acid synthases, and non-ribosomal peptide synthetases require posttranslational modification of their constituent acyl carrier protein domain(s) to become catalytically active . The inactive apoproteins are converted to their active holo-forms by posttranslational transfer of the 4'-phosphopantetheinyl (P-pant) moiety of coenzyme A to the sidechain hydroxyl of a conserved serine residue in each acyl carrier protein domain . The first P-pant transferase to be cloned and characterized was the recently reported Escherichia coli enzyme ACPS, responsible for apo to holo conversion of fatty acid synthase . Surprisingly, initial searches of sequence databases did not reveal any proteins with significant peptide sequence similarity with ACPS . RESULTS: Through refinement of sequence alignments that indicated low level similarity with the ACPS peptide sequence, we identified two consensus motifs shared among several potential ACPS homologs . This has led to the identification of a large family of proteins having 12-22 % similarity with ACPS, which are putative P-pant transferases . Three of these proteins, E . coli EntD and o195, and B . subtilis Sfp, have been overproduced, purified and found to have P-pant transferase activity, confirming that the observed low level of sequence homology correctly predicted catalytic function . Three P-pant transferases are now known to be present in E . coli (ACPS, EntD and o195); ACPS and EntD are specific for the activation of fatty acid synthase and enterobactin synthetase, respectively . The apo-protein substrate for o195 has not yet been identified . Sfp is responsible for the activation of the surfactin synthetase . CONCLUSIONS: The specificity of ACPS and EntD for distinct P-pant-requiring enzymes suggests that each P-pant-requiring synthase has its own partner enzyme responsible for apo to holo activation of its acyl carrier domains . This is the first direct evidence that in organisms containing multiple P-pant-requiring pathways, each pathway has its own posttranslational modifying activity.

Mol Microbiol, 1996 Nov, 22(3), 555 - 71
Degradation by proteases Lon, Clp and HtrA, of Escherichia coli proteins aggregated in vivo by heat shock; HtrA protease action in vivo and in vitro; Laskowska E et al.; Thermally aggregated, endogenous proteins of Escherichia coli form a distinct fraction, denoted S, which is separable by sucrose-density-gradient centrifugation . It was shown earlier that DnaK, DnaJ, IbpA and IbpB heat-shock proteins are associated with the S fraction . Comparison of the rise and decay of the S fraction in mutants defective for heat-shock proteases Lon (La), Clp, HtrA (DegP, Do) and in wild-type strains made studies of proteolysis and the function of the heat-shock response possible in vivo . Different timing and the extent of action of particular proteases was revealed by the initial size and decay kinetics of the S fraction . The proteases Lon, Clp, and HtrA all participated in removal of the aggregated proteins . Mutation in the gene encoding ClpB caused the most prominent effect (47% stabilization of the S fraction) . The correlation between the disappearance of the S fraction and proteolytic activity was supported by the result of the in vitro reaction . Approximately one third of the isolated S fraction was converted to trichloroacetic acid-soluble products by the purified HtrA protease . Mg2+ ions stimulated the reaction, in contrast to the reaction of the HtrA protease with casein . The digestion of the aggregated proteins, unlike the digestion of casein, by HtrA protease in vitro was inhibited by added DnaJ, which might reflect protection of the aggregated proteins in vivo by DnaJ from excessive degradation . One might expect that such an activity of DnaJ would promote denatured protein renaturation versus proteolysis . Moreover, among the aggregated proteins that are discernible by electrophoresis, none could be identified as being more susceptible than any other to HtrA degradation . The separation pattern of these proteins before and after the in vitro digestion did not show a difference corresponding to the loss of about 30% of constituting proteins . This was interpreted as recognition by the HtrA protease of a state of protein denaturation rather than specific amino acid sequences in particular proteins . We conclude that the fraction consisting of proteins heat-aggregated in vivo (i.e . the S fraction) contains endogenous substrates for the heat-shock proteases tested . Their use for in vitro reaction reveals information that is in some respects different from that obtained with exogenous substrates such as casein.

Mol Microbiol, 1996 Nov, 22(3), 459 - 71
Ligand-induced conformational change in the ferrichrome-iron receptor of Escherichia coli K-12; Moeck GS et al.; Ferrichrome-iron is actively transported across the outer membrane of Escherichia coli by the TonB-dependent receptor FhuA . To obtain FhuA in a form suitable for secondary-structure analyses, a hexahistidine tag was inserted into a surface-located site and the recombinant protein was purified by metal chelate chromatography . Functional studies indicated that the presence of the hexahistidine tag did not interfere with FhuA localization or with ligand-binding activity . Ferrichrome protected lysine 67 but not lysine 5 of purified recombinant FhuA from trypsinolysis . Results from trypsin digestion were interpreted as a conformational change in FhuA which had occurred upon ferrichrome binding, thereby preventing access of trypsin to lysine 67 . Circular dichroism and Fourier transform infrared spectroscopy revealed a predominance of beta-sheet structure for the purified protein . In the presence of ferrichrome, FhuA exhibited a secondary structure and a thermostability which were similar to FhuA without ligand . The addition of ferrichrome to purified FhuA reduced the ability of certain anti-FhuA monoclonal antibodies to bind to the receptor . All antibodies which could in this manner discriminate between FhuA and FhuA bound to ferrichrome had their determinants within a loop which is toward the N-terminus and which is exposed to the periplasm . These data indicate that the binding of ferrichrome induces a structural change that is propogated across the outer membrane and results in an altered conformation of a periplasmically exposed loop of FhuA . It is proposed that by such an alteration of FhuA conformation, TonB is triggered to energize the active transport of the bound ligand across the outer membrane.

J Interferon Cytokine Res, 1996 Nov, 16(11), 973 - 81
Characterization and biologic activities of recombinant rat soluble interleukin-6 receptor; Thibault V et al.; The soluble interleukin-6 receptor (sIL-6R) consists of the extracellular domain of the membrane-bound IL-6 receptor (gp80) found on many types of cells . Contrary to most other soluble cytokine receptors, it possesses in vitro agonistic properties, yet its physiologic role remains unknown . We have generated a cDNA encoding the rat sIL-6R and have expressed and purified the protein using Escherichia coli and baculovirus systems . Analysis of purified protein by electrophoresis and silver staining showed a single band migrating at 35 kDa for E . coli (nonglycosylated) and at 47 kDa for baculovirus-derived material . The purified protein is biologically active, as determined by the ability to convert human hepatoma cells (HepG2) from nonresponsive to responsive to rat IL-6 and induce acute-phase protein synthesis . Most important, we show that rat sIL-6R directly induces proliferation of the IL-6-dependent murine hybridoma cell line (B9) in an IL-6-like manner, with 50% proliferation induced by 100 ng/ml of baculovirus-derived receptor protein . Physiologic concentrations of sIL-6R dramatically enhance the sensitivity of B9 cells to IL-6, indicating that the bioassay for IL-6 is susceptible to modulation by the presence of sIL-6R in rodent serum samples . This sIL-6R-dependent B9 cell proliferation is fully abrogated by antibodies directed against rodent IL-6 and indicates autocrine production of low amounts of IL-6 by the B9 cell line.

Plant Physiol, 1996 Nov, 112(3), 1141 - 9
Evidence that barley 3-hydroxy-3-methylglutaryl-coenzyme a reductase kinase is a member of the sucrose nonfermenting-1-related protein kinase family; Barker JH et al.; A protein kinase was partially purified from barley (Hordeum vulgare L . cv Sundance) endosperm by ammonium sulfate fractionation, followed by ion-exchange, Reactive Blue, Mono-Q, and phosphocellulose chromatography . It was shown to phosphorylate Arabidopsis 3-hydroxy-3-methylglutaryl-coenzyme A (HMG-CoA) reductase and a synthetic peptide that was shown previously to act as a substrate for HMG-CoA reductase kinase purified from cauliflower, confirming it to be barley HMG-CoA reductase kinase . Sodium dodecyl sulfate-polyacrylamide gel electrophoresis of the partially purified preparation showed the presence of a polypeptide with an approximate relative molecular weight (M(r)) of 60,000, which is the size predicted for the barley sucrose nonfermenting-1 (SNF1)-related protein kinases BKIN2 and BKIN12 . Antisera were raised to a rye (Secale cereale L.) SNF1-related protein kinase (RKIN1) expressed in Escherichia coli as a fusion with maltose-binding protein and to a synthetic peptide with a sequence that is conserved in, and specific to, plant members of the SNF1-related protein kinase family . The maltose-binding protein-RKIN1 fusion protein antiserum recognized a doublet of polypeptides with an approximate M(r), of 60,000 in crude endosperm extracts and a single polypeptide in root extracts, which co-migrated with the smaller polypeptide in the endosperm doublet . Both antisera recognized a polypeptide with an approximate M(r) of 60,000 in the partially purified protein kinase preparation, suggesting strongly that barley HMG-CoA reductase kinase is a member of the SNF1-related protein kinase family.

J Cell Sci, 1996 Nov, 109 ( Pt 11), 2661 - 72
PTL-1, a microtubule-associated protein with tau-like repeats from the nematode Caenorhabditis elegans; Goedert M et al.; Tau, MAP2 and MAP4 are structural microtubule-associated proteins (MAPs) that promote the assembly and stability of microtubules . They share three or four imperfect tandem repeats of an amino acid motif, which is involved in the binding to microtubules . All sequences to data containing this motif are of mammalian origin . We report here the cloning and functional characterisation of a new member of this family of proteins from the nematode Caenorhabditis elegans . This protein exists as two isoforms of 413 and 453 amino acids with four or five tandem repeats that are 50% identical to the tau/MAP2/MAP4 repeats . Both isoforms bind to microtubules and promote microtubule assembly, with the five-repeat isoform being more effective at promoting assembly than the four-repeat isoform . When expressed in COS cells, the five-repeat isoform co-localises with microtubules and induces the formation of microtubule bundles, whereas its expression in Sf9 cells leads to the extension of long unipolar processes . In view of its length, amino acid sequence and functional characteristics, we have named this invertebrate structural MAP 'Protein with Tau-Like Repeats' (PTL-1) . In C . elegans PTL-1 is expressed in two places known to require microtubule function . It is first seen in the embryonic epidermis, when circumferentially oriented microtubules help to distribute forces generated during elongation . Later, it is found in mechanosensory neurons which contain unusual 15 protofilament microtubules required for the response to touch . These findings indicate that MAPs of the tau/MAP2/MAP4 family are found throughout much of the animal kingdom, where they may play a role in specialised processes requiring microtubules.

Protein Expr Purif, 1996 Nov, 8(3), 374 - 80
Purification and characterization of the hexokinase from Schistosoma mansoni, expressed in Escherichia coli; Armstrong RL et al.; The hexokinase (ATP;D-hexose 6-phosphotransferase, EC 2.7.1.1) of Schistosoma mansoni has been expressed in Escherichia coli as a fusion protein including an N-terminal polyhistidine tag and enterokinase cleavage site . The enzyme was purified by metal chelate chromatography to > 95% homogeneity, based on analysis by SDS-gel electrophoresis and isoelectric focusing . The absorbance at 280 nm was 0.54 for a 1 mg/ml solution (molar extinction coefficient 2.7 x 10(4) cm2 mol) . The pI of the S . mansoni hexokinase was 6.0-6.2, slightly more acidic than the rat Type I isozyme (pI 6.35) . The S . mansoni enzyme migrated as a single band of activity during nondenaturing cellulose acetate electrophoresis; the mobility was slightly greater than the rat Type I isozyme, consistent with the estimated pI . The Km values for substrates glucose and ATP were 128 +/- 10 and 927 +/- 41 microM, respectively . In accord with a previous report, the S . mansoni hexokinase exhibited moderate sensitivity to inhibition (competitive vs ATP) by the product, glucose 6-phosphate, with a Ki approximately 150 microM; the product analog, 1,5-anhydroglucitol 6-phosphate, was somewhat less effective as an inhibitor, with Ki approximately 500 microM . These kinetic properties were not altered by removal of the N-terminal fusion partner by enterokinase treatment . Immunological crossreactivity between the rat Type I isozyme and the S . mansoni hexokinase was demonstrated by immunoblotting, but this was markedly dependent on the preparation of antiserum used . The activity of the enzyme is apparently highly dependent on maintenance of free sulfhydryl groups . Activity was maintained during storage in the presence of monothioglycerol; activity lost during storage in the absence of monothioglycerol could be partially restored by treatment with this reagent.

Protein Expr Purif, 1996 Nov, 8(3), 365 - 73
Characterization of purified recombinant Bet v 1 with authentic N-terminus, cloned in fusion with maltose-binding protein; Spangfort MD et al.; A gene encoding the pollen major allergen Bet v 1 from Betula verrucosa (White Birch) has been cloned and expressed in Escherichia coli as a fusion with maltose-binding protein and a Factor Xa proteolytic cleavage site . A generally applicable cloning strategy based on polymerase chain reaction was designed to position the Factor Xa proteolytic site so that the authentic amino terminus of Bet v 1 was generated after cleavage . Fusion protein was isolated by amylose affinity chromatography and enzymatically cleaved by incubation with Factor Xa . Recombinant Bet v 1 was isolated by gel filtration and gave rise to a single band with apparent molecular weight of 17 kDa when analyzed by SDS-polyacrylamide gel electrophoresis . N-terminal sequencing of the first 20 amino acids showed complete agreement with the deduced Bet v 1 DNA sequence . Mass spectrometry showed that recombinant Bet v 1 has a molecular mass of 17,440 +/- 2 Da; 86% of the recombinant Bet v 1 amino acid sequence could be verified by digestion with Lys-C and mass spectrometric peptide mapping . The yield of purified recombinant Bet v 1 was 10 mg per liter E . coli cell culture . Two-dimensional gel electrophoresis of purified recombinant protein gave rise to one major protein spot and one or two minor spots focusing at slightly different pHs . The immunochemical properties of recombinant protein were indistinguishable from those of naturally occurring Bet v 1 when compared using a panel of murine monoclonal antibodies and serum IgE from birch pollen allergic patients . Furthermore, recombinant Bet v 1 elicited T-cell proliferation comparable to that of natural Bet v 1 . Thus, the methods used for bacterial expression and protein purification result in relatively high yields of folded recombinant Bet v 1 with correct N-terminal sequence and molecular mass . Furthermore, the B- and T-cell epitope structures of recombinant Bet v 1 closely resemble those of the natural protein from pollen.

Protein Expr Purif, 1996 Nov, 8(3), 358 - 64
Overexpression of human prothrombin in permanent cell lines using a dominant selection/amplification fusion marker; Herlitschka SE et al.; Human prothrombin was overexpressed in transformed eukaryotic cells using a dominant bifunctional selection and amplification marker . The marker consists of the murine wild-type dihydrofolate reductase (dhfr) cDNA and the Escherichia coli hygromycin phosphotransferase gene fused in frame . The gene of interest is connected by the encephalomyocarditis virus 5' untranslated region to the fusion marker gene, forming a dicistronic transcription unit . The human prothrombin gene (FII) was used to monitor expression after initial selection for hygromycin B resistance and DHFR activity . In Chinese hamster ovary (CHO) cells, 5-15 mU prothrombin/10(6) cells per 24 h was obtained; in human 293 kidney cells levels of 20-50 mU/ 10(6) cells per 24 h were obtained . Methotrexate-mediated amplification of the foreign gene in CHO cells resulted in a more than 10-fold increase in FII expression, while in the presence of methotrexate, 293 cells expressed 200-250 mU/10(6) cells per 24 h . The use of this fusion marker within a dicistronic transcription unit allowed efficient dominant selection of cell clones and amplification of the gene of interest . Stably transfected cell lines that were able to secrete high levels of processed gamma-carboxylated human prothrombin were thus obtained.

Protein Expr Purif, 1996 Nov, 8(3), 319 - 31
Expression and biological activity of genetic fusions between MalE, the maltose binding protein from Escherichia coli and portions of CD4, the T-cell receptor of the AIDS virus; Clement JM et al.; Hybrid molecules between MalE, the periplasmic maltose binding protein of Escherichia coli, and CD4, the human T-lymphocyte receptor for the AIDS virus HIV, have been constructed and purified . We show that CD4 can be fused as multiple repeats to both ends of a single MalE molecule . Hybrid proteins are exported into the periplasm of bacteria, bind monoclonal antibodies directed against CD4, bind HIV gp160, and inhibit HIV binding to CD4+ cells . MalE has been used as a scaffold to graft portions of CD4 . Deletion analysis allowed to define a minimal structural domain which folds in a way which is compatible with its biological activity . This minimal part was used to design compact hybrid molecules in which CD4 was inserted internally into MalE.

Protein Expr Purif, 1996 Nov, 8(3), 313 - 8
Expression, purification, and functional analysis of the DNA binding domain of the nuclear receptor Rev-erb beta; Terenzi H et al.; Rev-erb beta is a member of the nuclear receptor superfamily, which includes a group of transcription factors involved in the response to steroids, vitamin D, retinoic acids, and other lipophilic molecules . The Rev-erb nuclear receptors exist at least in two forms, namely alpha and beta, with a high degree of evolutionary conservation at the level of the DNA binding domain . Nevertheless, the exact type of DNA binding of these proteins is not fully understood . In order to get insight into this DNA binding mechanism we obtained a pure and functional homogeneous recombinant protein in bacteria corresponding to the DNA binding domain of Rev-erb beta (REDBD) . REDBD interacts with oligonucleotides containing an A/T-rich sequence preceding a single AGG-TCA site (Rev-RE) or with an AGGTCA direct repeat separated by 2 bp (Rev-DR2) . These results, and the affinity parameters of the interaction between REDBD and Rev-RE or Rev-DR2, indicate that our REDBD preparation is capable of specific and tight binding to DNA targets.

Protein Expr Purif, 1996 Nov, 8(3), 305 - 12
Expression of pig heart mitochondrial NADP-dependent isocitrate dehydrogenase in Escherichia coli; Soundar S et al.; Pig heart mitochondrial NADP-specific isocitrate dehydrogenase is the most extensively studied among the mammalian isocitrate dehydrogenases . The 1.2-kbp cDNA encoding this porcine mitochondrial NADP-specific enzyme has now been inserted into an expression vector, pMAL-c2, to be expressed as a fusion protein with maltose binding protein . Initially, the vector was constructed with a cleavage site for protease Factor Xa between the maltose binding protein and isocitrate dehydrogenase; however, since Factor Xa was also found to digest isocitrate dehydrogenase, a thrombin recognition site was substituted . The fusion protein was expressed in Escherichia coli by IPTG induction at 25 degrees C, and was separated from the endogenous E . coli isocitrate dehydrogenase by affinity chromatography on an amylose resin which adsorbs maltose binding protein and its fusion products . Cleavage of the fusion protein with thrombin generated pig heart NADP-specific isocitrate dehydrogenase, which was purified to homogeneity by affinity chromatography on Matrex Gel Red-A resin and gel filtration by FPLC . A 41-fold increase in specific activity to 37 enzyme units/mg with an approximate yield of 34% for the expressed enzyme was achieved by this purification procedure . This enzyme exhibits a single band (M(r) = 46,600) on polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate and, under standard assay conditions, has a Km for DL-isocitrate of 7.74 +/- 0.18 microM and a Km for NADP+ of 6.63 +/- 1.34 microM . These values are similar to the Kms measured for the enzyme purified from pig heart . The amino-terminal sequence of the expressed enzyme is identical with that of authentic porcine enzyme and distinguishable from the E . coli enzyme at 17 of the 18 residues determined . We conclude that this expression and purification system yields pure pig heart mitochondrial NADP-specific isocitrate dehydrogenase and should allow generation of wild-type and mutant enzymes in amounts suitable for their biochemical characterization and comparison.

Protein Expr Purif, 1996 Nov, 8(3), 299 - 304
Biochemical characterization of recombinant equine infectious anemia virus integrase; Engelman A; The integrase from equine infectious anemia virus (ELAV) was expressed in Escherichia coli as a polyhistidine fusion protein . The protein was purified under native and denaturing conditions using one-step nickel-affinity chromatography . The purified denatured protein was refolded in the presence of detergent . In vitro 3' processing and DNA strand transfer activities were analyzed under Mg(2+)- and Mn(2+)-dependent reaction conditions . Both protein preparations were similarly active . Only one viral DNA end was efficiently integrated during Mn(2+)-and Mg(2+)-dependent DNA strand transfer . Water was the predominant nucleophile for Mg(2+)- and Mn(2+)-dependent 3' processing activity . The results underscore functional similarities between EIAV integrase and the previously characterized HIV-1 enzyme.

Protein Expr Purif, 1996 Nov, 8(3), 295 - 8
High-level expression, purification, and crystallization of recombinant spinach glycolate oxidase in Escherichia coli; Stenberg K et al.; Glycolate oxidase is a flavin-dependent enzyme in the photorespiratory pathway in plants . Here we report the heterologous expression of glycolate oxidase in Escherichia coli and an isolation procedure which results in 4 mg pure protein per gram cell paste in only 1.5 days . This corresponds to a more than 50-fold improvement in yield compared to previously reported expression systems . The purified recombinant protein can be crystallized easily, and the crystals are isomorphous to those obtained from the protein isolated from spinach . The availability of large amounts of enzyme will be a great advantage in the 3D structural and biochemical studies of mutants and inhibitor complexes.

Protein Expr Purif, 1996 Nov, 8(3), 283 - 94
Expression and purification of the canine 54-kDa subunit of signal recognition particle as a his-tagged protein from Escherichia coli; Patel S et al.; The 54-kDa subunit of the signal recognition particle (SRP) binds nascent secretory polypeptides, binds the 7SL RNA (SRP RNA) component of SRP, and hydrolyzes GTP . Limited proteolysis of SRP 54-kDa suggests the protein has two domains, termed the G (GTP-binding) and M (methionine-rich) domains . The M domain is predicted to contain a number of amphiphilic helices, which provide a binding cleft for signal sequences . In order to obtain sufficient material for studies of relationships between structure and function, we have expressed the canine cDNA encoding the 54-kDa subunit in Escherichia coli using a T7 expression system . To aid purification, the protein was expressed with an amino-terminal extension encoding an initiating methionine and 10 histidine residues followed by an enterokinase cleavage site; 0.3mg of HIS-SRP 54-kDa was purified to give a single band on SDS-PAGE in 20% yield from 500 ml of cultured E . coli . Purified HIS-SRP 54-kDa was shown to be folded into the G and M domains, to inhibit the translocation of pre-prolactin into canine microsomes, and to bind mammalian SRP RNA only in the presence of the 19-kDa subunit of SRP.

Lipids, 1996 Nov, 31(11), 1173 - 8
Effects of growth temperature on the fatty acid composition of the free-living nematode Caenorhabditis elegans; Tanaka T et al.; The effects of growth temperature on the fatty acid compositions of the phosphatidylcholine (PC), phosphatidylethanolamine (PE), and total lipid (TL) fractions of the free-living nematode Caenorhabditis elegans were investigated . A reduction in growth temperature from 25 to 15 degrees C caused the proportions of eicosapentaenoic acid (20:5n-3) to increase from 23.6 to 32.5% in the PC, from 7.4 to 10.8% in the PE, and from 12.9 to 19.9% in the TL fractions . Conversely, the levels of dihomo-gamma-linolenic acid (20:3n-6) and arachidonic acid (20:4n-6) in these phospholipid fractions and the TL fraction both decreased with decreasing growth temperature . Analysis of the positional distribution of fatty acids in the PC fraction revealed that the change in the composition of C20 polyunsaturated fatty acid was obvious in position sn-2 . Lowering the growth temperature induced an increase in the level of the diacyl subclass of PE from 58% at 25 degrees C to 71% at 15 degrees C, with a concomitant decrease in the levels of the alkylacyl and alkenylacyl subclass of PE of C . elegans . These changes observed in the phospholipids of C . elegans might be one mechanism for adaptation to low temperature.

Invest Ophthalmol Vis Sci, 1996 Nov, 37(12), 2527 - 31
Experimental autoimmune uveoretinitis induced by the gamma-subunit of cyclic guanosine monophosphate phosphodiesterase in rats; Ren J et al.; PURPOSE: To investigate the capacity of the recombinant gamma-subunit (P gamma) of cyclic guanosine monophosphate phosphodiesterase to induce experimental autoimmune uveoretinitis in Lewis rats . METHODS: Bovine P gamma was expressed in Escherichia coli cells and purified by fast protein liquid chromatography . Lewis rats were immunized by a single footpad injection of P gamma emulsified in complete Freund's adjuvant . Clinical and histopathologic changes in the eye and pineal gland were examined . Lymphocytes were prepared from the lymph nodes of rats with uveitis and transferred by intraperitoneal injection to naive recipient rats . RESULTS: Immunization of rats with P gamma induced panuveitis and pinealitis with clinical and histopathologic changes similar to those induced by S-antigen . Lymphocytes from the lymph nodes of diseased rats transferred uveitis to naive recipients . CONCLUSIONS: P gamma, a retina-specific protein of molecular weight less than 10,000 kDa, is capable of inducing uveoretinitis in Lewis rats . The disease can be transferred adoptively to naive rats by injection of lymphocytes from donor rats with experimental autoimmune uveoretinitis . Inflammation of the pineal gland of immunized rats suggests that P gamma is not only localized to the retina but also to the pineal gland.

Poult Sci, 1996 Nov, 75(11), 1373 - 82
Effects of gonadal steroids and their antagonists on the humoral immune response of immune-selected broiler chicks; Leitner G et al.; The effects of gonadal hormones, testosterone (Te) and estrogen (E2) as factors in the development of the immune system in two lines, high response (HC) and low response (LC), of broiler chickens divergently selected for early or late immune maturation were studied . For this purpose, plasma Te and E2 levels were tested and correlated with immune response . Also, the effects of exogenous administration of gonadal steroids testosterone propionate (TP), dihydrotestosterone (DHT), and estradiol 3-benzoate (EB), and the nonsteroidal androgen antagonist flutomide (Flu) and anti-estrogen tamoxifen (Tam) on the immune system were studied . Male chicks of the LC line had a higher level of endogenous Te during first 30 d posthatch . The administration of TP or DHT had no noticeable effect on the humoral immune response, whereas DHT suppressed growth of the bursa of Fabricius of both sexes of HC line . No differences in the endogenous E2 level were observed between sexes in either line . Administration of EB inhibited comb and testicle growth and enhanced significantly the humoral immune response to Escherichia coli and sheep erythrocytes (SRBC) . The anti-androgen Flu and anti-estrogen Tam strongly inhibited humoral immune response to E . coli and SRBC antigen, whereas no effects on comb and testicle growth were observed . The experimental results suggest that gonadal hormones have similar principal posthatch effects in avian as in mammals; however, the gonadal steroids prehatch effects and the genetic-physiological-environmental effects require further study.

Poult Sci, 1996 Nov, 75(11), 1330 - 3
Effects of dietary taurine on growth and Escherichia coli resistance in chickens; Dunnington EA et al.; White Leghorn chicks selected for either high (HA) or low (LA) antibody response to SRBC were used in two experiments to ascertain the effects of differing levels of dietary taurine on growth, levels of taurine in blood plasma, heart, and brain, and response to Escherichia coli inoculation . A different level of dietary taurine was associated with maximal growth in each of the two selected populations . Line HA expressed greater growth with a relatively low level of taurine, whereas Line LA expressed greater growth with a relatively high level of taurine . Line LA chicks had a higher level of taurine in the heart than did Line HA chicks . Although increases in dietary taurine resulted in linear and curvilinear increases in plasma and heart taurine levels, they had no effect on brain levels . No apparent resistance or susceptibility to E . coli exposure was associated with stock or differing levels of dietary taurine.

Nucleic Acids Res, 1996 Nov 1, 24(21), 4369 - 71
Single locus microsatellites isolated using 5' anchored PCR; Fisher PJ et al.; Microsatellites are widely used as genetic markers because they are co-dominant, multiallelic, easily scored and highly polymorphic . A major drawback of microsatellite markers is the time and cost required to characterise them . We have developed a novel technique to reduce this cost by producing a microsatellite-rich PCR profile from genomic DNA which was cloned to yield a genomic library enriched for microsatellites . Sequence data and subsequent allele scoring within pedigrees revealed that these microsatellites retained their original repeat length and segregated normally . This technique permits genomic amplification with only one specific primer . Together with enrichment, the savings in primer costs reduces the cost of microsatellite characterisation considerably.

Nucleic Acids Res, 1996 Nov 1, 24(21), 4298 - 303
Drosophila ribosomal protein PO contains apurinic/apyrimidinic endonuclease activity; Yacoub A et al.; Drosophila ribosomal protein PO was overexpressed in Escherichia coli to allow for its purification, biochemical characterization and to generate polyclonal antibodies for Western analysis . Biochemical tests were originally performed to see if overexpressed PO contained DNase activity similar to that recently reported for the apurinic/apyrimidinic (AP) lyase activity associated with Drosophila ribosomal protein S3 . The overexpressed ribosomal protein was subsequently found to act on AP DNA, producing scissions that were in this case 5' of a baseless site instead of 3', as has been observed for S3 . As a means of confirming that the source of AP endonuclease activity was in fact due to PO, glutathione S-transferase (GST) fusions containing a Factor Xa cleavage site between GST and PO were constructed, overexpressed in an E.coli strain defective for the major 5'-acting AP endonucleases and the fusions purified using glutathione-agarose affinity column chromatography . Isolated fractions containing purified GST-PO fusion proteins were subsequently found to have authentic AP endonuclease activity . Moreover, glutathione-agarose was able to deplete AP endonuclease activity from GST-PO fusion protein preparations, whereas the resin was ineffective in lowering DNA repair activity for PO that had been liberated from the fusion construct by Factor Xa cleavage . These results suggested that PO was a multifunctional protein with possible roles in DNA repair beyond its known participation in protein translation . In support of this notion, tests were performed that show that GST-PO, but not GST, was able to rescue an E.coli mutant lacking the major 5'-acting AP endonucleases from sensitivity to an alkylating agent . We furthermore show that GST-PO can be located in both the nucleus and ribosomes . Its nuclear location can be further traced to the nuclear matrix, thus placing PO in a subcellular location where it could act as a DNA repair protein . Other roles beyond DNA repair seem possible, however, since GST-PO also exhibited significant nuclease activity for both single- and double-stranded DNA.

Nucleic Acids Res, 1996 Nov 1, 24(21), 4256 - 62
Different thermostabilities of FLP and Cre recombinases: implications for applied site-specific recombination; Buchholz F et al.; Genomic manipulations using site-specific recombinases rely on their applied characteristics in living systems . To understand their applied properties so that they can be optimally deployed, we compared the recombinases FLP and Cre in two assays . In both Escherichia coli and in vitro, FLP shows a different temperature optimum than Cre . FLP is more thermolabile, having an optimum near 30 degrees C and little detectable activity above 39 degrees C . Cre is optimally efficient at 37 degrees C and above . Consistent with FLP thermolability, recombination in a mammalian cell line mediated by a ligand- regulated FLP-androgen receptor fusion protein is more efficient at 35 degrees C than at higher temperatures . We also document a mutation in a commercially available FLP plasmid (FLP-F70L) which renders this recombinase even more thermolabile . The different temperature optima of FLP, FLP-F70L and Cre influence their strategies of usage . Our results recommend the use of Cre for applications in mice that require efficient recombination . The thermolabilities of FLP and FLP-F70L can be usefully exploited for gain of function and cell culture applications.

Nucleic Acids Res, 1996 Nov 1, 24(21), 4117 - 22
Investigation of some properties of oligodeoxynucleotides containing 4'-thio-2'-deoxynucleotides: duplex hybridization and nuclease sensitivity; Jones GD et al.; The thermal stabilities of the duplexes formed between 4'-thio-modified oligodeoxynucleotides and their DNA and RNA complementary strands were determined and compared with those of the corresponding unmodified oligodeoxynucleotides . A 16mer oligodeoxynucleotide containing 10 contiguous 4'-thiothymidylate modifications formed a less stable duplex with the DNA target (deltaTm/modification -1.0 degrees C) than the corresponding unmodified oligodeoxynucleotide . However, when the same oligodeoxynucleotide was bound to the corresponding RNA target, a small increase in Tm was observed (deltaTm/modification +0.16 degrees C) when compared with the unmodified duplex . A study to identify the specificity of an oligodeoxynucleotide containing a 4'-thiothymidylate modification when forming a duplex with DNA or RNA containing a single mismatch opposite the modification found the resulting Tms to be almost identical to the wild-type duplexes, demonstrating that the 4'-thio-modification in oligodeoxynucleotides has no deleterious effect on specificity . The nuclease stability of 4'-thio-modified oligodeoxynucleotides was examined using snake venom phosphodiesterase (SVPD) and nuclease S1 . No significant resistance to degradation by the exonuclease SVPD was observed when compared with the corresponding unmodified oligodeoxynucleotide . However, 4'-thio-modified oligodeoxynucleotides were found to be highly resistant to degradation by the endonuclease S1 . It was also demonstrated that 4'-thio-modified oligodeoxynucleotides elicit Escherichia coli RNase H hydrolysis of the RNA target only at high enzyme concentration.

J Bacteriol, 1996 Nov, 178(22), 6599 - 607
Sequence and transcript analysis of a novel Methanosarcina barkeri methyltransferase II homolog and its associated corrinoid protein homologous to methionine synthase; Paul L et al.; The sequence and transcript of the genes encoding a recently discovered coenzyme M methylase in Methanosarcina barkeri were analyzed . This 480-kDa protein is composed of two subunits in equimolar concentrations which bind one corrinoid cofactor per alphabeta dimer . The gene for the alphabeta polypeptide, mtsA, is upstream of that encoding the beta polypeptide, mtsB . The two genes are contiguous and overlap by several nucleotides . A 1.9-kb mRNA species which reacted with probes specific for either mtsA or mtsB was detected . Three possible methanogen consensus BoxA sequences as well as two sets of direct repeats were found upstream of mtsA . The 5' end of the mts transcript was 19 nucleotides upstream of the translational start site of mtsA and was positioned 25 bp from the center of the proximal BoxA sequence . The transcript was most abundant in cells grown to the late log phase on acetate but barely detectable in cells grown on methanol or trimethylamine . The amino acid sequence of MtsB was homologous to the cobalamin-binding fragment of methionine synthase from Escherichia coli and possessed the signature residues involved in binding the corrinoid, including a histidyl residue which ligates cobalt . The sequence of MtsA is homologous to the "A" and "M" isozymes of methylcobamide:coenzyme M methyltransferases (methyltransferase II), indicating that the alpha polypeptide is a new member of the methyltransferase II family of coenzyme M methylases . All three methyltransferase II homolog sequences could be aligned with the sequences of uroporphyrinogen decarboxylase from various sources . The implications of these homologies for the mechanism of corrinoid binding by proteins involved in methylotrophic methanogenesis are discussed.

J Bacteriol, 1996 Nov, 178(22), 6555 - 63
BfpB, an outer membrane lipoprotein required for the biogenesis of bundle-forming pili in enteropathogenic Escherichia coli; Ramer SW et al.; The bundle-forming pili (BFP) of enteropathogenic Escherichia coli are believed to play a role in pathogenesis by causing the formation of bacterial microcolonies that bind epithelial surfaces of the small intestine . This in vivo process is mimicked in vitro by the autoaggregation and localized adherence phenotypes . Expression of BFP, a member of the type IV pilus family, requires the enteroadherence factor (EAF) plasmid, which contains bfpA, the gene that encodes the principal structural subunit of BFP . Immediately downstream of bfpA are 13 open reading frames transcribed in the same direction as bfpA; together with bfpA, these compose the bfp gene cluster . Disruption of bfpB, the second open reading frame downstream of bfpA, was performed by allelic exchange . The resulting mutant, B171-8deltaB, did not exhibit the autoaggregation or localized adherence phenotype or produce BFP filaments . Thus, BfpB is required for pilus biogenesis . However, BfpA was produced at wild-type levels and processed normally by B171-8deltaB, indicating that BfpB acts at a step in the BFP biogenic pathway after production and processing of the structural subunit . Biochemical and cell fractionation studies showed that BfpB is a 58-kDa lipoprotein that is located primarily in the outer membrane . Assays of bfpA and bfpB mRNAs and protein expression showed that both genes are cotranscribed as part of an environmentally responsive operon that is regulated by growth phase and ammonium.

J Bacteriol, 1996 Nov, 178(22), 6508 - 17
Na+-induced transcription of nhaA, which encodes an Na+/H+ antiporter in Escherichia coli, is positively regulated by nhaR and affected by hns; Dover N et al.; nhaA encodes an Na+/H+ antiporter in Escherichia coli which is essential for adaptation to high salinity and alkaline pH in the presence of Na+ . We used Northern (RNA) analysis to measure directly the cellular levels of nhaA mRNA . NhaR belongs to the LysR family of regulatory proteins . Consistent with our previous data with an nhaA'-'lacZ fusion, NhaR was found to be a positive regulator and Na+ was found to be a specific inducer of nhaA transcription . In the nhaA'-'lacZ fusion, maximal induction was observed at alkaline pH . In contrast, in the nhaA+ strain both the level of nhaA expression and the induction ratio were lower at alkaline pH . This difference may be due to the activity of NhaA in the wild-type strain as NhaA efficiently excreted Na+ at alkaline pH and reduced the intracellular concentration of Na+, the signal for induction . We also showed that although the global regulator rpoS was not involved in nhaA regulation, the global regulator hns played a role . Thus, the expression of nhaA'-'lacZ was derepressed in strains bearing hns mutations and transformation with a low-copy-number plasmid carrying hns repressed expression and restored Na+ induction . The derepression in hns strains was nhaR independent . Most interestingly, multicopy nhaR, which in an hns+ background acted only as an Na+-dependent positive regulator, acted as a repressor in an hns strain in the absence of Na+ but was activated in the presence of the ion . Hence, an interplay between nhaR and hns in the regulation of nhaA was suggested.

J Bacteriol, 1996 Nov, 178(22), 6466 - 74
Transcriptional organization and regulation of expression of region 1 of the Escherichia coli K5 capsule gene cluster; Simpson DA et al.; The transcriptional organization and regulation of region 1 expression of the Escherichia coli K5 capsule gene cluster were studied . Region 1 was transcribed as an 8.0-kb polycistronic mRNA which was processed to form a separate 1.3-kb transcript encoding the 3'-most gene kpsS . Transcription of region 1 of the E . coli K5 capsule gene cluster was directed from a single promoter 225 bp upstream of a previously unidentified gene, kpsF . The promoter had -35 and -10 consensus sequences typical of an E . coli sigma 70 promoter, with no similarities to binding sites for other sigma factors . Two integration host factor (IHF) binding site consensus sequences were identified 110 bp upstream and 130 bp downstream of the transcription start site . In addition, two AT-rich regions separated by 16 bp identified upstream of the region 1 promoter were conserved upstream of the region 3 promoter . The kpsF gene was 98.8% identical with the kpsF gene identified in the E . coli K1 antigen gene cluster and confirms that the kpsF gene is conserved among group II capsule gene clusters . An intragenic Rho-dependent transcriptional terminator was discovered within the kpsF gene . No essential role for KpsF in the expression of the K5 antigen could be established . The temperature regulation of region 1 expression was at the level of transcription, with no transcription detectable in cells grown at 18 degrees C . Mutations in regulatory genes known to control temperature-dependent expression of a number of virulence genes had no effect on the temperature regulation of region 1 expression . Likewise, RfaH, which is known to regulate expression of E . coli group II capsules had no effect on the expression of region 1 . Mutations in the himA and himD genes which encode the subunits of the IHF led to a fivefold reduction in the expression of KpsE at 37 degrees C, confirming a regulatory role for IHF in the expression of region 1 genes.

Protein Sci, 1996 Nov, 5(11), 2276 - 86
The allosteric activator ATP induces a substrate-dependent alteration of the quaternary structure of a mutant aspartate transcarbamoylase impaired in active site closure; Baker DP et al.; Aspartate transcarbamoylase from Escherichia coli shows homotropic cooperativity for aspartate as well as heterotropic regulation by nucleotides . Structurally, it consists of two trimeric catalytic subunits and three dimeric regulatory subunits, each chain being comprised of two domains . Glu-50 and Ser-171 are involved in stabilizing the closed conformation of the catalytic chain . Replacement of Glu-50 or Ser-171 by Ala in the holoenzyme has been shown previously to result in marked decreases in the maximal observed specific activity, homotropic cooperativity, and affinity for aspartate (Dembowski NJ, Newton CJ, Kantrowitz ER, 1990, Biochemistry 29:3716-3723; Newton CJ, Kantrowitz ER, 1990, Biochemistry 29:1444-1451) . We have constructed a double mutant enzyme combining both mutations . The resulting Glu-50/ser-171-->Ala enzyme is 9-fold less active than the Ser-171-->Ala enzyme, 69-fold less active than the Glu-50-->Ala enzyme, and shows 1.3-fold and 1.6-fold increases in the {S}0.5Asp as compared to the Ser-171-->Ala and Glu-50-->Ala enzymes, respectively . However, the double mutant enzyme exhibits some enhancement of homotropic cooperativity with respect to aspartate, relative to the single mutant enzymes . At subsaturating concentrations of aspartate, the Glu-50/Ser-171 -->Ala enzyme is activated less by ATP than either the Glu-50-->Ala or Ser-171-->Ala enzyme, whereas CTP inhibition is intermediate between that of the two single mutants . As opposed to the wild-type enzyme, the Glu-50/Ser-171 -->Ala enzyme is activated by ATP and inhibited by CTP at saturating concentrations of aspartate . Structural analysis of the Ser-171-->Ala and Glu-50/Ser-171-->Ala enzymes by solution X-ray scattering indicates that both mutants exist in the same T quaternary structure as the wild-type enzyme in the absence of ligands, and in the same R quaternary structure in the presence of saturating N-(phosphonoacetyl)-L-aspartate . However, saturating concentrations of carbamoyl phosphate and succinate are unable to convert a significant fraction of either mutant enzyme population to the R quaternary structure, as has been observed previously for the Glu-50-->Ala enzyme . The curves for both the Ser-171-->Ala and Glu-50/Ser-171-->Ala enzymes obtained in the presence of substoichiometric amounts of PALA are linear combinations of the two extreme T and R states . The structural consequences of nucleotide binding to these two enzymes were also investigated . Most surprisingly, the direction and amplitude of the effect of ATP upon the double mutant enzyme were shown to vary depending upon the substrate analogue used.

Protein Sci, 1996 Nov, 5(11), 2236 - 47
Crystal structure analyses of uncomplexed ecotin in two crystal forms: implications for its function and stability; Shin DH et al.; Ecotin, a homodimeric protein composed of 142 residue subunits, is a novel serine protease inhibitor present in Escherichia coli . Its thermostability and acid stability, as well as broad specificity toward proteases, make it an interesting protein for structural characterization . Its structure in the uncomplexed state, determined for two different crystalline environments, allows a structural comparison of the free inhibitor with that in complex with trypsin . Although there is no gross structural rearrangement of ecotin when binding trypsin, the loops involved in binding trypsin show relatively large shifts in atomic positions . The inherent flexibility of the loops and the highly nonglobular shape are the two features essential for its inhibitory function . An insight into the understanding of the structural basis of thermostability and acid stability of ecotin is also provided by the present structure.

Protein Sci, 1996 Nov, 5(11), 2184 - 92
Cloning, expression, and spectroscopic characterization of Cucumis sativus stellacyanin in its nonglycosylated form; Nersissian AM et al.; The cDNA encoding the 182 amino acid long precursor stellacyanin from Cucumis sativus was isolated and characterized . The protein precursor consists of four sequence domains: I, a 23 amino acid hydrophobic N-terminal signal peptide with features characteristic of secretory proteins; II, a 109 amino acid copper-binding domain; III, a 26 amino acid hydroxyproline- and serine-rich peptide characteristic of motifs found in the extension family, extracellular structural glycoproteins found in plant cell walls; and IV, a 22 amino acid hydrophobic extension . Maturation of the protein involves posttranslational processing of domains I and IV . The copper-binding domain (domain II), which shares high sequence identity with other stellacyanins, has been expressed without its carbohydrate attachment sites, refolded from the Escherichia coli inclusion bodies, purified, and characterized by electronic absorption, EPR, ESEEM, and RR spectroscopy . Its spectroscopic properties are nearly identical to those of stellacyanin from the Japanese lacquer tree Rhus vernicifera, the most extensively studied and best characterized stellacyanin, indicating that this domain folds correctly, even in the absence of its carbohydrate moiety . The presence of a hydroxyproline- and serine-rich domain III suggests that stellacyanin may have a function other than that of a diffusible electron transfer protein, conceivably participating in redox reactions localized at the plant cell wall, which are known to occur in response to wounding or infection of the plant.

Protein Sci, 1996 Nov, 5(11), 2149 - 61
High-resolution X-ray structure of UDP-galactose 4-epimerase complexed with UDP-phenol; Thoden JB et al.; UDP-galactose 4-epimerase from Escherichia coli catalyzes the interconversion of UDP-glucose and UDP-galactose . In recent years, the enzyme has been the subject of intensive investigation due in part to its ability to facilitate nonstereospecific hydride transfer between beta-NADH and a 4-keto hexopyranose intermediate . The first molecular model of the epimerase from E . coli was solved to 2.5 A resolution with crystals grown in the presence of a substrate analogue, UDP-phenol (Bauer AJ, Rayment I, Frey PA, Holden HM, 1992, Proteins Struct Funct Genet 12:372-381) . There were concerns at the time that the inhibitor did not adequately mimic the sugar moiety of a true substrate . Here we describe the high-resolution X-ray crystal structure of the ternary complex of UDP-galactose 4-epimerase with NADH and UDP-phenol . The model was refined to 1.8 A resolution with a final overall R-factor of 18.6% . This high-resolution structural analysis demonstrates that the original concerns were unfounded and that, in fact, UDP-phenol and UDP-glucose bind similarly . The carboxamide groups of the dinucleotides, in both subunits, are displaced significantly from the planes of the nicotinamide rings by hydrogen bonding interactions with Ser 124 and Tyr 149 . UDP-galactose 4-epimerase belongs to a family of enzymes known as the short-chain dehydrogenases, which contain a characteristic Tyr-Lys couple thought to be important for catalysis . The epimerase/NADH/UDP-phenol model presented here represents a well-defined ternary complex for this family of proteins and, as such, provides important information regarding the possible role of the Tyr-Lys couple in the reaction mechanism.

Mol Biol Cell, 1996 Nov, 7(11), 1815 - 23
COPII coat subunit interactions: Sec24p and Sec23p bind to adjacent regions of Sec16p; Gimeno RE et al.; Formation of COPII-coated vesicles at the endoplasmic reticulum (ER) requires assembly onto the membrane of five cytosolic coat proteins, Sec23p, Sec24p, Sec13p, Sec31p, and Sar1p . A sixth vesicle coat component, Sec16p, is tightly associated with the ER membrane and has been proposed to act as a scaffold for membrane association of the soluble coat proteins . We previously showed that Sec23p binds to the C-terminal region of Sec16p . Here we use two-hybrid and coprecipitation assays to demonstrate that the essential COPII protein Sec24p binds to the central region of Sec16p . In vitro reconstitution of binding with purified recombinant proteins demonstrates that the interaction of Sec24p with the central domain of Sec16p does not depend on the presence of Sec23p . However, Sec23p facilitates binding of Sec24p to Sec16p, and the three proteins can form a ternary complex in vitro . Truncations of Sec24p demonstrate that the N-terminal and C-terminal regions of Sec24p display different binding specificities . The C terminus binds to the central domain of Sec16p, whereas the N terminus of Sec24p binds to both the central domain of Sec16p and to Sec23p . These findings define binding to Sec16p as a new function for Sec24p and support the idea that Sec16p organizes assembly of the COPII coat.

Mol Biol Cell, 1996 Nov, 7(11), 1805 - 13
A glutathione reductase mutant of yeast accumulates high levels of oxidized glutathione and requires thioredoxin for growth; Muller EG; A glutathione reductase null mutant of Saccharomyces cerevisiae was isolated in a synthetic lethal genetic screen for mutations which confer a requirement for thioredoxin . Yeast mutants that lack glutathione reductase (glr1 delta) accumulate high levels of oxidized glutathione and have a twofold increase in total glutathione . The disulfide form of glutathione increases 200-fold and represents 63% of the total glutathione in a glr1 delta mutant compared with only 6% in wild type . High levels of oxidized glutathione are also observed in a trx1 delta, trx2 delta double mutant (22% of total), in a glr1 delta, trx1 delta double mutant (71% of total), and in a glr1 delta, trx2 delta double mutant (69% of total) . Despite the exceptionally high ratio of oxidized/reduced glutathione, the glr1 delta mutant grows with a normal cell cycle . However, either one of the two thioredoxins is essential for growth . Cells lacking both thioredoxins and glutathione reductase are not viable under aerobic conditions and grow poorly anaerobically . In addition, the glr1 delta mutant shows increased sensitivity to the thiol oxidant diamide . The sensitivity to diamide was suppressed by deletion of the TRX2 gene . The genetic analysis of thioredoxin and glutathione reductase in yeast runs counter to previous studies in Escherichia coli and for the first time links thioredoxin with the redox state of glutathione in vivo.

Gen Comp Endocrinol, 1996 Nov, 104(2), 156 - 67
Production of recombinant insulin-like growth factor-II in the development of a radioimmunoassay in rainbow trout (Oncorhynchus mykiss); Gentil V et al.; Rainbow trout (Oncorhynchus mykiss) insulin-like growth factor-II (IGF-II) cDNA was amplified by reverse transcription-polymerase chain reaction and cloned into an expression vector (parHS3) for production by Escherichia coli . Recombinant rainbow trout IGF-II (rtIGF-II) was recovered from bacterial inclusion bodies, solubilized, and purified by gel filtration chromatography under reducing conditions . After refolding of the peptide by dialysis at basic pH, we obtained a final yield of 820 micrograms/liter culture medium . The rtIGF-II appeared as a single band estimated at 80% and with molecular mass of 7.5 kDa . A homologous radioimmunoassay (RIA) for the measurement of IGF-II in trout plasma has been developed using rtIGF-II as antigen, radiolabeled tracer, and standard . RIA sensitivity was 0.1 ng/ml and the ED50 was 0.75 +/- 0.06 ng/ml . Intra- and interassay coefficients of variation were 4.04% (n = 6) and 4.56% (n = 9), respectively, at ED50 levels . Cross-reactivities of 1 and 0.1% were found for recombinant coho salmon (Oncorhynchus kisutch) IGF-I and coho salmon insulin, respectively . No cross-reactivities were found for recombinant human IGF-I and IGF-II, porcine insulin, or a variety of salmonid pituitary hormones . The interference of IGF binding proteins (IGFBP) in the RIA has been tested with and without extraction (acid/ethanol extraction or C-25 acid chromatography) of fed and fasted trout plasma samples . Parallel displacement curves were obtained for unextracted and C-25-extracted plasma but not for acid/ethanol-extracted plasma . Comparison of IGF-II levels from these samples showed a possible interference of IGFBP in the RIA in fed (19.6 +/- 1.56 ng/ml unextracted and 25.25 +/- 1.21 ng/ml extracted) and fasted (21.58 +/- 1.02 ng/ml unextracted and 9.86 +/- 1.68 ng/ml extracted) trout plasma . Finally, the displacement curves for unextracted plasma from rainbow trout, Couch's sea bream (Pagrus pagrus), and carp (Cyprinus carpio) were parallel to that of the rtIGF-II standard and not strictly parallel for tilapia (Oreochromis niloticus) and catfish (Silurus glanis) plasma . No significant cross-reaction occurred with eel (Anguilla anguilla) plasma . The rainbow trout IGF-II RIA reported in this study has low variability and is highly sensitive . Therefore it is a reliable method for measuring IGF-II levels in salmonids and some nonsalmonid fish species.

Curr Genet, 1996 Nov, 30(5), 432 - 8
Deletion analysis of promoter elements of the Aspergillus oryzae agdA gene encoding alpha-glucosidase; Minetoki T et al.; The nucleotide sequence of a 1.5-kb fragment of the promoter region of the Aspergillus oryzae agdA gene encoding alpha-glucosidase was determined . A comparison with the promoter regions of other Aspergillus amylase genes indicated that there are three highly conserved sequences, designated Regions I, II and III, located at -670 nt, -596 nt and -544 nt relative to the start codon, respectively . The function of these consensus sequences in the agdA promoter was investigated by deletion analysis of a promoter fusion with the Escherichia coli uidA gene, using the niaD homologous-transformation system . Deletion of the upstream half of Region III (IIIa; -544 to -529) resulted in a more than 90% reduction in GUS activity and abolished maltose induction, suggesting that Region IIIa is a functionally essential element for high-level expression and maltose induction . Deletion of Region I and the downstream half of Region III (IIIb; -521 to -511) resulted in a significant reduction in GUS activity, but did not affect maltose induction . This suggested that these two elements most likely contain sequences involved in efficient expression in cooperation with Region IIIa . In addition, deletion of a 340-bp region between Region IIIb and the putative TATA box resulted in a 2-fold increase in activity.

Arch Microbiol, 1996 Nov, 166(5), 357 - 60
Fine surface structure of enterotoxemic Escherichia coli O139:K12 strains associated with swine edema disease; Meno Y et al.; The fine structure of the cell surface of seven enterotoxemic Escherichia coli (ETEEC) O139:K12 strains isolated from piglets with edema disease were examined electron microscopically using both the negative-staining method and the freeze-substitution fixation method . Densely packed, fine fibers were observed; they consisted of a capsule layer approximately 25 nm thick around the cell surfaces of strains 107/86, IW-2, ED-3, ED-43, and ED-61, all of which have a capacity to adhere strongly to HEp-2 cells . In contrast, no such structure was observed on the surface of strains RK-O139 or ED-1, both of which adhere only weakly to HEp-2 cells . These results suggest that the capsule structure might be associated with the ability to adhere to HEp-2 cells and, as a result, also potentially play some role in ETEEC infection.

Anal Biochem, 1996 Nov 1, 242(1), 90 - 4
A heterologous substrate assay for the HIV-1 protease engineered in Escherichia coli; Stebbins J et al.; A heterologous substrate assay for the human immunodeficiency virus type 1 (HIV-1) protease has been engineered in Escherichia coli . This assay detects the activity of the HIV-1 protease within intact bacterial cells and does not require biochemical purification of either the enzyme or the substrate . For this assay, nine HIV-1 protease specificity sites were genetically engineered into a heterologous protein (galactokinase) and the relative processing of these substrates by the wild-type and a substituted HIV-1 protease was determined . The results from these experiments revealed that the activity of the HIV-1 protease in the engineered heterologous substrate assay is consistent with previously reported in vitro assays and in vivo observations as well as a proposed catalytic specificity model.

Anal Biochem, 1996 Nov 1, 242(1), 26 - 30
The visualization of peroxisomal proteins containing a C-terminal targeting sequence on western blot by using the biotinylated PTS1-receptor; Fransen M et al.; A procedure to visualize proteins containing a C-terminal peroxisomal targeting signal (PTS1) in complex protein mixtures was developed using a bacterially expressed, biotinylated form of the human PTS1-receptor . The binding of this fusion product to purified PTS1-containing proteins that were separated by SDS-PAGE and blotted onto nitrocellulose was detected by means of streptavidin-alkaline phosphatase and shown to be both saturable and specific . When applied to total tissue extracts, in addition to PTS1-containing proteins various endogenous biotinylated proteins were visualized . Therefore, a two-step staining procedure was optimized whereby the endogenous biotinylated proteins were shielded with a blue precipitate, followed by incubation with the biotinylated receptor and detection of the resulting PTS1-receptor/PTS1-protein complexes with a phosphatase reaction coupled to the formation of a red-colored precipitate . This relatively inexpensive, simple, and fast technique enabled us to visualize a variety of PTS1-containing proteins . In addition, the information presented in this study can be used to facilitate the identification and characterization of receptor-ligand interaction in general and to eliminate interference by endogenous biotinylated proteins intrinsic to the streptavidin-biotin detection system.

J Med Virol, 1996 Nov, 50(3), 221 - 9
Characterisation of a panel of monoclonal antibodies raised against recombinant HCV core protein; Ferns RB et al.; Hepatitis C virus (HCV) has as yet no practical culture system so any antigen or antibody studies must be carried out using recombinant antigens . In this study, HCV core sequence was amplified by PCR, inserted into pRSET, and expressed in E . coli . The resultant core protein was purified using nickel affinity chromatography which bound the six histidine tag attached to the N-terminus of the protein . After elution in imidazole buffer, the core protein was used to immunise Balb/c mice and monoclonal antibodies against HCV core were raised . Six monoclonals were examined in a variety of assays . All of them recognised the p27 kDa protein which they were raised against and 2D2 and 3D7 recognised the core component of an HCV Recombinant Immunoblot Assay (RIBA) . None of the antibodies recognised the linear peptides in an Innolia HCV assay . 2D2 showed cytoplasmic granular staining in 1-5% of cells in frozen section of HCV infected livers . Cross-competition assays between themselves and human anti-HCV core positive sera divided the antibodies into two main groups (I and II), with a sub-division of group I into a and b . Group I antibodies were unable to be inhibited by human anti-HCV sera whereas group II antibodies were inhibited by these sera (up to 62%) . Epitopes recognised by all the monoclonals were probably conformational with the group I epitope being located within the first 105 amino acids of the core sequence and the group II epitope between amino acids 105 and 160.

Biotechniques, 1996 Nov, 21(5), 898 - 903
Protection of DNA during preparative agarose gel electrophoresis against damage induced by ultraviolet light; Grundemann D et al.; Preparative gel electrophoresis of double-stranded DNA usually includes staining the gel with ethidium bromide followed by illumination with ultraviolet (UV-B) light . In this report, DNA isolated from agarose gels was found to be a poor substrate for in vitro transcription, transformation of E . coli and PCR . Inhibition was not caused by enzyme-inhibiting impurities in the agarose gel, but was induced by a standard transilluminator fitted with 312-nm tubes . Interestingly, it was possible to protect the DNA against UV damage by the addition of cytidine or guanosine to the electrophoresis buffer.

J Gen Virol, 1996 Nov, 77 ( Pt 11), 2827 - 31
Stimulation of vaccinia virion DNA helicase I8R, but not A18R, by a vaccinia core protein L4R, an ssDNA binding protein; Bayliss CD et al.; Four DNA-dependent ATPases were purified from vaccinia virions and tested for DNA helicase activity on two dsDNA substrates . ATPases D6R and D11L were inactive on both substrates, A18R unwound the substrate with a short 20 bp duplex region and 18R unwound both substrates . In addition, the 18R protein was stimulated to unwind longer DNA duplexes by a 25 kDa protein purified from vaccinia virions, representing the cleaved product of the L4R gene, an ssDNA binding protein . Purified recombinant 25 kDa L4R protein also stimulated 18R DNA helicase activity and maximum activity was observed only when there were < 13 nucleotides of DNA per molecule of L4R protein . The DNA helicase activity of the A18R protein was not stimulated by either recombinant 25 kDa L4R protein or by an E . coli ssDNA binding protein.

J Gen Virol, 1996 Nov, 77 ( Pt 11), 2807 - 18
The latent membrane protein 2 gene of Epstein-Barr virus is important for efficient B cell immortalization; Brielmeier M et al.; The viral latent membrane proteins 2 (LMP2) of Epstein-Barr virus (EBV) were analysed genetically to evaluate their role in B cell immortalization . LMP2 is transcribed as two differently spliced mRNAs which code for the LMP2A and -B proteins, also called terminal protein-1 and -2 . LMP2A and -B are found in latently infected, growth-transformed B lymphocytes in vitro, in different human tumours, and in latently infected B cells in vivo . Two different approaches were used to generate EBV mutants in which the second, third and part of the fourth exon of the LMP2 gene were deleted by insertion of a marker gene . Initially, conventional homologous recombination in a Burkitt's lymphoma cell line (P3HR1) between the endogenous EBV genome and an introduced plasmid was used to generate EBV mutants . This experiment identified LMP2 as dispensable for B cell immortalization as has been reported . In a second approach, the same LMP2 mutant gene was analysed in the context of a mini-EBV plasmid . These are E . coli constructs that are sufficient when packaged into an EBV coat both to initiate and to maintain proliferation of infected B cells . In comparison with a fully competent mini-EBV, LMP2- mini-EBVs were found to be greatly reduced in their capacity to yield immortalized B cell clones . This finding confirmed the initially observed bias against LMP2- B cell clones, most of which were found to be coinfected with complementing P3HR1 virus . These results indicate that LMP2 contributes to the efficiency of B cell immortalization and that the LMP2s phenotype is auxiliary in nature.

J Gen Virol, 1996 Nov, 77 ( Pt 11), 2795 - 805
Cloning and expression of the Epstein-Barr virus-encoded dUTPase: patients with acute, reactivated or chronic virus infection develop antibodies against the enzyme; Sommer P et al.; The gene encoding the Epstein-Barr virus (EBV)-specific dUTPase was amplified from virus DNA by PCR . The active enzyme was expressed in Escherichia coli and in insect cells as a non-fusion protein . The protein from E . coli specifically converted dUTP to dUMP and did not react with other dNTPs or NTPs . Preliminary experiments yielded a Km value of about 0.8 microM for dUTP . MAbs against the dUTPase reacted with a protein of approximately 31 kDa in 12-O-tetradecanoyl-phorbol-13-acetate (TPA)-stimulated B cells harbouring either type 1 or type 2 EBV . The protein was found in untreated cells at low levels, whereas induction of the lytic replication cycle by TPA treatment or by providing the immediate early transactivator BZLF1 in trans resulted in increased expression . We demonstrated that the virus dUTPase isolated from EBV-infected cells is a phosphoprotein . The protein expressed in insect cells was used to test for the presence of specific antibodies in sera from normal, healthy carriers and from patients with various diseases . While the sera of EBV-negative individuals (0/3) or healthy carriers (0/33) did not contain detectable levels of antibodies, patients with mononucleosis (5/18), chronic EBV infection (2/7), EBV reactivation (7/20) and human immunodeficiency virus infection (5/24) showed elevated antibody titres against the enzyme . This indicated that the dUTPase is expressed during EBV replication and reactivation . The enzyme might therefore be a potential target for drug therapy under conditions of active DNA replication.

J Cell Biol, 1996 Nov, 135(4), 1163 - 77
Distinct regions control transcriptional activation of the alpha1(VI) collagen promoter in different tissues of transgenic mice; Braghetta P et al.; To identify regions involved in tissue specific regulation of transcription of the alpha1(VI) collagen chain, transgenic mice were generated carrying various portions of the gene's 5'-flanking sequence fused to the E . coli beta-galactosidase gene . Analysis of the transgene expression pattern by X-gal staining of embryos revealed that: (a) The proximal 0.6 kb of promoter sequence activated transcription in mesenchymal cells at sites of insertion of superficial muscular aponeurosis into the skin; tendons were also faintly positive . (b) The region between -4.0 and -5.4 kb from the transcription start site was required for activation of the transgene in nerves . It also drove expression in joints, in intervertebral disks, and in subepidermal and vibrissae mesenchyme . (c) The fragment comprised within -6.2 and -7.5 kb was necessary for high level transcription in skeletal muscle and meninges . Positive cells in muscle were mostly mononuclear and probably included connective tissue elements, although staining of myoblasts was not ruled out . This fragment also activated expression in joints, in intervertebral disks, and in subepidermal and vibrissae mesenchyme . (d) beta-Galactosidase staining in vibrissae induced by the sequences -4.0 to -5.4 and -6.2 to -7.5 was not coincident: with the latter sequence labeled nuclei were found mainly in the ventral and posterior quadrant, and, histologically, in the outer layers of mesenchyme surrounding and between the follicles, whereas with the former the remaining quadrants were positive and expressing cells were mostly in the inner layers of the dermal sheath . (e) Other tissues, notably lung, adrenal gland, digestive tract, which produce high amounts of collagen type VI, did not stain for beta-galactosidase . (f) Central nervous system and retina, in which the endogenous gene is inactive, expressed the lacZ transgene in most lines . The data suggest that transcription of alpha1(VI) in different tissues is regulated by distinct sequence elements in a modular arrangement, a mechanism which confers high flexibility in the temporal and spatial pattern of expression during development.

J Cell Biol, 1996 Nov, 135(4), 1043 - 57
Preferential MyoD homodimer formation demonstrated by a general method of dominant negative mutation employing fusion with a lysosomal protease; Li FQ et al.; We report on a general strategy for engineering dominant negative mutations that, in principle, requires neither extensive structural or functional knowledge of the targeted protein . The approach consists of fusing the lysosomal protease cathepsin B (CB) to a subunit of a multimeric protein . The CB fusion polypeptide can proteolytically digest the multimer and/or detour the multimer from its usual subcellular destination to the lysosome . We first demonstrate the general validity of the approach with CB fusion to E . coli lacZ, encoding tetrameric beta-galactosidase . Cotransfection of NIH 3T3 cells with a vector expressing a CB-lacZ fusion inhibits the beta-galactosidase activity produced by transfection of lacZ alone . We infer that the dominant negative inhibition results from both direct proteolysis of the beta-galactosidase tetramer by the fusion subunit and detour of the tetramer to the lysosome . In a specific application of this strategy, we have fused CB to the dimeric bHLH skeletal muscle transcription factor MyoD . The CB-MyoD fusion protein localizes to the cytoplasm, presumably the lysosome, demonstrating the dominance of lysosomal localization to nuclear localization . The CB-MyoD fusion appears to divert homodimerizing native MyoD from its usual nuclear destination, consequently inhibiting MyoD-mediated transactivation and in vitro differentiation of C2C12 myoblasts . Surprisingly, the CB-MyoD fusion fails to interact with the bHLH heterodimerization partners, E12 and E47, suggesting preferential MyoD homodimer formation, at least in the prenuclear cellular compartments.

J Cell Biol, 1996 Nov, 135(4), 1027 - 42
Characterization of pinin, a novel protein associated with the desmosome-intermediate filament complex; Ouyang P et al.; We have identified a protein named pinin that is associated with the mature desmosomes of the epithelia (Ouyang, P., and S.P . Sugrue . 1992 . J . Cell Biol . 118:1477-1488) . We suggest that the function of pinin is to pin intermediate filaments to the desmosome . Therefore, pinin may play a significant role in reinforcing the intermediate filament-desmosome complex . cDNA clones coding for pinin were identified, using degenerative oligonucleotide probes that were based on the internal amino acid sequence of pinin for the screening of a cDNA library . Immunoblotting of expressed recombinant proteins with the monoclonal 08L antibody localized the 08L epitope to the carboxyl end of the protein . Polyclonal antibodies directed against fusion proteins immunoidentified the 140-kD protein in tissue extracts . Immunofluorescence analysis, using the antifusion protein antibody, demonstrated pinin at lateral epithelial boundaries, which is consistent with desmosomal localization . The conceptual translation product of the cDNA clones contained three unique domains: (a) a serine-rich domain; (b) a glutamine-proline, glutamine-leucine repeat domain; and (c) an acidic domain rich in glutamic acid . Although the 3' end of the open reading frame of the clone for pinin showed near identity to a partial cDNA isolated for a pig neutrophil phosphoprotein (Bellavite, P., F . Bazzoni, et al . 1990 . Biochem . Biophys . Res . Commun . 170:915-922), the remaining sequence demonstrated little homology to known protein sequences . Northern blots of mRNA from chicken corneal epithelium, MDCK cells, and various human tissues indicated that pinin messages exhibit tissue-specific variation in size, ranging from 3.2 to 4.1 kb . Genomic Southern blots revealed the existence of one gene for pinin, suggesting alternative splicing of the mRNA . Expression of the full-length cDNA clones in human 293 cells and monkey COS-7 cells demonstrated that a 140-kD immunoreactive species on Western blots corresponded to pinin . Pinin cDNA transfected into the transformed 293 cells resulted in enhanced cell-cell adhesion . Immunofluorescence staining revealed that the expressed pinin protein was assembled to the lateral boundaries of the cells in contact, which is consistent with the staining pattern of pinin in epithelial cells.

J Cell Biol, 1996 Nov, 135(4), 913 - 24
Rab11 regulates recycling through the pericentriolar recycling endosome; Ullrich O et al.; Small GTPases of the rab family are crucial elements of the machinery that controls membrane traffic . In the present study, we examined the distribution and function of rab11 . Rab11 was shown by confocal immunofluorescence microscopy and EM to colocalize with internalized transferrin in the pericentriolar recycling compartment of CHO and BHK cells . Expression of rab11 mutants that are preferentially in the GTP- or GDP-bound state caused opposite effects on the distribution of transferrin-containing elements; rab11-GTP expression caused accumulation of labeled elements in the perinuclear area of the cell, whereas rab11-GDP caused a dispersion of the transferrin labeling . Functional studies showed that the early steps of uptake and recycling for transferrin were not affected by overexpression of rab11 proteins . However, recycling from the later recycling endosome was inhibited in cells overexpressing the rab11-GDP mutant . Rab5, which regulates early endocytic trafficking, acted before rab11 in the transferrin-recycling pathway as expression of rab5-GTP prevented transport to the rab11-positive recycling endosome . These results suggest a novel role for rab11 in controlling traffic through the recycling endosome.

Biochem J, 1996 Nov 1, 319 ( Pt 3), 947 - 51
Characterization of a novel pterin intermediate formed in the catalytic cycle of tyrosine hydroxylase; Almas B et al.; A novel pterin intermediate, in addition to the expected 4a-hydroxytetrahydrobiopterin (4a-OH-BH4) and quinonoid dihydrobiopterin, was generated during catalytic turnover of tyrosine hydroxylase (TH) with tetrahydrobiopterin as the cofactor . Based on chromatographic, spectroscopic and stability properties its structure is proposed to be similar to the product formed by the non-enzymic conversion of synthetic 4a-OH-BH4 {Bailey, Rebrin, Boerth and Ayling (1995) J . Am . Chem . Soc . 117, 10203-10211} . This compound was tentatively described as a 4a-adduct of a side-chain hydroxy group, i.e . the O2', 4a-cyclic-tetrahydrobiopterin (4a-Cyc-BH4) . The intermediate generated in the TH reaction has a UV spectrum which is similar to that of 4a-OH-BH4, but elutes with a longer retention time (tR = 1.69 min compared with 1.06 min) on reversed-phase chromatography . Its conversion into quinonoid dihydrobiopterin is catalysed by pterin-4a-carbinolamine dehydratase (EC 4.2.1.96), although 4a-OH-BH4 is the preferred substrate for that enzyme . A precursor-product relationship was demonstrated between 4a-OH-BH4 and the putative 4a-Cyc-BH4 intermediate . The apparent stability of this compound is dependent on pH as well as on the nature of the buffer ions . At pH 8.0 a large amount was generated in Hepes and Tris, but little in phosphate buffer . At pH 7.0 in Hepes (standard assay conditions) and Tris buffer the putative 4a-Cyc-BH4, but no 4a-OH-BH4, was observed . None of the intermediates was observed at pH 6.0 . The accumulation of these intermediates in the absence of dehydratase has important implications for the assay of TH and phenylalanine hydroxylase activities, and is also compatible with a possible physiological role of the dehydratase in the synthesis of catecholamines in vivo.

Biochem J, 1996 Nov 1, 319 ( Pt 3), 713 - 6
Expression of recombinant rat myo-inositol 1,4,5-trisphosphate 3-kinase B suggests a regulatory role for its N-terminus; Thomas S et al.; We have expressed rat myo-inositol 1,4,5-trisphosphate (IP3) 3-kinase B as both a full-length, recombinant, non-fusion protein and a full-length, recombinant, fusion protein with maltose-binding protein (MBP) in Escherichia coli . The fusion protein with MBP is soluble, binds calmodulin and is enzymically active whereas the non-fusion protein is insoluble and does not bind calmodulin unless co-expressed with bacterial chaperone proteins (either GroES and GroEL, or DnaK, DnaJ and GrpE) . However, soluble, calmodulin-binding non-fusion IP3 3-kinase B is enzymically inactive . The catalytic domain of the enzyme has previously been shown to reside near the C-terminus; the results we present suggest an auto-regulatory role for the N-terminus.

Ann Gastroenterol Hepatol (Paris), 1996 Nov-Dec, 31(6), 333 - 6
{Liver abscess: diagnosis and treatment . Study of a series of 22 cases}; Benazzouz M et al.; Twenty-two cases of abscess of the liver are reported . Eighteen were due to pyogenic organisms and four to amebas . The diagnosis was established based on clinical and laboratory evaluations and, above all, on ultrasonography with aspiration of the lesion . The causative organism was recovered from the aspirate in 33.3% of cases . Seventeen patients were treated by percutaneous aspiration . Two patients required insertion of a drain because of a biliary fistula . The success rate of percutaneous aspiration with or without drainage was 88.2% in our series . The two patients who had surgery had loculated abscesses with thick pus . In conclusion, the diagnosis and treatment of hepatic abscesses have benefited from advances in imaging techniques; in particular, aspiration or drainage can be performed simply under ultrasonographic guidance.

J Med Microbiol, 1996 Nov, 45(5), 353 - 8
Serotyping and categorisation of Escherichia coli strains isolated between 1958 and 1992 from diarrhoeal diseases in Asia; Tamura K et al.; A total of 3065 strains of Escherichia coli isolated between 1958 and 1992 from patients with diarrhoea in different countries were examined for virulence factors by hybridisation with biotinylated DNA probes for genes that coded for production of heat-labile and heat-stable enterotoxins, enteroinvasiveness, production of verotoxins and attaching-and-effacing factor and were serotyped . Of the 3065 strains, 1998 were placed into one of four pathogenic categories by their virulence factors: 1057 enterotoxigenic E . coli (ETEC) comprising 30 O-groups, 73 serovars and 137 untypable strains; 132 enteroinvasive E . coli (EIEC) comprising 11 O-groups and 13 serovars; 64 verotoxin-producing E . coli (VTEC) comprising 11 O-groups, 17 serovars and 13 untypable strains; and 745 enteropathogenic E . coli (EPEC) comprising 34 O-groups, 92 serovars and 91 untypable strains . The remaining 1067 strains did not hybridise with any of the DNA probes used . About half the number of O-groups recognised were not restricted to a single pathogenic category, although the combinations of O- and H-antigens were different in each category.

J Med Microbiol, 1996 Nov, 45(5), 331 - 7
Comparative cytotoxicity of purified Shiga-like toxin-IIe on porcine and bovine aortic endothelial and human colonic adenocarcinoma cells; Valdivieso-Garcia A et al.; Porcine and bovine aortic endothelial cells and human colonic adenocarcinoma cells were compared for their susceptibility to the toxic effect of purified Shiga-like toxin IIe (SLT-IIe), measured by the neutral red cytotoxicity assay . Cytotoxicity correlated with toxin binding as indicated by fluorescence activated cell sorter analysis and with the globotriosylceramide (Gb3) and globotetraosylceramide (Gb4) content of cells determined by high pressure liquid chromatography . One line of porcine aortic endothelial cells was 1400-fold more susceptible than the line of bovine aortic endothelial cells that was tested, but a second line of porcine aortic endothelial cells was highly refractory to SLT-IIe . Human colonic adenocarcinoma cells lacked detectable levels of Gb4 and were least susceptible to SLT-IIe.

J Mol Biol, 1996 Nov 1, 263(3), 475 - 85
Roles of alpha 114 and beta 87 amino acid residues in the polymerization of hemoglobin S: implications for gene therapy; Ho C et al.; Three novel recombinant mutants of sickle hemoglobin (Hb S, beta 6Glu-->Val) have been constructed to assess the role of proline at alpha 114 and threonine at beta 87 in the polymerization of deoxygenated Hb S . Using the hemoglobin expression system (pHE2) designed in our laboratory, four plasmids were expressed separately in Escherichia coli to produce the four recombinant hemoglobins: r Hb S (beta 6Glu-->Val); r Hb S-Chiapas (beta 6Glu-->Val, alpha 114Pro-->Arg); r Hb S-D-Ibadan (beta 6Glu-->Val, beta 87Thr-->Lys); and r Hb S-Chiapas-D-Ibadan (beta 6Glu-->Val, alpha 114Pro-->Arg, beta 87Thr-->Lys) . The structural features of these four recombinant hemoglobins were analyzed by proton nuclear magnetic resonance spectroscopy, and were found to be similar to those of human normal adult hemoglobin (Hb A) under identical conditions . The recombinant hemoglobins were further investigated by measuring the oxygen-binding properties, which were found to be comparable to those of Hb A . Delay-time gelation studies of the three mutants of r Hb S were carried out in 1.8 M potassium phosphate (pH 7.34) by a temperature jump from 4 degrees C to 30 degrees C and an increase in delay time over that of r Hb S was observed, as well as an overall decrease in the polymerization of these three mutants of Hb S . A more detailed and quantitative investigation has also been carried out to determine the equilibrium solubility (Csat) in 0.1 M potassium phosphate (pH 7.35) at 25 degrees C of the three Hb S mutants as well as of mixtures of these mutants with Hb S versus mixtures of fetal hemoglobin (Hb F) and Hb A with Hb S . The inhibition of polymerization demonstrated in these experiments suggests that the interactions involving the two amino acid residues alpha 114Pro and beta 87Thr are very important to the formation of Hb S polymer, and modification of these amino acids results in an anti-sickling potential . Of particular interest is the inhibitory effect of alpha 114Pro-->Arg, which offers a novel opportunity to use an alpha-chain construct, in addition to a beta-chain construct in the same vector, in gene therapy for sickle cell anemia, with the objective of modifying a larger number of hemoglobin tetramers at a given level of expression.

J Mol Biol, 1996 Nov 1, 263(3), 396 - 410
Analysis of the ribosome large subunit assembly and 23 S rRNA stability in vivo; Liiv A et al.; The ability of mutant 23 S ribosomal RNA to form particles with proteins of the large ribosomal subunit in vivo was studied . A series of overlapping deletions covering the entire 23 S rRNA, were constructed in the plasmid copy of an E . coli 23 S rRNA gene . The mutant genes were expressed in vivo using an inducible tac promoter . Mutant species of 23 S rRNA, containing deletions between positions 40 and 2773, were incorporated into stable ribonucleoprotein particles . In contrast, if one end of the 23 S rRNA was deleted, the mutant rRNA was unstable and did not form ribosomal particles . Protein composition of the mutant particles was specific; the presence of the primary rRNA-binding proteins corresponded to their known binding sites . Furthermore, several previously unknown ribosomal protein binding sites in 23 S rRNA were identified . Implications of the results on ribosome assembly are discussed.

Virology, 1996 Nov 1, 225(1), 191 - 5
The open reading frame 2 product of cacao swollen shoot badnavirus is a nucleic acid-binding protein; Jacquot E et al.; The function of the open reading frame 2 product (p2) of cacao swollen shoot virus (CSSV) and of other badnaviruses is not yet determined . Their carboxyl-termini are lysine and proline rich and also contain alanine residues, amino acids present at the C-termini of histone-like proteins . Full-length CSSV p2 (132 amino acids) or versions truncated at the C-terminus (128, 113, 103, or 101 amino acids) were expressed in Escherichia coli and partially purified . When assayed in nucleic acid-binding tests, p2 was able to interact with CSSV and other double-stranded DNAs and with CSSV and other single-stranded RNA transcripts in sequence-nonspecific manner . Moreover, this binding activity was progressively lost as the C-terminus was gradually deleted.

EMBO J, 1996 Nov 1, 15(21), 5999 - 6008
Chromosomal replication incompatibility in Dam methyltransferase deficient Escherichia coli cells; Lobner-Olesen A et al.; Dam methyltransferase deficient Escherichia coli cells containing minichromosomes were constructed . Free plasmid DNA could not be detected in these cells and the minichromosomes were found to be integrated in multiple copies in the origin of replication (oriC) region of the host chromosome . The absence of the initiation cascade in Dam- cells is proposed to account for this observation of apparent incompatibility between plasmid and chromosomal copies of oriC . Studies using oriC-pBR322 chimeric plasmids and their deletion derivatives indicated that the incompatibility determinant is an intact and functional oriC sequence . The seqA2 mutation was found to overcome the incompatability phenotype by increasing the cellular oriC copy number 3-fold thereby allowing minichromosomes to coexist with the chromosome . The replication pattern of a wild-type strain with multiple integrated minichromosomes in the oriC region of the chromosome, led to the conclusion that initiation of DNA replication commences at a fixed cell mass, irrespective of the number of origins contained on the chromosome.

EMBO J, 1996 Nov 1, 15(21), 5796 - 803
The nucleotide exchange factor MGE exerts a key function in the ATP-dependent cycle of mt-Hsp70-Tim44 interaction driving mitochondrial protein import; Schneider HC et al.; Import of preproteins into the mitochondrial matrix is driven by the ATP-dependent interaction of mt-Hsp70 with the peripheral inner membrane import protein Tim44 and the preprotein in transit . We show that Mge1p, a co-chaperone of mt-Hsp70, plays a key role in the ATP-dependent import reaction cycle in yeast . Our data suggest a cycle in which the mt-Hsp70-Tim44 complex forms with ATP: Mge1p promotes assembly of the complex in the presence of ATP . Hydrolysis of ATP by mt-Hsp70 occurs in complex with Tim44 . Mge1p is then required for the dissociation of the ADP form of mt-Hsp70 from Tim44 after release of inorganic phosphate but before release of ADP . ATP hydrolysis and complex dissociation are accompanied by tight binding of mt-Hsp70 to the preprotein in transit . Subsequently, the release of mt-Hsp70 from the polypeptide chain is triggered by Mge1p which promotes release of ADP from mt-Hsp70 . Rebinding of ATP to mt-Hsp70 completes the reaction cycle.

Am J Vet Res, 1996 Nov, 57(11), 1662 - 7
Endotoxin stimulates in vitro pituitary growth hormone release in eicosanoid-dependent manner; Coleman ES et al.; OBJECTIVE: To investigate the signal transduction pathways by which endotoxin stimulates in vitro pituitary cell growth hormone (GH) release . ANIMALS: Pituitary cell cultures derived from 6 sheep . PROCEDURE: Signal transduction pathways involved in endotoxin-mediated GH release from sheep pituitary cell cultures were evaluated by the use of specific blockers of arachidonic acid and its metabolites, extracellular calcium, protein kinase C, and protein kinase A . Cell cultures were exposed to the specific blockers in the presence or absence of endotoxin (Escherichia coli O55:B5, 10 micrograms/ml) for 24 hours . In addition, effects of endotoxin on GH cell content and GH mRNA values were determined . RESULTS: Nordihydroquairetic acid (lipoxygenase blocker, 10 microM, 30 microM) and eicosatetraynoic acid (arachidonic acid competitor, 10 microM) decreased endotoxin-stimulated GH release . The calcium channel blocker verapamil (25 microM) decreased baseline and endotoxin-stimulated GH release . Phorbol myristate acetate-induced down-regulation of protein kinase C, indomethacin, or the protein kinase A blocker H89 did not alter endotoxin-stimulated GH release . Endotoxin increased GH mRNA values by 50.1 +/- 6.0%, but the cell content of GH was not affected . CONCLUSIONS: A direct effect of endotoxin on the pituitary gland to stimulate GH secretion was evident, an effect mediated predominantly by arachidonic acid and its metabolites through the lipoxygenase pathway . Endotoxin-stimulated GH release requires extracellular calcium and is associated with increased cell GH mRNA content . CLINICAL RELEVANCE: A better understanding of the signal transduction pathways involved in endotoxin-mediated effects will allow more appropriate therapeutic intervention in clinical cases of endotoxemia.

Arch Biochem Biophys, 1996 Nov 1, 335(1), 152 - 60
Interconversion of the androstenedione hydroxylase specificities of cytochromes P450 2B4 and 2B5 upon simultaneous site-directed mutagenesis of four key substrate recognition residues; He YQ et al.; Based on recent studies of single reciprocal mutants of cytochrome P450 2B4 and the highly related P450 2B5 at positions 114, 294, 363, and 367 {G . D . Szklarz, Y . Q . He, K . M . Kedzie, J . R . Halpert, and V . L . Burnett (1996) Arch . Biochem . Biophys . 327,308-318}, a number of multiple mutants were constructed, expressed in Escherichia coli, and assayed with androstenedione, progesterone, and benzyloxyresorufin . Simultaneous substitutions of Ile-114, Ser-294, Ile-363, and Val-367 in cytochrome P450 2B4 with Phe, Thr, Val, and Ala, respectively from 2B5, resulted in a marked increase in androstenedione 15alpha- and 16alpha-hydroxylation compared with the wild-type enzyme and yielded a metabolite profile indistinguishable from that of cytochrome P450 2B5 . Likewise, the reciprocal P450 2B5 quadruple mutant exhibited the specificity for 16beta-hydroxylation characteristic of the 2B4 wild type . The two reciprocal quadruple mutants of P450 2B4 and 2B5 also displayed benzyloxyresorufin dealkylase activities similar to those of the wild-type P450 2B5 and 2B4, respectively . However, the progesterone metabolite profile of P450 2B5 was not identical to that of the 2B4 quadruple mutant or of a quintuple mutant in which residue 370 was also mutated to the 2B5 residue . Therefore, the 17beta-acetyl group on progesterone as opposed to the oxo group on androstenedione may lead to interaction with additional amino acid residues.

Arch Biochem Biophys, 1996 Nov 1, 335(1), 73 - 81
Specificity of aspartokinase III from Escherichia coli and an examination of important catalytic residues; Keng YF et al.; Aspartokinase III (AK III) has been purified from a plasmid-containing strain of Escherichia coli . The enzyme shows broad specificity for the phosphoryl acceptor substrate . Structural analogs of aspartic acid with a derivatized alpha-carboxyl group are accepted as alternative substrates by the enzyme . Derivatives at the alpha-amino group are also tolerated by AK III but with diminished catalytic activity . As has been previously observed with aspartokinase I (T . S . Angeles and R . E . Viola, 1992, Biochemistry 31, 799), derivatization of the beta-carboxyl group, which serves as the phosphoryl acceptor, does not prevent catalytic activity . These beta-derivatized analogs are capable of productive binding to these enzymes through a reversal of regiospecificity, making the alpha-carboxyl group available as the phosphoryl acceptor . Chemical modification and pH profile studies have identified the functional groups of cysteine and histidine as being involved in the catalytic activity of AK III.

Arch Biochem Biophys, 1996 Nov 1, 335(1), 61 - 72
S-glutathiolated hepatocyte proteins and insulin disulfides as substrates for reduction by glutaredoxin, thioredoxin, protein disulfide isomerase, and glutathione; Jung CH et al.; The disulfide-reducing activities of glutaredoxin, thioredoxin, protein disulfide isomerase, glutathione, and cysteine were directly compared with a mixture of hepatocyte 35S-glutathiolated proteins as the substrate . Dethiolation of individual 35S-labeled protein bands from the mixture of hepatocyte proteins was analyzed by SDS-PAGE . All of the 35S-labeled protein bands could be completely dethiolated by glutaredoxin, thioredoxin, protein disulfide isomerase, glutathione, or cysteine . On a molar basis glutaredoxin was over 10 times more effective than either thioredoxin or protein disulfide isomerase . Dethiolation rates of individual proteins varied in minor ways . For example, glutaredoxin dethiolated the 15-, 30-, and 48-kDa protein bands 3 to 4 times faster than the 27-, 28-, and 77-kDa bands . Glutaredoxins from pig liver or from bovine heart had the same specificity and similar activity . The rate of dethiolation by glutathione alone was low compared to the glutaredoxin-catalyzed process, but all 35S-labeled protein bands could be reduced by glutathione, cysteine, or dithiothreitol . Glutathione was clearly more effective than cysteine when these two thiols were compared on the basis of the concentration of thiolate anion available at neutral pH . Therefore, glutathione is a more specific reductant of S-glutathiolated proteins than is cysteine but it is much less effective than glutaredoxin . Since glutaredoxin activity in cells is 10 times higher than the concentration used in these experiments, ample activity is available to account for substantial rates of dethiolation in vivo . Thioredoxin is quite inefficient as a reductant of S-glutathiolated proteins, but it was reasoned that it might first reduce glutaredoxin, which then could reduce the S-glutathiolated protein . A combination of thioredoxin and glutaredoxin was effective . It is proposed that glutaredoxin is the principal agent responsible for protein dethiolation in vivo . The effectiveness of glutaredoxin, thioredoxin, and protein disulfide isomerase as reductants for protein disulfide bonds was examined with insulin as the substrate . Protein disulfide isomerase was very effective and thioredoxin was nearly as effective . Human thioredoxin was similar to Escherichia coli thioredoxin in reactivity and specificity . Glutaredoxin did not facilitate insulin reduction at equal concentrations . Thus, protein disulfide isomerase and thioredoxin are more effective than glutaredoxin as reductants of insulin protein disulfides . The apparent reduction potential of pig liver glutaredoxin (-0.159 +/- 0.004 V) was determined by measuring the amount of reduced glutaredoxin in equilibrium with mixtures of glutathione and glutathione disulfide . Glutaredoxin was a weaker reductant than E . coli thioredoxin (-0.260 V) and was similar to protein disulfide isomerase (-0.11 to -0.19 V) . The role of these proteins as disulfide reductants is not determined solely by thermodynamic considerations . A glutathione binding site at the dithiol region of glutaredoxin may be of primary importance for its function in protein dethiolation, while a different specific peptide binding site in thioredoxin may be more suited to certain protein disulfide structures.

Clin Diagn Lab Immunol, 1996 Nov, 3(6), 663 - 8
Modification of Escherichia coli B glutathione synthetase with polyethylene glycol for clinical application to enzyme replacement therapy for glutathione deficiency; Inoue Y et al.; Glutathione synthetase of Escherichia coli B was modified with polyethylene glycol, and the properties of the resultant modified enzyme were investigated . The thermal stability of the modified enzyme and its resistance against several proteases increased compared with those of the native enzyme . The modified enzyme was injected intravenously via the rat tail vein, and the circulating life of the enzyme in plasma was monitored . The half-life of the native enzyme was 50 min, whereas that of the modified enzyme was approximately 24 h . The systemic anaphylaxis reaction was tested by using rats intravenously injected with the native and modified enzymes . For the native enzyme, strong reactions such as dyspnea and tumble were observed; however, no symptom or only a very weak reaction, such as scratching, was observed with the modified enzyme.

Antimicrob Agents Chemother, 1996 Nov, 40(11), 2592 - 7
Efficient technique for screening drugs for activity against Trypanosoma cruzi using parasites expressing beta-galactosidase; Buckner FS et al.; A new drug screening method was devised utilizing Trypanosoma cruzi cells that express the Escherichia coli beta-galactosidase gene . Transfected parasites catalyze a colorimetric reaction with chlorophenol red beta-D-galactopyranoside as substrate . Parasite growth in the presence of drugs in microtiter plates was quantitated with an enzyme-linked immunosorbent assay reader . The assay was performed with the mammalian form of T . cruzi that requires intracellular growth on a monolayer of fibroblast cells . To determine if selective toxicity to the parasites was occurring, the viability of the host cells in the drug was assayed with AlamarBlue . The drugs benznidazole, fluconazole, and amphotericin B were shown to inhibit the parasites at concentrations similar to those previously reported . Several compounds were tested that are inhibitors of glyceraldehyde-3-phosphate dehydrogenase of the related organisms Leishmania mexicana and Trypanosoma brucei . One of these compounds, 2-guanidino-benzimidazole, had an 50% inhibitory concentration of 10 microM in our assay . Two derivatives of this compound were identified with in vitro activity at even lower concentrations . In addition, the assay was modified for testing compounds for lytic activity against the bloodstream form of the parasite under conditions used for storing blood products . Thus, an assay with beta-galactosidase-expressing T . cruzi greatly simplifies screening drugs for selective anti-T . cruzi activity, and three promising new compounds have been identified.

J Am Coll Surg, 1996 Nov, 183(5), 450 - 6
The greater omentum is the primary site of neutrophil exudation in peritonitis; Fukatsu K et al.; BACKGROUND: Peritonitis remains a major infectious problem . Neutrophil influx into the peritoneal cavity is one of the most important host defense mechanisms . However, no studies have focused on the site of neutrophil exudation . This study examined the primary anatomic site of neutrophil exudation in bacterial peritonitis . STUDY DESIGN: Fifty-five rats were injected intraperitoneally with saline solution (control group) or 10(7) Escherichia coli (peritonitis group) . In experiment 1, 1 x 10(6) fluorescein-labeled neutrophils were infused 3 hours after the challenge . Then, peritoneal-lavaged fluids and peritoneal tissues (the greater omentum, mesentery, parietal peritoneum, colon, and ileum) were obtained . Subpopulations of peritoneal exudative cells and numbers of labeled neutrophils in tissues were counted . In experiment 2, labeled neutrophils were infused at 10 minutes and at 1 and 5 hours after the challenge . Peritoneal tissues were also harvested . The number of labeled neutrophils in each tissue was determined . RESULTS: In experiment 1, numbers of labeled peritoneal neutrophils and exudative neutrophils were higher in the peritonitis group than in the control group . Numbers of exudative neutrophils showed a positive correlation with numbers of labeled peritoneal neutrophil . In experiment 2, at 1 and 5 hours after the challenge, the number of labeled neutrophils was higher in the peritonitis group than in the control group . The number of neutrophils in the omentum was higher than the number in other peritoneal tissues . CONCLUSIONS: Our fluorescence microscopic method is useful for detecting neutrophil adhesion . Neutrophil exudation into the peritoneal cavity was most marked in the omentum . The greater omentum may play an important role in host defense as a source of exudative neutrophils.

Arch Surg, 1996 Nov, 131(11), 1222 - 8
Effect of heat shock and endotoxin stress on enterocyte viability apoptosis and function varies based on whether the cells are exposed to heat shock or endotoxin first; Xu DZ et al.; BACKGROUND: Stress-gene responses, including the heat shock (HS) response and the acute phase response, are protective mechanisms for cells after exposure to stress . Both responses cannot occur simultaneously, and, in endothelial cells, the sequence of stress-gene expression seems to be a critical factor in whether cellular protection or injury occurs . OBJECTIVE: To determine if the sequence of stress-gene expression affects cellular protection or injury in epithelial cells . DESIGN: Randomized controlled in vitro study . SETTING: University research laboratory . SUBJECTS: Rat intestinal epithelial cell-6 (IEC-6) cells were grown on 35-mm culture dishes, chamber slides, or in a bicameral system to confluence or until tight junction integrity was established . INTERVENTIONS: Rat IEC-6 cells were examined for viability, apoptosis, and bacterial translocation (BT) after exposure to 25-micrograms/mL lipopolysaccharide (LPS) for 18 hours to HS (43 degrees C) for 90 minutes, to LPS followed by HS, or to HS followed by LPS . MAIN OUTCOME MEASURES: The IEC-6 cells were stained for viability and apoptosis using trypan blue and a direct immunoperoxidase detection of digoxigenin-labeled genomic DNA (Apop Tag Plus In Situ Apoptosis Detection Kit, Oncor, Gaithersburg, Md), respectively . Bacterial translocation was measured by culturing the bacteria (ie, Escherichia coli) that crossed the IEC-6 cell monolayer in the bicameral system . RESULTS: Control cells (medium only) and cells exposed to LPS alone, HS alone, or HS followed by LPS had a viability from 92% to 98%, and the percentage of apoptotic cells ranged from 2.2% to 5.7% . In contrast, IEC-6 cells exposed to LPS followed by HS had a significantly lower viability (83%, P < .05 vs all other groups) and a higher percentage of apoptotic cells (12.2%, P < .01) . At 3 hours after challenge with E coli, the LPS-exposed IEC-6 cell monolayers had significantly increased BT vs control monolayers (P < .05), while the IEC-6 cell monolayers exposed to HS followed by LPS had decreased BT (P < .05) . Conversely, cells exposed to LPS followed by HS had the highest magnitude of BT (P < .01 vs all other groups) . CONCLUSIONS: These results indicate that preinduction of HS response can diminish LPS-induced cell injury, while induction of HS response after the LPS challenge (ie, the acute phase response) may lead to decreased enterocyte viability, increased apoptosis, and cellular dysfunction as manifested by BT.

Arch Surg, 1996 Nov, 131(11), 1148 - 53; discussion 1153-4
Relative contribution of endothelial cell and polymorphonuclear neutrophil activation in their interactions in systemic inflammatory response syndrome; Chen X et al.; OBJECTIVE: To examine the relative contribution of polymorphonuclear neutrophil (PMN) vs endothelial cell (EC) activation on the adherence and subsequent killing of ECs by PMNs . DESIGN: In vitro comparative studies of PMN-EC adherence and cytotoxicity . SETTING: Research laboratory and the surgical intensive care unit of a tertiary-level university hospital . PATIENTS: Patients with systemic inflammatory response syndrome admitted to the surgical intensive care unit and hospitalized preoperative noninfected surgical patients . INTERVENTION: None . METHODS: Polymorphonuclear neutrophils were isolated from 21 healthy volunteers, 22 preoperative patients, and 30 patients from the surgical intensive care unit with systemic inflammatory response syndrome . The PMNs were activated with lipopolysaccharide, 100 ng/mL (Escherichia coli 0111:b4), for 40 minutes at 37 degrees C before the adherence and cytotoxicity assays . Human umbilical vein endothelial monolayers were stimulated with tumor necrosis factor alpha, 25 ng/mL, and interleukin 1 beta, 15 U/mL, for 3 hours . The PMNs or EC cells were labeled with sodium chromate Cr 51 and used in a standard adherence or killing assay as required . RESULTS: Control and preoperative patient PMN treatment with lipopolysaccharide produced a modest increase in adherence . The PMNs from patients with systemic inflammatory response syndrome showed moderately increased human umbilical vein endothelial cell adherence, and this could not be augmented further with lipopolysaccharide stimulation . There was a marked increase in PMN adherence to EC after EC activation in all study groups (P < .001) . Similar to the adherence data, human umbilical vein endothelial cell cytotoxicity was significantly increased in all groups after human umbilical vein endothelial cell activation (P < .01) but not after PMN stimulation with lipopolysaccharide . CONCLUSION: These data suggest that stimulation of ECs is far more important in producing increased adherence and cytotoxicity of EC than PMN stimulation with lipopolysaccharide in all study groups . Therapeutic efforts in patients with systemic inflammatory response syndrome should be focused on the EC.

J Biol Chem, 1996 Nov 1, 271(44), 27927 - 30
An aspartate/insulin receptor chimera mitogenically activates fibroblasts; Biemann HP et al.; A gene encoding the ligand-binding domain of the Escherichia coli aspartate receptor fused to the cytoplasmic domain of the insulin receptor tyrosine kinase to produce the chimeric aspartate insulin receptor (AIR) was expressed in mammalian cells . A murine fibroblast transfectant line designated CA3 was generated that stably expressed the AIR receptor . This 70,000 Mr receptor containing the tyrosine kinase of the insulin receptor was recognized by aspartate receptor-specific antisera . When isolated in cellular membrane preparations, AIR was found to be capable of autophosphorylation and phosphorylation of histone H2B on tyrosine . The receptor was found to be predominately cytoplasmic and to be situated in the endoplasmic reticulum and Golgi membranes by immunofluorescence imaging of CA3 cells . Mitogenic effects of AIR were observed; CA3 cells continued DNA synthesis under serum deprivation conditions that prevented parental cells from cycling . These results demonstrate that a chimeric receptor containing procaryotic transmembrane sequences is expressed by a eucaryotic cell in intracellular membranes and functionally couples to cellular signaling pathways.

J Biol Chem, 1996 Nov 1, 271(44), 27795 - 801
Roles of the FabA and FabZ beta-hydroxyacyl-acyl carrier protein dehydratases in Escherichia coli fatty acid biosynthesis; Heath RJ et al.; There are two genes, fabA and fabZ, encoding beta-hydroxyacyl-acyl carrier protein (ACP) dehydratases that function in the dissociated, type II fatty acid synthase system of Escherichia coli . We have investigated their roles in fatty acid synthesis by purifying the two proteins and reconstituting cycles of fatty acid synthesis in vitro using five other purified proteins . FabA and FabZ exhibited broad, overlapping chain length specificities . The FabZ dehydratase efficiently catalyzed the dehydration of short chain beta-hydroxyacyl-ACPs and long chain saturated and unsaturated beta-hydroxyacyl-ACPs . FabA was most active on intermediate chain length beta-hydroxyacyl-ACPs and also possessed significant activity toward both short and long chain saturated beta-hydroxyacyl-ACPs . Significantly, FabA was virtually inactive in the dehydration of long chain unsaturated beta-hydroxyacyl-ACP . The introduction of the double bond at the 10-carbon stage of fatty acid synthesis by FabA was only detected in the presence of beta-ketoacyl-ACP synthase I (FabB) . A yeast two-hybrid analysis failed to detect an interaction between FabA and FabB, therefore the channeling of intermediates toward unsaturated fatty acid synthesis by FabB was attributed to the affinity of the condensing enzyme for cis-decenoyl-ACP . The broad substrate specificity of FabZ coupled with the inactivity of FabA toward a long chain unsaturated beta-hydroxyacyl-ACP provides a biochemical explanation for the phenotypes of cells with genetically altered levels of the two dehydratases.

J Biol Chem, 1996 Nov 1, 271(44), 27730 - 8
ATP-dependent degradation of CcdA by Lon protease . Effects of secondary structure and heterologous subunit interactions; Van Melderen L et al.; CcdA, the antidote protein of the ccd post-segregational killing system carried by the F plasmid, was degraded in vitro by purified Lon protease from Escherichia coli . CcdA had a low affinity for Lon (Km >/=200 microM), and the peptide bond turnover number was approximately 10 min-1 . CcdA formed tight complexes with purified CcdB, the killer protein encoded in the ccd operon, and fluorescence and hydrodynamic measurements suggested that interaction with CcdB converted CcdA to a more compact conformation . CcdB prevented CcdA degradation by Lon and blocked the ability of CcdA to activate the ATPase activity of Lon, suggesting that Lon may recognize bonding domains of proteins exposed when their partners are absent . Degradation of CcdA required ATP hydrolysis; however, CcdA41, consisting of the carboxyl-terminal 41 amino acids of CcdA and lacking the alpha-helical secondary structure present in CcdA, was degraded without ATP hydrolysis . Lon cleaved CcdA primarily between aliphatic and hydrophilic residues, and CcdA41 was cleaved at the same peptide bonds, indicating that ATP hydrolysis does not affect cleavage specificity . CcdA lost alpha-helical structure at elevated temperatures (Tm approximately 50 degrees C), and its degradation became independent of ATP hydrolysis at this temperature . ATP hydrolysis may be needed to disrupt interactions that stabilize the secondary structure of proteins allowing the disordered protein greater access to the proteolytic active sites.

J Biol Chem, 1996 Nov 1, 271(44), 27670 - 6
Cloning and identification of rat deoxyuridine triphosphatase as an inhibitor of peroxisome proliferator-activated receptor alpha; Chu R et al.; Peroxisome proliferator-activated receptors (PPARs) are members of the nuclear receptor superfamily that transcriptionally regulate responsive genes by binding to the peroxisome proliferator response elements . Protein(s) interacting with PPAR isoforms (alpha, delta, and gamma) may modulate the PPAR-mediated transcriptional activation . Using a yeast two-hybrid system to screen a rat liver cDNA library, we have identified rat deoxyuridine-triphosphatase (dUTPase, EC 3.6 . 1.23) as a PPARalpha-interacting protein . This cDNA encodes a polypeptide of 203 amino acids; the C-terminal 141-amino acid segment of this protein corresponds to the full-length human enzyme, which exhibits 92% identity with human dUTPase; the N-terminal extra 62-amino acid residue region is arginine-rich . In vitro binding assays indicate that rat dUTPase interacts with all three isoforms of mouse PPAR, but not with retinoid X receptor and thyroid hormone receptor . Interaction of PPARalpha with dUTPase is with the N-terminal 62-amino acid segment of rat dUTPase . Full-length rat dUTPase prevents PPAR-retinoid X receptor heterodimerization resulting in an inhibition of PPAR activity in a ligand-independent manner . Immunostaining of human kidney tsA201 cells, transiently expressing dUTPase showed that this protein is present predominantly in the cytoplasm but translocates into the nucleus with PPARalpha when PPARalpha is coexpressed with dUTPase . Northern blot hybridization shows that rat dUTPase is encoded by an abundant 1kilobase mRNA species present in all rat tissues . The identification of dUTPase as a PPAR-interacting protein suggests a possible link between tumorigenic peroxisome proliferators and the enzyme system involved in the maintenance of DNA fidelity.

J Biol Chem, 1996 Nov 1, 271(44), 27438 - 44
Lack of electron transfer from cytochrome b5 in stimulation of catalytic activities of cytochrome P450 3A4 . Characterization of a reconstituted cytochrome P450 3A4/NADPH-cytochrome P450 reductase system and studies with apo-cytochrome b5; Yamazaki H et al.; Many catalytic activities of cytochrome P450 (P450) 3A4, the major human liver P450 enzyme, require cytochrome b5 (b5) for optimal rates . The stimulatory effect of b5 on P450 reactions has generally been thought to be due to transfer of electrons from ferrous b5 to the ferrous P450-O2-substrate complex . We found that apo-b5, devoid of heme, could substitute for b5 in stimulating two prototypic activities, testosterone 6beta hydroxylation and nifedipine oxidation . The stimulatory effect was not seen with albumin, hemoglobin, catalase, or cytochrome c . Apo-b5 could not substitute for b5 in a testosterone 6beta hydroxylation system composed of NADH-b5 reductase and P450 3A4 . Rates of electron transfer from NADPH-P450 reductase to ferric P450 3A4 were too slow (<2 min-1) to support testosterone 6beta hydroxylation ( approximately 14 min-1) unless b5 or apo-b5 was present, when rates of approximately 700 min-1 were measured . The oxidation-reduction potential (Em,7) of the ferric/ferrous couple of P450 3A4 was unchanged ( approximately -310 mV) under different conditions in which the kinetics of reduction were altered by the addition of substrate and/or apo-b5 . Rapid reduction of P450 3A4 required substrate and a preformed complex of P450 3A4, NADPH-P450 reductase, and b5; the rates of binding of the proteins to each other were 2-3 orders of magnitude less than reduction rates . We conclude that b5 can facilitate some P450 3A4-catalyzed oxidations by complexing with P450 3A4 and enhancing its reduction by NADPH-P450 reductase, without directly transferring electrons to P450.

J Biol Chem, 1996 Nov 1, 271(44), 27339 - 45
Consequences of removal of a molybdenum ligand (DmsA-Ser-176) of Escherichia coli dimethyl sulfoxide reductase; Trieber CA et al.; We have used site-directed mutagenesis and EPR spectroscopy to examine the consequences of altering the molybdenum ligand in Escherichia coli dimethyl sulfoxide (Me2SO) reductase (DmsABC) . Mutagenesis of DmsA-Ser-176 to Ala, Cys, or His abolishes both respiratory growth on Me2SO and in vitro benzyl viologen:Me2SO oxidoreductase activity . EPR spectroscopy reveals changes in the line shape and the gav of the Mo(V) signals of the S176A and S176C enzymes . The midpoint potentials (Em,7) of the Mo(VI)/Mo(V) and Mo(V)/Mo(IV) couples in DmsABC are -15 and -175 mV . The Em,7 of the Mo(V)/Mo(IV) couple in the S176A mutant is 35 mV; however, the Mo(V) species could not be further oxidized with ferricyanide . Titration of the S176C mutant produced several overlapping Mo(V) species occurring at Eh > -150 mV, suggesting heterogeneity in the molybdenum environment . A Mo(V) spectrum was not visible in S176H membranes poised between -435 to 350 mV or oxidized with 200 microM ferricyanide . No differences were detected in the EPR spectra of the reduced {4Fe-4S} clusters of DmsABC and the S176A and S176H mutant enzymes; however, the S176C mutation altered the EPR line shape of one of the reduced {4Fe-4S} clusters.

J Biol Chem, 1996 Nov 1, 271(44), 27311 - 20
Structural studies on folding intermediates of serine hydroxymethyltransferase using fluorescence resonance energy transfer; Cai K et al.; Previous studies have demonstrated that the in vitro folding pathway of Escherichia coli serine hydroxymethyltransferase has both monomer and dimer intermediates that are stable for periods of minutes to hours at 4 degrees C (Cai K., Schirch, D., and Schirch, V . (1995) J . Biol . Chem . 270, 19294-19299) . Single Trp mutant enzymes were constructed and used in combination with other methods to show that on the folding pathway of this enzyme two domains rapidly fold to form a monomer in which the amino-terminal 55 amino acid residues and a segment around the active site region of Lys229 remain in a largely disordered form . This partially folded enzyme can form dimers and slowly undergoes a rate-determining conformational change in which the unstructured segments assume their native state (Cai, K . , and Schirch, V . (1996) J . Biol . Chem . 271, 2987-2994) . To further assess the kinetics and structural details of the intermediates during folding, fluorescence energy transfer and fluorescence anisotropy measurements were made of the three Trp residues and pyridoxal 5'-phosphate, attached covalently to the active site by reduction to a secondary amine by sodium cyanoborohydride . These studies confirmed that the basic kinetic folding pathway remained the same in the reduced enzyme as compared to the earlier studies with the apoenzyme . Both equilibrium and kinetic intermediates were identified and their structural characteristics determined . The results show that the active site Lys229-bound pyridoxyl 5'-phosphate remains more than 50 angstroms from any Trp residues until the final rate-determining conformational change when it approaches each Trp residue at the same rate . The environment of each Trp residue and the pyridoxyl phosphate in both an equilibrium folding intermediate and a kinetic folding intermediate are described.

J Biol Chem, 1996 Nov 1, 271(44), 27274 - 9
Synthesis of GDP-L-fucose by the human FX protein; Tonetti M et al.; FX is a homodimeric NADP(H)-binding protein of 68 kDa, first identified in human erythrocytes, from which it was purified to homogeneity . Its function has been unrecognized despite partial structural and genetic characterization . Recently, on the basis of partial amino acid sequence, it proved to be the human homolog of the murine protein P35B, a tumor rejection antigen . In order to address the biochemical role of FX, its primary structure was completed by cDNA sequencing . This sequence revealed a significant homology with many proteins from different organisms . Specifically, FX showed a remarkable similarity with a putative Escherichia coli protein, named Yefb, whose gene maps in a region of E . coli chromosome coding for enzymes involved in synthesis and utilization of GDP-D-mannose . Accordingly, a possible role of FX in this metabolism was investigated . The data obtained indicate FX as the enzyme responsible for the last step of the major metabolic pathway resulting in GDP-L-fucose synthesis from GDP-D-mannose in procaryotic and eucaryotic cells . Specifically, purified FX apparently catalyzes a combined epimerase and NADPH-dependent reductase reaction, converting GDP-4-keto-6-D-deoxymannose to GDP-L-fucose . This is the substrate of several fucosyltranferases involved in the correct expression of many glyconjugates, including blood groups and developmental antigens.

J Biol Chem, 1996 Nov 1, 271(44), 27193 - 6
Crystallization and preliminary diffraction data of a major pollen allergen . Crystal growth separates a low molecular weight form with elevated biological activity; Bufe A et al.; Group V major allergen Phl p 5b of timothy grass pollen induces allergic rhinitis and bronchial asthma in 90% of grass pollen-allergic patients . In addition to its allergenicity ribonuclease activity has recently been attributed to this 29-kDa protein . The allergen was expressed in Escherichia coli and subsequently purified . Spontaneous conversion of these preparations to a mixture of various forms with molecular sizes between 10 and 29 kDa was consistently observed . Surprisingly, crystals could be grown from this heterogenous preparation . Single crystals, redissolved and analyzed by SDS-polyacrylamide gel electrophoresis and immunoblot, yielded one distinct low molecular weight protein, which was identified by amino acid sequencing as the C-terminal 13-kDa portion of the allergen . Histamine release assays with single crystal solutions using basophils of an allergic patient demonstrated allergenicity comparable with that of the holo-allergen . By contrast, RNase activity of the crystallized C-terminal form was 23 times higher than that of the full-length parent allergen . Crystals were used to collect preliminary diffraction data; the space group was evaluated to I4122 with cell dimensions of a = 87.7 A, b = 87.7 A, and c = 59.6 A . We conclude that preferential crystal growth of the 13-kDa form is indicative of a compact conformation of this particular C-terminal portion of the allergen . Thus, we show here that protein crystallization is not only a prerequisite for structural analyses, but it also can provide a unique separation technique to localize the functional domain of a major allergen.

J Cell Physiol, 1996 Nov, 169(2), 290 - 9
Hypoxia primes endotoxin-induced tissue factor expression in human monocytes and endothelial cells by a PAF-dependent mechanism; Herbert JM et al.; Tissue factor (TF) is a glycoprotein which acts as a trigger of the coagulation cascade . TF expression may be induced at the surface of monocytes and endothelial cells by several stimuli including bacterial endotoxin (LPS) and cytokines (IL 1 beta, TNF alpha) and there is a large body of evidence for the involvement of hypoxia as a primaring factor in the process leading to thrombosis . To define the molecular basis underlying this phenomenon, we evaluated the relative role of platelet activating factor (PAF) . PAF primed human monocytes and human umbilical vein endothelial cells (HUVEC) for TF expression following exposure to E coli LPS but was unable to enhance the induction of TF expression by IL 1 beta . The priming effect of PAF with regard to LPS occurred in a time- and dose-dependent manner and was inhibited by the PAF receptor antagonist SR 27417 . When HUVEC or monocytes were exposed to an hypoxic environment, a significant rise in LPS-induced TF expression was observed . Hypoxia had no effect on IL 1-induced TF expression . The enhanced LPS-induced TF expression in both cell types was mediated by PAF as indicated by the inhibition obtained with SR 27417, added during hypoxia . Although the importance of hypoxia in the etiology of venous thrombosis has been acknowledged for a long time, evaluation of the relative importance of PAF in the process leading to thrombus formation is still lacking . Stasis-induced thrombosis performed in the rabbit jugular vein was enhanced in a dose-dependent manner by the prior i.v . administration of LPS (0.05 to 100 micrograms/ kg, i.v.) . SR 27417 administered simultaneously with LPS prevented thrombus formation with an ED50 value of 0.1 +/- 0.04 mg/kg . These results therefore show that hypoxia promotes LPS-induced TF expression in HUVEC and human monocytes through a PAF-dependent mechanism in vitro and in vivo.

Hepatology, 1996 Nov, 24(5), 1224 - 9
Uncoupling of biliary lipid from bile acid secretion by formyl-methionyl-leucyl-phenylalanine in the rat; Mizuno K et al.; A neutrophil chemotactic factor N-formyl-methionyl-leucyl-phenylalanine (fMLP), produced by Escherichia coli under conditions of intestinal inflammation, is reported to circulate enterohepatically in the presence of experimental colitis, but its effect on bile secretion is unclear . Therefore, we investigated the effect of fMLP on bile secretion in a single-pass isolated perfused rat liver system . Infusion of fMLP at different concentrations (2 micromol/L, 10 micromol/L, and 20 micromol/L) into the portal vein resulted in excretion into bile in the native form, independent of sodium taurocholate (1 micromol/min) infusion . Excretion of fMLP increased dose dependently, and approximately 12% of the infused dose was detected at each concentration . With constant infusion of sodium taurocholate (1 micromol/min), fMLP (20 micromol/L) increased bile flow but decreased phospholipid and cholesterol secretion . Bile acid secretion was not affected . Phospholipid/bile acid molar ratios decreased from 0.069 +/- 0.002 to 0.038 +/- 0.002, and cholesterol/bile acid molar ratios decreased from 0.0074 +/- 0.0009 to 0.0029 +/- 0.0008 . Thus, administration of fMLP resulted in the uncoupling of biliary excretion of phospholipid and cholesterol from that of bile acids; this effect proved reversible . The increase in bile flow caused by fMLP infusion appeared to result from osmotic choleresis . When 25 mg of horseradish peroxidase, a conventional marker of transcytotic vesicle transport pathway, was infused for 1 minute as a pulse load into the portal vein after continuous infusion of taurocholate, its late peak excretion was reduced by fMLP (10 micromol/L) from 9.59 +/- 1.09 to 6.05 +/- 0.66 (ng/g liver) . Gel-permeation chromatography of bile showed a specific association of fMLP with bile acids . These results suggest an uncoupling of biliary lipids from bile acids by fMLP because of inhibition of transcellular vesicle transport and interaction between fMLP and bile acid micelles in the bile canaliculus.

RNA, 1996 Nov, 2(11), 1100 - 9
A conserved RNA motif involved in divalent cation utilization by nuclear RNase P; Pagan-Ramos E et al.; Catalytic RNAs are metalloenzymes that require precise coordination of divalent cation cofactors . In RNase P RNA, a conserved structural subdomain that has been implicated in magnesium coordination contains the consensus sequence acAGaRA . Randomization mutagenesis of the analogous sequence in the Saccharomyces cerevisiae nuclear RNase P RNA gene, RPR1, gave viable sequence variants that confer magnesium-correctable growth defects and are defective in magnesium cofactor utilization by the RNase P holoenzyme in vitro . Kinetic analysis of the defective holoenzymes suggests that the primary effects were on catalytic rate, rather than substrate recognition . The possible involvement of this RNA subdomain in catalysis is discussed.

Free Radic Res, 1996 Nov, 25(5), 369 - 84
The role of iron-sulfur clusters in in vivo hydroxyl radical production; Liochev SL; The in vivo production of HO- requires iron ions, H2O2 and O2- or other oxidants but probably does not occur through the Haber-Weiss reaction . Instead oxidants, such as O2-, increase free iron by releasing Fe(II) from the iron-sulfur clusters of dehydratases and by interfering with the iron-sulfur clusters reassembly . Fe(II) then reduces H2O2, and in turn Fe(III) and the oxidized cluster are re-reduced by cellular reductants such as NADPH and glutathione . In this way, SOD cooperates with cellular reductants in keeping the iron-sulfur clusters intact and the rate of HO . production to a minimum . O2- and other oxidants can release iron from Fe(II)-containing enzymes as well as copper from thionein . The released Fe(III) and Cu(II) are then reduced to Fe(II) and Cu(I) and can then participate in the Fenton reaction . In mammalian cells oxidants are able to convert cytosolic aconitase into active IRE-BP, which increases the "free" iron concentration intracellularly both by decreasing the biosynthesis of ferritin and increasing biosynthesis of transferrin receptors . The biological role of the soxRS regulon of Escherichia coli, which is involved in the adaptation toward oxidative stress, is presumably to counteract the oxidative inactivation of the iron clusters and the subsequent release of iron with consequent increased rate of production of HO.

J Lab Clin Med, 1996 Nov, 128(5), 515 - 9
Comparison of endothelial activation during endotoxic and posttraumatic conditions by serum analysis of soluble E-selectin in nonhuman primates; Kneidinger R et al.; Because the presence of E-selectin, which is one of the adhesion molecules on the endothelium, cannot be determined directly in vivo except by invasive biopsy techniques, the only available kinetic information concerning its activity is the serum level of the soluble form . Therefore we tried to measure soluble E-selectin levels and compare the degree of endothelial activation in a trauma and endotoxic situation, where endothelial activation is supposed to occur . To perform these studies, an enzyme-linked immunosorbent assay with two monoclonal antibodies was set up and used in (1) a model of endotoxic shock in baboons (n = 8) (1.5 mg/kg Escherichia coli endotoxin as a 10-minute intravenous infusion), (2) a hemorrhagic traumatic shock model in baboons (n = 6), where trauma was simulated by infusion of zymosan-activated serum and hemorrhage . Soluble E-selectin was released in vivo after the application of endotoxin, and it reached a peak level after 24 hours (13.82 +/- 5.38 ng/ml) . In baboons with hemorrhagic shock, much lower levels (5.03 +/- 1.98 ng/ml) of soluble E-selectin were found . A lower soluble E-selectin level indicates a lower level of endothelial activation after experimental hemorrhagic traumatic shock (with endotoxin translocation from the gut) as compared with endotoxic shock probably caused by much lower endotoxin levels in traumatic shock.

Am J Hum Genet, 1996 Nov, 59(5), 1048 - 56
Molecular analysis of a GM2-activator deficiency in two patients with GM2-gangliosidosis AB variant; Schepers U et al.; Lysosomal degradation of ganglioside GM2 by beta-hexosaminidase A (hex A) requires the presence of the GM2 activator protein (GM2AP) as an essential cofactor . A deficiency of the GM2 activator causes the AB variant of GM2 gangliosidosis, a recessively inherited disorder characterized by excessive neuronal accumulation of GM2 and related glycolipids . Two novel mutations in the GM2 activator gene (GM2A) have been identified by the reverse-transcriptase-PCR method--a three-base deletion, AAG262-264, resulting in a deletion of Lys88, and a single-base deletion, A410, that causes a frameshift . The latter results in substitution of 33 amino acids and the loss of another 24 amino acid residues . Both patients are homoallelic for their respective mutations inherited from their parents, who are heteroallelic at the GM2A locus . Although the cultured fibroblasts of both patients produce normal levels of activator mRNA, they lack a lysosomal form of GM2AP . Pulse/chase labeling of cultured fibroblasts of the patients, in presence and absence of brefeldin A, indicates a premature degradation of both--mutant and truncated--GM2APs in the endoplasmic reticulum or Golgi . These results were supported by in vitro translation experiments and expression of the mutated proteins . When the mutated GM2APs were expressed in Escherichia coli, both mature GM2AP forms turned proved to exhibit only residual activities in an in vitro assay.

Am J Hum Genet, 1996 Nov, 59(5), 1006 - 11
Gene structure and mutations of glutaryl-coenzyme A dehydrogenase: impaired association of enzyme subunits that is due to an A421V substitution causes glutaric acidemia type I in the Amish; Biery BJ et al.; The structure of the human glutaryl coenzyme A dehydrogenase (GCD) gene was determined to contain 11 exons and to span approximately 7 kb . Fibroblast DNA from 64 unrelated glutaric acidemia type I (GA1) patients was screened for mutations by PCR amplification and analysis of SSCP . Fragments with altered electrophoretic mobility were subcloned and sequenced to detect mutations that caused GA1 . This report describes the structure of the GCD gene, as well as point mutations and polymorphisms found in 7 of its 11 exons . Several mutations were found in more than one patient, but no one prevalent mutation was detected in the general population . As expected from pedigree analysis, a single mutant allele causes GA1 in the Old Order Amish of Lancaster County, Pennsylvania . Several mutations have been expressed in Escherichia coli, and all produce diminished enzyme activity . Reduced activity in GCD encoded by the A421V mutation in the Amish may be due to impaired association of enzyme subunits.

FEMS Microbiol Lett, 1996 Nov 1, 144(2-3), 259 - 66
Role of cytochrome b562 in the archaeal aerobic respiratory chain of Sulfolobus sp . strain 7; Iwasaki T et al.; The role of cytochrome b562, a fragile constituent of the respiratory terminal oxidase supercomplex of the thermoacidophilic archaeon, Sulfolobus sp . strain 7, was investigated spectroscopically in the membrane-bound state . Cytochrome b562 did not react with CO or cyanide in the membrane-bound state, while it was irreversibly modified to a CO-reactive form (b59) upon solubilization in the presence of cholate and LiCl . Cyanide titration analyses with the succinate-reduced membrane suggested that cytochrome b562 was upstream of both the "gy = 1.89' Rieske FeS cluster and the a-type cytochromes . These results show that the b-type cytochrome functions as an intermediate electron transmitter in the terminal oxidase supercomplex.

FEMS Microbiol Lett, 1996 Nov 1, 144(2-3), 235 - 40
Duplication of genes encoding the immunodominant 38 kDa antigen in Mycobacterium intracellulare; Thangaraj HS et al.; Mycobacterium avium is a causative agent of mycobacterioses in systemically immunocompromised individuals, whereas Mycobacterium intracellulare is responsible for causing infections in relatively immunocompetent hosts . In an attempt to identify components that could be involved in virulence, we characterised the 38 kDa-encoding gene of M intracellulare that is absent in M . avium . This antigen cross reacts immunologically with a major 38 kDa antigen of M . tuberculosis, and both antigens are homologues of the phosphate transport subunit S (PstS) of the pst complex of Escherichia coli . Unlike the M . tuberculosis complex the M . intracellulare coding gene was found to be duplicated . We also identified and characterised other pst genes that may constitute an operon . Considering that multiple isoforms of PstS are present in mycobacteria the possible role of pstS1 genes for pathogenesis is discussed.

FEMS Microbiol Lett, 1996 Nov 1, 144(2-3), 229 - 33
MetR-mediated repression of the glyA gene in Escherichia coli; Lorenz E et al.; Inactivation of either of the two MetR binding sites centered at bp -143 and 121 relative to the +1 transcription start site results in reduced glyA-lacZ expression in a wild-type strain below the level seen in a metR mutant . This reduced expression is dependent on the side of the DNA helix MetR binds relative to the RNA polymerase binding site . Thus, a single MetR dimer bound to the DNA may play a physiological role in maintaining appropriate glyA gene expression, functioning as a repressor under low MetR conditions.

FEMS Microbiol Lett, 1996 Nov 1, 144(2-3), 197 - 202
Cloning and nucleotide sequence of the mphB gene for macrolide 2'-phosphotransferase II in Escherichia coli; Noguchi N et al.; Macrolide 2'-phosphotransferase II {MPH(2')II} inactivates macrolide antibiotics . The mphB gene for MPH(2')II was cloned from Escherichia coli and sequenced . Analysis of the nucleotide sequence indicated that mphB encoded a protein of 302 amino acids with a molecular mass of 34483 Da . The carboxy terminal region of the deduced protein contained a sequence that resembled a conserved functional domain in aminoglycoside phosphotransferases.

FEMS Microbiol Lett, 1996 Nov 1, 144(2-3), 171 - 6
Mapping of noninvasion TnphoA mutations on the Escherichia coli O18:K1:H7 chromosome; Bloch CA et al.; The most virulent newborn meningitis-associated Escherichia coli are of the serotype O18:K1:H7 . We previously isolated a large number of E . coli O18:K1:H7 mutants resulting from transposon TnphoA mutagenesis that fail to invade brain microvascular endothelial cells . We have now determined Ic locations of 45 independent insertions . Twelve were localized to the 98 min region, containing a 120 kb segment that is characteristic of E . coli O18:K1:H7 . Another, the previously described insertion ibe-10::TnphoA, was localized to the 87 min region, containing a 20 kb segment found in this E . coli . These noninvasion mutations may define new O18:K1:H7 pathogenicity islands carrying genes for penetration of the blood-brain barrier of newborn mammals.

FEMS Microbiol Lett, 1996 Nov 1, 144(2-3), 145 - 50
Involvement of calcium in interactions between gingival epithelial cells and Porphyromonas gingivalis; Izutsu KT et al.; Porphyromonas gingivalis, a periodontal pathogen can invade primary cultures of gingival epithelial cells . This invasion was significantly inhibited (74-81%) by thapsigargin and 1,2-bis(2-aminophenoxy)ethane-N,N,N1,N1-tetraacetic acid, acetoxymethyl ester (BAPTA/AM), but not by EDTA or amiloride . Release of Ca2+ from an intracellular store and the subsequent increase in cytosolic {Ca2+} may, therefore, be involved in the invasion process, while Ca2+ influx is not . Moreover, cytosolic {Ca2+} was found to increase transiently in about 30% of gingival epithelial cells acutely exposed to P . gingivalis, but not in unexposed cells, or in cells exposed to noninvasive Escherichia coli . These findings indicate that P . gingivalis invasion of epithelial cells is correlated with activation of {Ca2+}-dependent host cell signaling systems.

Appl Environ Microbiol, 1996 Nov, 62(11), 4296 - 8
Aspergillus nidulans stcP encodes an O-methyltransferase that is required for sterigmatocystin biosynthesis; Kelkar HS et al.; The Aspergillus nidulans stcP gene was previously identified as a transcribed region associated with a cluster of genes proposed to be involved in sterigmatocystin biosynthesis (D . W . Brown, J.-H . Yu, H . S . Kelkar, M . Fernandes, T . C . Nesbitt, N . P . Keller, T . H . Adams, and T . J . Leonard, Proc . Natl . Acad . Sci . USA 93:1418-1422, 1996) . stcP was predicted to encode a methyltransferase responsible for conversion of demethylsterig-matocystin to sterigmatocystin . Here we demonstrate that disruption of stcP in A . nidulans results in strains that accumulate demethylsterigmatocystin.

Appl Environ Microbiol, 1996 Nov, 62(11), 4256 - 9
Cloning, sequencing, and expression of a Thermomonospora fusca protease gene in Streptomyces lividans; Lao G et al.; The major Thermomonospora fusca YX extracellular protease gene (tfpA) was cloned into Escherichia coli and Streptomyces lividans and was sequenced . The open reading frame encoded 375 residues, including a 31-residue potential signal sequence, an N-terminal prosequence containing 150 residues, and the 194-residue mature protease that belongs to the chymotrypsin family . The protease was secreted by S . lividans, but evidence suggested that it was bound to an extracellular protease inhibitor . An inhibitor-deficient mutant was selected to produce protease for purification.

Appl Environ Microbiol, 1996 Nov, 62(11), 4114 - 20
Death of the Escherichia coli K-12 strain W3110 in soil and water; Bogosian G et al.; Whether Escherichia coli K-12 strain W3110 can enter the "viable but nonculturable" state was studied with sterile and nonsterile water and soil at various temperatures . In nonsterile river water, the plate counts of added E . coli cells dropped to less than 10 CFU/ml in less than 10 days . Acridine orange direct counts, direct viable counts, most-probable-number estimates, and PCR analyses indicated that the added E . coli cells were disappearing from the water in parallel with the number of CFU . Similar results were obtained with nonsterile soil, although the decline of the added E . coli was slower . In sterile water or soil, the added E . coli persisted for much longer, often without any decline in the plate counts even after 50 days . In sterile river water at 37 degrees C and sterile artificial seawater at 20 and 37 degrees C, the plate counts declined by 3 to 5 orders of magnitude, while the acridine orange direct counts remained unchanged . However, direct viable counts and various resuscitation studies all indicated that the nonculturable cells were nonviable . Thus, in either sterile or nonsterile water and soil, the decline in plate counts of E . coli K-12 strain W3110 is not due to the cells entering the viable but nonculturable state, but is simply due to their death.

Appl Environ Microbiol, 1996 Nov, 62(11), 4003 - 8
Hypochlorous acid activates the heat shock and soxRS systems of Escherichia coli; Dukan S et al.; A series of plasmids, containing fusions of different stress promoters to lux reporter genes, was used in an attempt to monitor the defense circuits activated upon exposure of Escherichia coli to sublethal doses of free chlorine . A significant level of activation was exhibited by promoters of three heat shock genes (grpE, dnaK, and lon), in an rpoH-dependent manner . The promoter of micF, a gene under the control of the soxRS regulon, was also strongly induced, but not in a soxR mutant . This induction was not affected by sodA and sodB mutations, implying that it did not involve oxygen radical activity . Free-chlorine activation of both heat shock and soxRS regulons required an exposure of less then I s in duration . The oxyR or the SOS regulons were apparently not induced by free chlorine (as judged by lack of activation of katG and recA, respectively), and neither was the universal stress (uspA) protein.

J Clin Microbiol, 1996 Nov, 34(11), 2853 - 5
The recombinant 120-kilodalton protein of Ehrlichia chaffeensis, a potential diagnostic tool; Yu XJ et al.; DNA encoding two repeat units of 120-kDa protein of Ehrlichia chaffeensis was cloned into the expression vector pGEX and expressed in Escherichia coli . The sensitivity and specificity of a dot blot assay for detection of human antibodies with the recombinant protein were 86 and 100%, respectively, compared with an indirect immunofluorescence assay.

J Clin Microbiol, 1996 Nov, 34(11), 2819 - 21
Use of an alkaline phosphatase-conjugated oligonucleotide probe for the gene encoding the bundle-forming pilus of enteropathogenic Escherichia coli; Nagayama K et al.; An alkaline phosphatase-conjugated 29-base oligonucleotide probe was developed to detect the gene encoding the bundle-forming pilus of enteropathogenic Escherichia coli . The sensitivity and specificity of the probe versus the results of localized adherence in the HEp-2 cell assay and fluorescent actin staining assay positivity were 95.7 and 100%, respectively.

J Clin Microbiol, 1996 Nov, 34(11), 2812 - 4
Rapid detection and isolation of shiga-like toxin (verocytotoxin)-producing Escherichia coli by direct testing of individual enterohemolytic colonies from washed sheep blood agar plates in the VTEC-RPLA assay; Beutin L et al.; By combining the enterohemolysin test and the VTEC-RPLA test (specific for the detection of Shiga-like toxin I {SLT-I}, SLT-II, and SLT-IIc), single colonies of SLT-producing Escherichia coli were found to constitute between 0.03 and 68.1% of the coliform flora in human stool cultures and were isolated and characterized within 72 to 96 h.

J Clin Microbiol, 1996 Nov, 34(11), 2728 - 30
Evaluation of 16S rRNA gene PCR with primers Hp1 and Hp2 for detection of Helicobacter pylori; Chong SK et al.; The PCR primer set Hp1-Hp2, which amplifies a 109-bp fragment of the 16S rRNA gene of Helicobacter pylori, has been widely used for the detection of H . pylori in clinical specimens . We have examined 34 stool samples and 50 human tissue samples from H . pylori-infected and uninfected patients, five human leukocyte samples, and one human cell line by this PCR method . All of these specimens produced a 109-bp PCR product . When Escherichia coli DNA was used as the template, several nonspecific bands, but not the 109-bp band, were observed . No PCR products were generated when DNA samples from five different fungi were used as templates . These results indicate that this 109-bp PCR product was amplified from the human genome . The 109-bp PCR product generated from various clinical specimens also hybridized with the probe pHp, corresponding to a region internal to the PCR product of Hp1-Hp2 . We conclude that the 16S rRNA gene PCR with the primer set Hp1-Hp2 is not specific and cannot be used to detect H . pylori in clinical specimens.

J Infect Dis, 1996 Nov, 174(5), 1124 - 6
Prevalence of enteropathogenic, enteroaggregative, and diffusely adherent Escherichia coli among isolates from children with diarrhea in new Caledonia; Germani Y et al.; The clinical significance of HEp-2-adherent Escherichia coli in children with diarrhea in New Caledonia has been examined by testing isolates from stools of ill children and matched controls in a HEp-2 cell binding assay and by hybridizing the same clones with DNA probes identifying the enteropathogenic (EPEC), enteroaggregative (EAggEC), and diffusely adherent (DAEC) E . coli . From the 100 patient-control pairs, 35 HEp-2-adherent strains were isolated; 24 were identified as the only pathogen in stools of ill children, and 11 were from controls . EPEC strains were significantly associated with diarrheal disease (P < .008) in children in the first 2 years of life . For the DAEC strains, the difference in rate of isolation between patients and controls was significant only when the presence of afa/daa sequences in the strains was considered (P = .03, Fisher's exact test) . The afa/daa-positive DAEC isolates were characterized from children 2-6 years old . EAggEC strains were isolated equally in patients and controls.

Parasitology, 1996 Nov, 113 ( Pt 5), 457 - 64
Expression of NF-Y nuclear factor in Schistosoma mansoni; Serra E et al.; The A subunit of NF-Y nuclear factor from Schistosoma mansoni was expressed in E . coli fused to a histidine tag and purified by affinity chromatography using a Ni(2+)-Agarose matrix . Antibodies against the recombinant protein were prepared and used for Western blot and immunolocalization . The presence of SMNF-YA in all stages of the parasite life-cycle was determined by RT-PCR and Western blot analysis . The immunolocalization of SMNF-YA showed the presence of this factor in a parenchymal cell population of cercariae and adult worms and in embryos within eggs . The expression of SMNF-YA was demonstrated to decrease in maturating spermatozoites whereas an accumulation of this factor was observed in the nucleus from oocytes during their maturation processes.

J Virol, 1996 Nov, 70(11), 8169 - 74
ATPase, GTPase, and RNA binding activities associated with the 206-kilodalton protein of turnip yellow mosaic virus; Kadare G et al.; The 206-kDa protein of turnip yellow mosaic virus belongs to an expanding group of proteins containing a domain which includes the consensus nucleotide binding site GxxxxGKS/T . A portion of this protein (amino acids {aa} 916 to 1259) was expressed in Escherichia coli and purified by affinity chromatography to near homogeneity . In the absence of any other viral factors, it exhibited ATPase and GTPase activities in vitro . A mutant protein with a single amino acid substitution in the consensus nucleotide binding site (Lys-982 to Ser) exhibited only low levels of both activities, implying that Lys-982 is important for nucleoside triphosphatase activity . The protein also possessed nonspecific RNA binding capacity . Deletion mutants revealed that an N-terminal domain (aa 916 to 1061) and a C-terminal domain (aa 1182 to 1259) participate in RNA binding . The results presented here provide the first experimental evidence that turnip yellow mosaic virus encodes nucleoside triphosphatase and RNA binding activities.

J Virol, 1996 Nov, 70(11), 8155 - 9
Polycistronic (tri- or bicistronic) phytoreoviral segments translatable in both plant and insect cells; Suzuki N et al.; Genomic segment S12 of rice dwarf virus and segment S9 of wound tumor virus, both members of the genus Phytoreovirus, have small out-of-phase overlapping open reading frames (ORFs) . Western blot (immunoblot) analysis revealed that rice dwarf virus S12 mRNA specified translation products from the large ORF and two overlapping small ORFs both in rice plant hosts and in Spodoptera frugiperda insect cells . These results provide the first example of a tricistronic mRNA for a segmented double-stranded RNA virus . Similarly, wound tumor virus S9 mRNA was found to direct the synthesis of protein products from both the large ORF and small out-of-frame ORF in S . frugiperda cells . Results of site-specific and deletion mutagenesis studies were consistent with a leaky scanning translation mechanism for the synthesis of the small ORFs.

J Virol, 1996 Nov, 70(11), 7878 - 84
Synthesis, processing, and oligomerization of bovine herpesvirus 1 gE and gI membrane proteins; Whitbeck JC et al.; This study reports the identification and initial characterization of the precursors, modified forms, and oligomers of bovine herpesvirus 1 (BHV-1) gI and gE proteins with polyvalent rabbit serum specific for gI or gE . Our experiments used the Colorado strain of BHV-1 and mutant viruses with insertions of the Escherichia coli lacZ gene into the predicted gE and gI reading frames . We also translated the gE and gI open reading frames in vitro and expressed them in uninfected cells using eukaryotic expression vectors . Precursor-product relationships were established by pulse-chase analysis and endoglycosidase H and glycopeptidase F digestions . Like the homologous glycoproteins of herpes simplex virus type 1, pseudorabies virus, and varicella-zoster virus, BHV-1 gI and gE are modified by N-linked glycosylation and associate with each other soon after synthesis, forming a noncovalent complex in infected and transfected cells . An analysis of mutant and wild-type-virus-infected cells and transfected COS cells expressing gE or gI alone suggested that gE-gI complex formation is necessary for efficient processing of the gE precursor to its mature form . One new finding was that unlike the other alphaherpesvirus gI homologs, a fraction of pulse-labeled gI synthesized in BHV-1-infected cells apparently is cleaved into two relatively stable fragments 2 to 4 h after the pulse . Finally, we incubated BHV-1-infected cell extracts with nonimmune mouse, rabbit, horse, pig, and calf sera and found no evidence that gE or gI functioned as Fc receptors as reported for the herpes simplex virus type 1 and varicella-zoster virus homologs.

J Virol, 1996 Nov, 70(11), 7669 - 77
De novo synthesis of the early transcription factor 70-kilodalton subunit is required for morphogenesis of vaccinia virions; Hu X et al.; Vaccinia virus early transcription factor (VETF) is a heterodimeric protein that is packaged in virus particles for expression of early genes during the next round of infection . To investigate additional roles of VETF, we constructed a conditionally lethal recombinant vaccinia virus in which the D6R gene, encoding the 70-kDa subunit of VETF, is under stringent Escherichia coli lac operator control . When cells were infected with the recombinant virus in the absence of an inducer, synthesis of the 70-kDa protein was undetectable and the yield of infectious virus was severely reduced . Under these nonpermissive conditions, DNA replication and synthesis of viral proteins other than the one encoded by D6R occurred, suggesting that de novo synthesis of VETF is not required for expression of early or late genes during the virus growth cycle . Electron microscopy, however, revealed that immature virus particles and masses of electron-dense material accumulated in the absence of an inducer . We concluded that VETF has a direct role in virion morphogenesis or is required for expression of a novel subset of genes that have such a role.

J Bacteriol, 1996 Nov, 178(21), 6389 - 93
In vitro transcriptional analysis of TyrR-mediated activation of the mtr and tyrP+3 promoters of Escherichia coli; Yang J et al.; In order to understand the mechanism by which the TyrR protein activates transcription from the mtr and tyrP+3 promoters, we have carried out in vitro transcription experiments with supercoiled DNA templates . We have shown that addition of the histone-like protein HU or integration host factor (IHF) greatly inhibited the transcription from the mtr and tyrP+3 promoters . In the presence of phenylalanine, the wild-type TyrR protein, but not a mutant TyrR protein (activation negative), was able to relieve the HU- or IHF-mediated inhibition of transcription . In contrast, the alleviation of the HU- or IHF-mediated transcription inhibition by the wild-type TyrR protein did not occur when a mutant RNA polymerase with a C-terminally truncated alpha subunit was used to carry out the transcription reaction.

J Bacteriol, 1996 Nov, 178(21), 6378 - 81
The Tra2 core of the IncP(alpha) plasmid RP4 is required for intergeneric mating between Escherichia coli and Streptomyces lividans; Giebelhaus LA et al.; Escherichia coli cells and Streptomyces mycelia are able to form close contacts in the absence of a conjugative system which might facilitate intergeneric plasmid transfer without the genes required for mating pair formation (Tra2) of the RP4 plasmid . The same Tra2 genes found to be essential for RP4 plasmid transfer, RSF1010 mobilization, and donor-specific phage propagation in E . coli were also required for intergeneric transfer between E . coli and Streptomyces lividans.

J Bacteriol, 1996 Nov, 178(21), 6352 - 6
Escherichia coli proteins synthesized during recovery from starvation; Siegele DA et al.; Proteins synthesized in Escherichia coli during recovery from starvation were resolved by two-dimensional polyacrylamide gel electrophoresis . Nine outgrowth-specific proteins, which appeared in two kinetic groups, that were not detected in either starved or exponential-phase cells were synthesized . Five other proteins whose rate of synthesis during outgrowth was > or = 5-fold higher than during exponential growth were observed.

J Bacteriol, 1996 Nov, 178(21), 6348 - 51
The temperature-sensitive growth and survival phenotypes of Escherichia coli cydDC and cydAB strains are due to deficiencies in cytochrome bd and are corrected by exogenous catalase and reducing agents; Goldman BS et al.; The cydDC operon of Escherichia coli encodes an ATP-dependent transporter of unknown function that is required for cytochrome bd synthesis . Strains containing defects in either the cydD or cydC gene also demonstrate hypersensitivity to growth at high temperatures and the inability to exit the stationary phase at 37 degrees C . We wished to determine what is responsible for these hypersensitive phenotypes and whether they are due to a lack of the CydDC proteins or a defect of the cytochrome bd encoded by the cydAB genes . Using both K-12- and B-type strains of E . coli, we have compared the phenotypes of isogenic cydAB mutants and cydC mutants . In both K-12- and B-type backgrounds, the hypersensitive phenotypes are due to defects of cytochrome bd activity and not defects of the cydDC genes . We also found that the temperature-sensitive growth phenotypes can be suppressed by exogenous reducing agents, such as glutathione and cysteine . Strikingly, even the enzymes catalase and superoxide dismutase, when added exogenously, can correct the temperature-sensitive and stationary phase arrest phenotypes . We propose that the temperature sensitive growth phenotypes are due to a buildup of diffusible oxygen radicals brought on by the absence of cytochrome bd.

J Bacteriol, 1996 Nov, 178(21), 6338 - 47
Use of heme reporters for studies of cytochrome biosynthesis and heme transport; Goldman BS et al.; Strains of Escherichia coli containing mutations in the cydDC genes are defective for synthesis of the heme proteins cytochrome bd and c-type cytochromes . The cydDC genes encode a putative heterodimeric ATP-binding cassette transporter that has been proposed to act as an exporter of heme to the periplasm . To more fully understand the role of this transporter (and other factors) in heme protein biosynthesis, we developed plasmids that produce various heme proteins (e.g., cytochrome b5, cytochrome b562, and hemoglobin) in the periplasm of E . coli . By using these reporters, it was shown that the steady-state levels of polypeptides of heme proteins known to be stable without heme (e.g., cytochrome b5 and hemoglobin apoprotein) are significantly reduced in a cydC mutant . Exogenous addition of hemin to the cydC mutant still resulted in < 10% of wild-type steady-state levels of apohemoglobin in the periplasm . Since the results of heme reporter studies are not consistent with lower heme availability (i.e., heme export) in a cydC mutant, we analyzed other properties of the periplasm in cydC mutants and compared them with those of the periplasm in cydAB (encoding cytochrome bd) mutants and wild-type cells . Our results led us to favor a hypothesis whereby cydDC mutants are defective in the reduction environment within the periplasmic space . Such an imbalance could lead to defects in the synthesis of heme-liganded proteins . The heme reporters were also used to analyze strains of E . coli with a defect in genes encoding homologs of a different ABC transporter (helABC) . The helABC genes have previously been shown to be required for the assembly of c-type cytochromes in Rhodobacter capsulatus (R . G . Kranz, J . Bacteriol . 171:456-464, 1989; D . L . Beckman, D . R . Trawick, and R . G . Kranz, Genes Dev . 6:268-283, 1992) . This locus was shown to be essential in E . coli for endogenous cytochrome c biogenesis but not cytochrome b562 synthesis . Consistent with these and previous results, it is proposed that the HelABC transporter is specifically involved in heme export for ligation (hel) . This class of periplasmic cytochromes is proposed to require heme liganding before undergoing correct folding.

J Bacteriol, 1996 Nov, 178(21), 6310 - 8
The DnrN protein of Streptomyces peucetius, a pseudo-response regulator, is a DNA-binding protein involved in the regulation of daunorubicin biosynthesis; Furuya K et al.; DnrN, a protein essential for the transcription of the dnrI gene, which in turn activates transcription of the daunorubicin biosynthesis genes in Streptomyces peucetius, was overproduced in Escherichia coli and S . peucetius . The cell-free extract from E . coli was used to conduct DNA-binding assays . The results of gel mobility shift analysis showed that DnrN binds specifically to the dnrI promoter region with a high affinity (Kd = 50 nM) . Neither acetyl phosphate nor ATP affected the binding ability, and there was no difference in binding between wild-type DnrN and a mutant form (D-55-->N) lacking the putative phosphorylation site (aspartate 55) of a response regulator protein . Therefore, phosphorylation of DnrN apparently is not necessary for DNA binding . DNase I footprinting analysis indicated binding regions at 37 to 55 bp and 62 to 100 bp upstream of the transcriptional start point of dnrI . Interestingly, the sequence of these regions includes consecutive overlapping triplets {5'-(A/T)GC, 5'-(A/T)CG, 5'-(A/T)C(A/T)} that have been shown to be the preferential binding site of daunorubicin (J . B . Chaires and J . E . Herrera, Biochemistry 29:6145-6153, 1990) . This may explain why daunorubicin appeared to inhibit the binding of DnrN to the dnrI promoter, which could result in feedback repression of daunorubicin production . The results of Western blotting (immunoblotting) analysis with His-tagged DnrN antiserum showed that dnrN expression is coincident with daunorubicin production and that the maximum level of DnrN is 0.01% of total protein in the wild-type S . peucetius strain . Since the level of DnrN was lowered in mutant strains that do not produce daunorubicin, we speculate that dnrN and dnrI expression are regulated by daunorubicin.

J Bacteriol, 1996 Nov, 178(21), 6275 - 80
In vivo and in vitro characterization of Escherichia coli protein CheZ gain- and loss-of-function mutants; Sanna MG et al.; Bacterial chemotaxis results from the ability of flagellated bacteria to control the frequency of switching between smooth-swimming and tumbling episodes in response to changes in concentration of extracellular substances . High levels of phosphorylated CheY protein are the intracellular signal for inducing the tumbling mode of swimming . The CheZ protein has been shown to control the level of phosphorylated CheY by regulating its rate of dephosphorylation . To identify functional domains in the CheZ protein, we made mutants by random mutagenesis of the cheZ gene and constructed a series of deletions . The map position and the in vivo and in vitro activity of the resulting gain- or loss-of-function mutant proteins define separate functional domains of the CheZ protein.

J Bacteriol, 1996 Nov, 178(21), 6250 - 7
Characterization of a cytoplasmic trehalase of Escherichia coli; Horlacher R et al.; Escherichia coli can synthesize trehalose in response to osmotic stress and is able to utilize trehalose as a carbon source . The pathway of trehalose utilization is different at low and high osmolarity . At high osmolarity, a periplasmic trehalase (TreA) is induced that hydrolyzes trehalose in the periplasm to glucose . Glucose is then taken up by the phosphotransferase system . At low osmolarity, trehalose is taken up by a trehalose-specific enzyme II of the phosphotransferase system as trehalose-6-phosphate and then is hydrolyzed to glucose and glucose-6-phosphate . Here we report a novel cytoplasmic trehalase that hydrolyzes trehalose to glucose . treF, the gene encoding this enzyme, was cloned under ara promoter control . The enzyme (TreF) was purified from extracts of an overexpressing strain and characterized biochemically . It is specific for trehalose exhibiting a Km of 1.9 mM and a Vmax of 54 micromol of trehalose hydrolyzed per min per mg of protein . The enzyme is monomeric, exhibits a broad pH optimum at 6.0, and shows no metal dependency . TreF has a molecular weight of 63,703 (549 amino acids) and is highly homologous to TreA . The nonidentical amino acids of TreF are more polar and more acidic than those of TreA . The expression of treF as studied by the expression of a chromosomal treF-lacZ fusion is weakly induced by high osmolarity of the medium and is partially dependent on RpoS, the stationary-phase sigma factor . Mutants producing 17-fold more TreF than does the wild type were isolated.

J Bacteriol, 1996 Nov, 178(21), 6233 - 7
H+/e- stoichiometry for NADH dehydrogenase I and dimethyl sulfoxide reductase in anaerobically grown Escherichia coli cells; Bogachev AV et al.; Anaerobically grown Escherichia coli cells were shown to acidify the reaction medium in response to oxygen or dimethyl sulfoxide (DMSO) pulses, with the H+/e- stoichiometry being close to 2.5 and 1.5, respectively . In the presence of the NADH dehydrogenase I (NDH-I) inhibitor 8-methyl-N-vanillyl-6-nonenamide (capsaicin) or in mutants lacking NDH-I, this ratio decreased to 1 for O2 and to 0 for DMSO . These data suggest that (i) the H+/e- stoichiometry for E . coli NDH-I is at least 1.5 and (ii) the DMSO reductase does not generate a proton motive force.

J Bacteriol, 1996 Nov, 178(21), 6184 - 91
Tn7 transposition as a probe of cis interactions between widely separated (190 kilobases apart) DNA sites in the Escherichia coli chromosome; DeBoy RT et al.; We have used the bacterial transposon Tn7 to examine communication between widely separated DNA sites in the Escherichia coli chromosome . Using Tn7 target immunity, a regulatory feature of transposition which influences target selection, we have evaluated (i) how the presence of Tn7 sequences at one DNA site affects Tn7 insertion into another site in the same DNA molecule and (ii) the nucleotide distances over which the two sites are able to communicate . We demonstrate that Tn7 sequences at one chromosomal site act at a distance to inhibit insertion of Tn7 elsewhere in that DNA as far away as 190 kb, reflecting effective long-range cis interactions . We have found that while target immunity is effective over a substantial region of the chromosome, insertion of Tn7 into a more distant site 1.9 Mb away in the same DNA is not inhibited; this observation provides evidence that target immunity relies on DNA spacing . We also find that within the region of the chromosome affected by target immunity, the magnitude of the immune effect is greater at close DNA sites than DNA sites farther away, suggesting that target immunity is distance dependent . We also extend the characterization of the Tn7 end-sequences involved in transposition and target immunity and describe how Tn7 target immunity can be used as a tool for probing bacterial chromosome structure.

J Bacteriol, 1996 Nov, 178(21), 6145 - 50
Hypochlorous acid stress in Escherichia coli: resistance, DNA damage, and comparison with hydrogen peroxide stress; Dukan S et al.; We have investigated the mechanisms of killing of Escherichia coli by HOCl by identifying protective functions . HOCl challenges were performed on cultures arrested in stationary phase and in exponential phase . Resistance to HOCl in both cases was largely mediated by genes involved in resistance to hydrogen peroxide (H2O2) . In stationary phase, a mutation in rpoS, which controls the expression of starvation genes including those which protect against oxidative stress, renders the cells hypersensitive to killing by HOCl . RpoS-regulated genes responsible for this sensitivity were dps, which encodes a DNA-binding protein, and, to a lesser extent, katE and katG, encoding catalases; all three are involved in resistance to H2O2 . In exponential phase, induction of the oxyR regulon, an adaptive response to H2O2, protected against HOCl exposure, and the oxyR2 constitutive mutant is more resistant than the wild-type strain . The genes involved in this oxyR-dependent resistance have not yet been identified, but they differ from those primarily involved in resistance to H2O2, including katG, ahp, and dps . Pretreatment with HOCl conferred resistance to H2O2 in an OxyR-independent manner, suggesting a specific adaptive response to HOCl . fur mutants, which have an intracellular iron overload, were more sensitive to HOCl, supporting the generation of hydroxyl radicals upon HOCl exposure via a Fenton-type reaction . Mutations in recombinational repair genes (recA or recB) increased sensitivity to HOCl, indicative of DNA strand breaks . Sensitivity was visible in the wild type only at concentrations above 0.6 mg/liter, but it was observed at much lower concentrations in dps recA mutants.

J Bacteriol, 1996 Nov, 178(21), 6123 - 32
Cloning and characterization of the genes encoding nitrilotriacetate monooxygenase of Chelatobacter heintzii ATCC 29600; Knobel HR et al.; A 6.2-kb DNA fragment containing the genes for the nitrilotriacetate (NTA) monooxygenase of Chelatobacter heintzii ATCC 29600 was cloned and characterized by DNA sequencing and expression studies . The nucleotide sequence contained three major open reading frames (ORFs) . Two of the ORFs, which were oriented divergently with an intergenic region of 307 bp, could be assigned to the NTA monooxygenase components A and B . The predicted N-terminal amino acid sequences of these ORFs were identical with those determined for the purified components . We therefore named these genes ntaA (for component A of NTA monooxygenase) and ntaB (for component B) . The ntaA and ntaB genes could be expressed in Escherichia coli DH5alpha, and the gene products were visualized after Western blotting (immunoblotting) and incubation with polyclonal antibodies against component A or B . By mixing overproduced NtaB from E . coli and purified component A from C . heintzii ATCC 29600, reconstitution of a functional NTA monooxygenase complex was possible . The deduced gene product of ntaA showed only significant homology to SoxA (involved in dibenzothiophene degradation) and to SnaA (involved in pristamycin synthesis); that of ntaB shared weak homologies in one domain with other NADH:flavine mononucleotide oxidoreductases . These homologies provide no conclusive answer as to the possible evolutionary origin of the NTA monooxygenase . The deduced gene product of the third ORF (ORF1) had homology in the N-terminal region with the GntR class of bacterial regulator proteins and therefore may encode a regulator protein, possibly involved in regulation of ntaA and ntaB expression.

J Bacteriol, 1996 Nov, 178(21), 6116 - 22
Extragenic suppression of motA missense mutations of Escherichia coli; Garza AG et al.; The MotA and MotB proteins are thought to comprise elements of the stator component of the flagellar motor of Escherichia coli . In an effort to understand interactions among proteins within the motor, we attempted to identify extragenic suppressors of 31 dominant, plasmid-borne alleles of motA . Strains containing these mutations were either nonmotile or had severely impaired motility . Four of the mutants yielded extragenic suppressors mapping to the FlaII or FlaIIIB regions of the chromosome . Two types of suppression were observed . Suppression of one type (class I) probably results from increased expression of the chromosomal motB gene due to relief of polarity . Class I suppressors were partial deletions of Mu insertion sequences in the disrupted chromosomal motA gene . Class I suppression was mimicked by expressing the wild-type MotB protein from a second, compatible plasmid . Suppression of the other type (class II) was weaker, and it was not mimicked by overproduction of wild-type MotB protein . Class II suppressors were point mutations in the chromosomal motB or fliG genes . Among 14 independent class II suppressors characterized by DNA sequencing, we identified six different amino acid substitutions in MotB and one substitution in FliG . A number of the strongest class II suppressors had alterations of residues 136 to 138 of MotB . This particular region within the large, C-terminal periplasmic domain of MotB has previously not been associated with a specific function . We suggest that residues 136 to 138 of MotB may interact directly with the periplasmic face of MotA or help position the N-terminal membrane-spanning helix of MotB properly to interact with the membrane-spanning helices of the MotA proton channel.

J Bacteriol, 1996 Nov, 178(21), 6097 - 104
Influence of impaired chaperone or secretion function on SecB production in Escherichia coli; Muller JP; The efficient export of proteins through the cytoplasmic membrane of Escherichia coli requires chaperones to maintain protein precursors in a translocation-competent conformation . In addition to SecB, the major chaperone facilitating export of particular precursors, heat shock-induced chaperones DnaK-DnaJ and GroEL-GroES are also involved in this process . By use of secB'-lacZ gene fusions and immunoprecipitation experiments, SecB production was studied in E . coli strains containing conditional lethal mutations in chaperone or sec genes . While the loss of heat shock chaperones resulted in an increased production of SecB, mutations in sec genes showed only minor effects on SecB synthesis . Neither the plasmid-mediated overexpression of precursors of exoproteins nor the overexpression of secB altered the synthesis of SecB . These results suggest that under conditions where chaperones become depleted, E . coli responds by raising the expression of secB . These data confirm the supposed synergy of different chaperones involved in protein export.

J Bacteriol, 1996 Nov, 178(21), 6091 - 6
The stationary-phase-exit defect of cydC (surB) mutants is due to the lack of a functional terminal cytochrome oxidase; Siegele DA et al.; The surB gene was identified as a gene product required for Escherichia coli cells to exit stationary phase at 37 degrees C under aerobic conditions . surB was shown to be the same as cydC, whose product is required for the proper assembly and activity of cytochrome d oxidase . Cytochrome d oxidase, encoded by the cydAB operon, is one of two alternate terminal cytochrome oxidases that function during aerobic electron transport in E . coli . Mutations inactivating the cydAB operon also cause a temperature-sensitive defect in exiting stationary phase, but the phenotype is not as severe as it is for surB mutants . In this study, we examined the phenotypes of surB1 delta(cydAB) double mutants and the ability of overexpression of cytochrome o oxidase to suppress the temperature-sensitive stationary-phase-exit defect of surB1 and delta(cydAB) mutants and analyzed spontaneous suppressors of surB1 . Our results indicate that the severe temperature-sensitive defect in exiting stationary phase of surB1 mutants is due both to the absence of terminal cytochrome oxidase activity and to the presence of a defective cytochrome d oxidase . Membrane vesicles prepared from wild-type, surB1, and delta(cydAB) strains produced superoxide radicals at the same rate in vitro . Therefore, the aerobic growth defects of the surB1 and delta(cydAB) strains are not due to enhanced superoxide production resulting from the block in aerobic electron transport.

Infect Immun, 1996 Nov, 64(11), 4876 - 81
Attaching and effacing enteropathogenic Escherichia coli O18ab invades epithelial cells and causes persistent diarrhea; Scaletsky IC et al.; A case of persistent diarrhea following Escherichia coli O18ab gastroenteritis is reported . Electron microscopy of a biopsy of the small intestine showed effacement of the brush border, attachment of bacteria to the epithelial cells with pedestal formation, and bacteria within the enterocytes . The bacterial isolate was an enteropathogenic E . coli isolate which did not contain the adherence factor (EAF) but possessed the attaching-effacing eae gene, was able to invade HeLa cells in a gentamicin invasion assay, and also invaded rabbit intestinal cells . Results suggest that E . coli organisms of the O18ab serotype may cause diarrhea by an as yet unknown pathogenic mechanism, involving attaching to and effacing of enterocytes followed by invasion of the epithelial cells.

Infect Immun, 1996 Nov, 64(11), 4820 - 5
Enteropathogenic Escherichia coli markedly decreases the resting membrane potential of Caco-2 and HeLa human epithelial cells; Stein MA et al.; It is presumed, but not proven, that enteropathogenic Escherichia coli (EPEC) causes secretory diarrhea by altering ion transport in enterocytes . In this study we used the whole-cell, current clamp variant of the patch clamp technique to demonstrate that EPEC infection of HeLa and Caco-2 human epithelial cells reduces cell resting membrane potential . The observed reduction of resting membrane potential in HeLa cells results from EPEC-mediated signal transduction to the host cell but is not dependent upon EPEC-mediated elevation of levels of intracellular free calcium . These findings indicate that EPEC can directly alter the relative distribution of ions across epithelial host cell membranes . This may be relevant to the etiology of diarrhea caused by EPEC infection.

Infect Immun, 1996 Nov, 64(11), 4761 - 8
T84 cells in culture as a model for enteroaggregative Escherichia coli pathogenesis; Nataro JP et al.; Enteroaggregative Escherichia coli (EAEC) is an important cause of persistent diarrhea in many developing parts of the world, yet the pathogenetic mechanisms of EAEC diarrhea are unknown . Experiments with animal models suggest that EAEC strains damage the intestinal mucosa, and a putative cytotoxin has been described . To characterize the mucosal effects of EAEC, we studied strain 042, which we have shown to cause diarrhea in adult volunteers . Strain 042 was incubated in an in vitro organ culture model with biopsy-derived normal intestinal mucosa from pediatric patients . Strain 042 adhered strongly to samples of jejunal, ileal, and colonic mucosa . In addition, scanning electron microscopic examination of in vitro-infected intestinal biopsies revealed cytotoxic effects marked by exfoliation of mucosal epithelial cells . To develop an in vitro model to study these effects, we incubated 042 with polarized monolayers of the human intestinal epithelial cell lines Caco-2 and T84 . Strain 042 adhered strongly to T84 cells but not to Caco-2 cells . T84 cells infected with 042 displayed marked toxic effects, most prominently in areas where bacteria were adhering . The apical membrane of damaged cells exhibited vesiculation and shedding of microvilli . The cytoplasm of affected cells displayed subnuclear vacuolization, and in some cases, nuclei of affected cells became separated from the surrounding cytoplasm . Severely affected cells ruptured, releasing their nuclei . Vacuolated remnant cells were seen throughout the monolayer . Strain 042 was not internalized by T84 cells . We concluded that EAEC strain 042 alters intestinal cell morphology, ultimately leading to cell death . Although the factor(s) required for this effect remains to be elucidated, T84 cells may serve as a valuable model in EAEC pathogenesis studies.

Infect Immun, 1996 Nov, 64(11), 4751 - 60
Adhesion of enteroaggregative Escherichia coli to pediatric intestinal mucosa in vitro; Hicks S et al.; Organ cultures of small- and large-intestinal mucosa from children were used to examine the interactions of enteroaggregative Escherichia coli (EAEC) with human intestine . Mucosae from patients aged between 3 and 190 months were cultured with five EAEC strains isolated from infants with diarrhea in the United Kingdom and with two well-described prototype EAEC strains, 17-2 and 221 . The prototype strains adhered to jejunal, ileal, and colonic mucosae . The wild-type strains also adhered to this tissue but showed a variable pattern of adhesion: two adhered to all intestinal levels, one adhered to jejunum and ileum, one adhered to ileum only, and one adhered to ileum and colon . Adherence was in an aggregative or stacked-brick pattern, resembling that seen on HEp-2 cells . Electron microscopy of infected small intestinal mucosa revealed bacteria in association with a thick mucus layer above an intact enterocyte brush border, which contained extruded cell fragments . This mucus layer was not present on controls . EAEC adherence to colonic mucosa was associated with cytotoxic effects including microvillous vesiculation (but without evidence of an attaching/effacing lesion), enlarged crypt openings, the presence of intercrypt crevices, and increased epithelial cell extrusion . These results demonstrate that in vitro organ culture of intestinal mucosa from children can be used to investigate EAEC pathogenesis in childhood directly . EAEC strains appear able to colonize many regions of the gastrointestinal tract, without overt changes to small intestinal mucosa but with cytotoxic effects on colonic mucosa.

Infect Immun, 1996 Nov, 64(11), 4638 - 42
Lipopolysaccharide binding proteins on polymorphonuclear leukocytes: comparison of adult and neonatal cells; Qing G et al.; We have previously shown that polymorphonuclear leukocytes (PMN) from cord blood of normal full-term infants have a decreased priming response to lipopolysaccharide (LPS) compared with PMN of adults . Because the reason for this difference is poorly understood, we compared LPS binding on PMN from adults and newborns by using a photoactivatable iodinated LPS (from Escherichia coli O111:B4), coupled to 2-(p-azidosalicylamido)-1,3'-dithopropionate (LPS-ASD) to covalently link LPS to the PMN membrane . We incubated 2 x 10(4) adult or neonatal PMN with 125I-ASD-LPS (100 ng/ml) together with unlabelled LPS (0 to 100,000 ng/ml) for 20 min at 4 degrees C . The maximum total 125I-ASD-LPS binding to newborn PMN (1,004 +/- 103 cpm) was lower than that binding to adult PMN (3,583 +/- 444 cpm; P < 0.01 with respect to newborn PMN) . However, the concentration of unlabelled LPS that displaced 50% of the maximum specifically bound 125I-ASD-LPS was similar for PMN from adult and newborn infants (-4.85 +/- 0.04 and -5.13 +/- 0.14 log g of LPS per ml, respectively; P > 0.05) . We further assessed the membrane binding of 125I-ASD-LPS to PMN by using membrane extracts analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and autoradiography . LPS binding proteins were found at approximately 73, 55 to 57, and 25 kDa in both adult and neonatal PMN . However, PMN from newborn infants had markedly lower membrane-associated 125I-ASD-LPS at the 55- to 57- and 25-kDa protein bands as indicated by the intensity of the autoradiograph . Binding of LPS at these bands was specific for the lipid A portion of LPS, since purified unlabelled lipid A displaced 125I-ASD-LPS in a dose-dependent manner . Thus, PMN from newborn infants bind less LPS than do PMN from adults, even though the sites for LPS membrane binding appear to be the same.

Infect Immun, 1996 Nov, 64(11), 4508 - 13
Identification of a cross-reactive continuous B-cell epitope in enterotoxigenic Escherichia coli colonization factor antigen I; Rudin A et al.; Enterotoxigenic Escherichia coli (ETEC) colonizes the intestine by means of several antigenically distinct colonization factors (CFs) . Several of these CFs have very significant amino acid sequence similarity or identity, particularly in the N-terminal end . We have previously shown that a monoclonal antibody (MAb) raised against the subunits of colonization factor antigen I (CFA/I) fimbriae, which reacts with a peptide corresponding to the 25 N-terminal amino acids of such subunits, can inhibit attachment to intestinal cells of ETEC expressing heterologous as well as homologous CFs, with related amino acid sequences . In this study we have, by means of Pepscan analysis, determined the sequence of the MAb-specific linear epitope to be 15IDLLQ19 . Parenteral immunization of rabbits with an N-terminal 25-mer synthetic peptide of CFA/I fimbrial subunit, either covalently coupled to bovine serum albumin or uncoupled, induced high titers of specific antibodies against this peptide as well as against CFA/I fimbriae . Increased titers against several heterologous CF fimbriae with a related N-terminal sequence were also induced, whereas no increase was seen against fimbriae with an unrelated sequence . Neither antisera against the coupled peptide nor antisera against the uncoupled peptide inhibited binding of CF-expressing bacteria to the human intestinal cell line Caco-2 in spite of high titers . The difference in the inhibitory capabilities of the antipeptide sera and the MAb might be due to slightly different epitope specificities . Thus, whereas the antipeptide sera bound to several continuous epitopes in the N-terminal end, none of them reacted specifically with the epitope 15IDLLQ19.

Infect Immun, 1996 Nov, 64(11), 4480 - 7
Attachment of a noninvasive enteric pathogen, enteropathogenic Escherichia coli, to cultured human intestinal epithelial monolayers induces transmigration of neutrophils; Savkovic SD et al.; An intense inflammatory cell infiltrate, consisting primarily of polymorphonuclear leukocytes (PMN), accompanies enteric infection by enteropathogenic Escherichia coli (EPEC) . The mechanism(s) by which this pathogen elicits PMN recruitment has not been studied . To determine whether EPEC infection of intestinal epithelial cells induces PMN to transmigrate, an in vitro model consisting of cultured human intestinal epithelial monolayers (T84), a human EPEC strain (E2348/69), and isolated human PMN was used . Results of these studies showed that EPEC attachment to T84 monolayers stimulated the transepithelial migration of PMN in a dose-dependent fashion . This event was not attributable to the classic bacterial chemoattractants, n-formylated peptides, or other soluble bacterial factors . A nonadherent EPEC strain, JPN15, was unable to cause PMN to cross the epithelial monolayer . Epithelial protein synthesis was required for maximum EPEC-induced PMN transmigration to occur . Transfer assays demonstrated the presence of a chemokine in sterilized medium from infected monolayers . Neutralizing antibodies to interleukin 8 ablated approximately 50% of the chemotactic activity . Studies with EPEC mutant strains revealed that the eaeB gene, required for the activation of signal transduction pathways in host cells, was crucial for eliciting PMN transmigration . These data show for the first time that attachment of a noninvasive enteric pathogen to intestinal epithelial cells induces PMN transmigration . These findings strongly suggest that EPEC attachment to target host cells activates a signal transduction cascade which ultimately leads to the expression and release of an epithelium-derived chemotactic factor(s) for PMN.

Infect Immun, 1996 Nov, 64(11), 4415 - 23
Channel-forming activity and channel size of the RTX toxins ApxI, ApxII, and ApxIII of Actinobacillus pleuropneumoniae; Maier E et al.; The determinants of the Actinobacillus pleuropneumoniae RTX toxins ApxI, ApxII, and ApxIII were expressed in an Escherichia coli strain . The toxins were concentrated from the supernatants of cell cultures . The addition of the toxins to the aqueous-phase-bathing lipid bilayer membranes resulted in an increase in the membrane conductance when membranes made of asolectin or phosphatidylethanolamine were used . The toxins were relatively inactive in membranes made of other lipids . The membrane activity (i.e., the number of channels formed at a given Apx concentration) was different for each of the three Apx toxins . That of ApxI, which has the strongest cytotoxic activity, was highest, followed by that of ApxIII and ApxII, which is the least cytotoxic . The conductance increases of ApxIII and ApxII were smaller by factors of 10 and 50, respectively, than that of ApxI under otherwise identical conditions . Single-channel experiments demonstrated that all three Apx toxins formed ion-permeable channels of different conductances . The major open state was approximately the same for the two hemolytic toxins ApxI and ApxII (540 and 620 pS in 0.15 M KCI), whereas the single-channel conductance of the nonhemolytic ApxIII was approximately one-fifth of that of the other two toxins (95 pS) . Experiments with different salts suggested that the Apx channels of A . pleuropneumoniae were exclusively cation selective because of negative charges localized at the channel mouth . Analysis of the single-channel data using the Renkin correction factor suggested that the Apx toxins formed aqueous channels with different diameters for the three toxins . Pore-forming properties of the Apx toxins were compared with those of other RTX toxins . All of these toxins have common features and form channels that are transient but have different sizes as judged from the different single-channel conductances.

J Neurochem, 1996 Nov, 67(5), 2200 - 3
Cloning of CDP-diacylglycerol synthase from a human neuronal cell line; Heacock AM et al.; A critical step in the supply of substrate for the phosphoinositide signal transduction pathway is the formation of the liponucleotide intermediate, CDP-diacylglycerol, catalyzed by CDP-diacylglycerol synthase . Further insight into the regulation of phosphoinositide biosynthesis was sought by cloning of the gene for the vertebrate enzyme . Sequence of the corresponding gene from Drosophila was used to prepare a probe for screening of a human neuronal cell cDNA library . A cDNA was isolated with a predicted open reading frame of 1,332 bases, encoding a protein of 51 kDa . The amino acid sequence showed 50% identity (75% similarity) to that of Drosophila eye CDP-diacylglycerol synthase and substantial similarity to the Saccharomyces cerevisiae and Escherichia coli homologues . Northern blot analysis, with human cDNA riboprobes, suggested that the corresponding mRNA was expressed in all human tissues examined . Expression of the human cDNA in COS cells resulted in a more than fourfold increase in CDP-diacylglycerol synthase activity . Knowledge of the sequence of vertebrate CDP-diacylglycerol synthase should facilitate further investigations into its regulation and the possible existence of distinct isoforms.

Transgenic Res, 1996 Nov, 5(6), 385 - 95
FLP-mediated site-specific recombination in microinjected murine zygotes; Ludwig DL et al.; The FLP recombinase of yeast catalyses site-specific recombination between repeated FLP recombinase target (FRT) elements in yeast and in heterologous systems (Escherichia coli, Drosophila, mosquito and cultured mammalian cells) . In this report, it is shown that transient FLP recombinase expression can recombine and activate an extrachromosomal silent reporter gene following coinjection into fertilized one-cell mouse eggs . Furthermore, it is demonstrated that introduction of a FLP-recombinase expression vector into transgenic one-cell fertilized mouse eggs induces a recombination event at a chromosomal FRT target locus . The resulting event occurred at the one-cell stage and deleted a chromosomal tandem array of a FRT containing lacZ expression cassette down to one or two copies . These results demonstrate that the FLP recombinase can be utilized to manipulate the genome of transgenic animals and suggest that FLP recombinase-mediated plasmid-to-chromosome targeting is feasible in microinjected eggs.

Gene, 1996 Oct 31, 178(1-2), 71 - 4
Inclusion of an upstream transcriptional terminator in phage display vectors abolishes background expression of toxic fusions with coat protein g3p; Krebber A et al.; Expression of toxic gene products affects bacterial cell growth and phage display, causing a strong selection against plasmid maintenance and integrity . During phage propagation steps, in particular, phagemid instability can dramatically affect diversity of antibody libraries or even lead to the deletion of antibody genes . We constructed a modified phage display vector by introducing a strong transcriptional terminator upstream of the lac promoter, which together with glucose suppression of its CAP-dependent activation, very efficiently represses product formation before induction.

J Immunol Methods, 1996 Oct 30, 198(1), 101 - 10
Generation of soluble recombinant human acute phase serum amyloid A2 (A-SAA2) protein and its use in development of an A-SAA specific ELISA; McCormack CC et al.; Human acute phase serum amyloid A (the A-SAA2 isoform) was expressed at high levels using the pGEX bacterial expression system . A-SAA2 protein was expressed in E . coli NM544 as part of a fusion protein facilitating rapid purification . A-SAA2 was cleaved from the fusion moiety in the presence of a non-ionic detergent (Triton X-100) to release a soluble A-SAA2 . Further purification using ion exchange chromatography yielded a pure A-SAA2 (3 mg per litre of culture) . Antibodies generated against recombinant A-SAA2 were specific for the acute phase SAAs, A-SAA1 and A-SAA2 and showed no cross-reactivity with the constitutively expressed SAA (C-SAA) . These antibodies were used to develop a rapid enzyme-linked immunosorbent assay (ELISA) specific for the measurement of A-SAA in serum.

Mol Biochem Parasitol, 1996 Oct 30, 81(2), 225 - 37
Recombinant Plasmodium falciparum dihydrofolate reductase-based in vitro screen for antifolate antimalarials; Brobey RK et al.; We describe the system for screening the effective antifolate antimalarials that uses the recombinant Plasmodium falciparum DHFR domain of the bifunctional DHFR-TS expressed in Escherichia coli, and were designed with amino acid alterations found in the DHFR genes of the antifolate resistant strains . The validity of the screen was verified by the subsequent examination of several substituted pyrrolo{2,3-d}pyrimidines for their antimalarial activity . Among the 120 chemical derivatives, 5 compounds were identified by their preferential inhibition of the drug sensitive pfDHFR to that of the mammalian isoenzyme . As compared to the sensitive enzyme, the decrease in response of the cycolguanil-resistant and pyrimethamine-resistant enzymes to the selected compounds were relatively moderate . This gave folds decrease in sensitivity of 0.8-7.5 and 3.6-29, respectively, while those for cycloguanil and pyrimethamine were 400 and 308 . The compounds inhibited the growth of drug-sensitive cultured P . falciparum with 50% effective concentrations of the ranged 0.17-30 nM . As contrasted with the sensitive strain, the fold decrease in sensitivity of the resistant parasites were 0.9-2 and 15-50 in the case of the test compounds, while those for cycloguanil and pyrimethamine were 690 and 20,500 . Moreover, the most selective pyrrolo-pyrimidine (P-1) showed in vivo activity against P . berghei in mice.

Mol Biochem Parasitol, 1996 Oct 30, 81(2), 137 - 49
Molecular cloning of a myoD-like gene from the parasitic nematode, Trichinella spiralis; Connolly B et al.; The infective larval stage of the nematode Trichinella spiralis is an intracellular parasite of skeletal muscle cells . Infection with T . spiralis results in dedifferentiation of the host cell and the formation of a host/parasite complex . A gene encoding a T . spiralis Helix-Loop-helix (HLH) protein with homology to the myogenic transcription factor, MyoD, and to the Caenorhabditis elegans protein, CeMyoD, has been identified and partially characterized . The tsmyd-1 gene is expressed constitutively during the muscle-larval and adult stages . A purified recombinant Tsmyd-1 protein, expressed in Escherichia coli, binds to a high affinity mouse MyoD DNA binding site in vitro . The present study describes the first HLH gene to be identified in Trichinella.

Proc Natl Acad Sci U S A, 1996 Oct 29, 93(22), 12423 - 7
Gene expression and cell fusion analyzed by lacZ complementation in mammalian cells; Mohler WA et al.; Complementing reporter genes provide biological indicators of coincident expression of proteins in cells . We have adapted intracistronic complementation of the Escherichia coli lacZ gene for use in mammalian cells . Enzymatic activity detectable by quantitative biochemical assay, flow cytometry, or microscopy is produced upon convergent expression of two distinct mutant lacZ peptides within single cells, or upon fusion of cells expressing such mutants . A novel fluorescent substrate for beta-galactosidase (Fluor-X-Gal) increases detection and permits simultaneous microscopic visualization of other fluorescent markers . The enzymatic complementation described here should facilitate studies of cell fusion, cell lineage, and signal transduction, by producing activity only when two proteins are expressed at the same time and place in intact cells.

Proc Natl Acad Sci U S A, 1996 Oct 29, 93(22), 12400 - 5
Intron insertion facilitates amplification of cloned virus cDNA in Escherichia coli while biological activity is reestablished after transcription in vivo; Johansen IE; Insertion of introns into cloned cDNA of two isolates of the plant potyvirus pea seedborne mosaic virus facilitated plasmid amplification in Escherichia coli . Multiple stop codons in the inserted introns interrupted the open reading frame of the virus cDNA, thereby terminating undesired translation of virus proteins in E . coli . Plasmids containing the full-length virus sequences, placed under control of the cauliflower mosaic virus 35S promoter and the nopaline synthase termination signal, were stable and easy to amplify in E . coli if one or more introns were inserted into the virus sequence . These plasmids were infectious when inoculated mechanically onto Pisum sativum leaves . Examination of the cDNA-derived viruses confirmed that intron splicing of in vivo transcribed pre-mRNA had occurred as predicted, reestablishing the virus genome sequences . Symptom development and virus accumulation of the cDNA derived viruses and parental viruses were identical . It is proposed that intron insertion can be used to facilitate manipulation and amplification of cloned DNA fragments that are unstable in, or toxic to, E . coli . When transcribed in vivo in eukaryotic cells, the introns will be eliminated from the sequence and will not interfere with further analysis of protein expression or virus infection.

Proc Natl Acad Sci U S A, 1996 Oct 29, 93(22), 12251 - 5
The cellulose-binding domain of the major cellobiohydrolase of Trichoderma reesei exhibits true reversibility and a high exchange rate on crystalline cellulose; Linder M et al.; Cellulose-binding domains (CBDs) bind specifically to cellulose, and form distinct domains of most cellulose degrading enzymes . The CBD-mediated binding of the enzyme has a fundamental role in the hydrolysis of the solid cellulose substrate . In this work we have investigated the reversibility and kinetics of the binding of the CBD from Trichoderma reesei cellobiohydrolase I on microcrystalline cellulose . The CBD was produced in Escherichia coli, purified, and radioactively labeled by reductive alkylation with 3H . Sensitive detection of the labeled CBD allowed more detailed analysis of its behavior than has been possible before, and important novel features were resolved . Binding of the CBD was found to be temperature sensitive, with an increased affinity at lower temperatures . The interaction of the CBD with cellulose was shown to be fully reversible and the CBD could be eluted from cellulose by simple dilution . The rate of exchange measured for the CBD-cellulose interaction compares well with the hydrolysis rate of cellobiohydrolase I, which is consistent with its proposed mode of action as a processive exoglucanase.

Proc Natl Acad Sci U S A, 1996 Oct 29, 93(22), 12217 - 22
Resolution of Holliday junctions in genetic recombination: RuvC protein nicks DNA at the point of strand exchange; Bennett RJ et al.; The RuvC protein of Escherichia coli catalyzes the resolution of recombination intermediates during genetic recombination and the recombinational repair of damaged DNA . Resolution involves specific recognition of the Holliday structure to form a complex that exhibits twofold symmetry with the DNA in an open configuration . Cleavage occurs when strands of like polarity are nicked at the sequence 5'-WTT decreases S-3' (where W is A or T and S is G or C) . To determine whether the cleavage site needs to be located at, or close to, the point at which DNA strands exchange partners, Holliday structures were constructed with the junction points at defined sites within this sequence . We found that the efficiency of resolution was optimal when the cleavage site was coincident with the position of DNA strand exchange . In these studies, junction targeting was achieved by incorporating uncharged methyl phosphonates into the DNA backbone, providing further evidence for the importance of charge-charge repulsions in determining DNA structure.

Proc Natl Acad Sci U S A, 1996 Oct 29, 93(22), 12206 - 11
Coordinating DNA replication initiation with cell growth: differential roles for DnaA and SeqA proteins; Boye E et al.; We describe here the development of a new approach to the analysis of Escherichia coli replication control . Cells were grown at low growth rates, in which case the bacterial cell cycle approximates that of eukaryotic cells with G1, S, and G2 phases: cell division is followed sequentially by a gap period without DNA replication, replication of the single chromosome, another gap period, and finally the next cell division . Flow cytometry of such slowly growing cells reveals the timing of replication initiation as a function of cell mass . The data show that initiation is normally coupled to cell physiology extremely tightly: the distribution of individual cell masses at the time of initiation in wild-type cells is very narrow, with a coefficient of variation of less than 9% . Furthermore, a comparison between wild-type and seqA mutant cells shows that initiation occurs at a 10-20% lower mass in the seqA mutant, providing direct evidence that SeqA is a bona fide negative regulator of replication initiation . In dnaA (Ts) mutants the opposite is found: the mass at initiation is dramatically increased and the variability in cell mass at initiation is much higher than that for wild-type cells . In contrast to wild-type and dnaA(Ts) cells, seqA mutant cells frequently go through two initiation events per cell division cycle, and all the origins present in each cell are not initiated in synchrony . The implications for the complex interplay amongst growth, cell division, and DNA replication are discussed.

Proc Natl Acad Sci U S A, 1996 Oct 29, 93(22), 12183 - 8
The "allosteric three-site model" of elongation cannot be confirmed in a well-defined ribosome system from Escherichia coli; Semenkov YP et al.; For the functional role of the ribosomal tRNA exit (E) site, two different models have been proposed . It has been suggested that transient E-site binding of the tRNA leaving the peptidyl (P) site promotes elongation factor G (EF-G)-dependent translocation by lowering the energetic barrier of tRNA release {Lill, R., Robertson, J . M . & Wintermeyer, W . (1989) EMBO J . 8, 3933-3938} . The alternative "allosteric three-site model" {Nierhaus, K.H . (1990) Biochemistry 29, 4997-5008} features stable, codon-dependent tRNA binding to the E site and postulates a coupling between E and aminoacyl (A) sites that regulates the tRNA binding affinity of the two sites in an anticooperative manner . Extending our testing of the two conflicting models, we have performed translocation experiments with fully active ribosomes programmed with heteropolymeric mRNA . The results confirm that the deacylated tRNA released from the P site is bound to the E site in a kinetically labile fashion, and that the affinity of binding, i.e., the occupancy of the E site, is increased by Mg2+ or polyamines . At conditions of high E-site occupancy in the posttranslocation complex, filling the A site with aminoacyl-tRNA had no influence on the E site, i.e., there was no detectable anticooperative coupling between the two sites, provided that second-round translocation was avoided by removing EF-G . On the basis of these results, which are entirely consistent with our previous results, we consider the allosteric three-site model of elongation untenable . Rather, as proposed earlier, the E site-bound state of the leaving tRNA is a transient intermediate and, as such, is a mechanistic feature of the classic two-state model of the elongating ribosome.

Biochemistry, 1996 Oct 29, 35(43), 13709 - 15
NACP, a protein implicated in Alzheimer's disease and learning, is natively unfolded; Weinreb PH et al.; The "non-A beta component of Alzheimer's disease amyloid plaque" (NAC) is a minor peptide component of the insoluble fibrillar core of the Alzheimer's disease (AD) neuritic plaque . NAC amyloid fibrils seed the polymerization of A beta 1-40, the major AD amyloid protein . NAC is derived from a 14 kDa precursor protein, designated NACP, a member of a highly conserved family of heat-stable brain-specific acidic proteins which have been suggested to be involved in synapse formation and/or stabilization . NACP has also been suggested to play a role in AD . We present herein a conformational analysis of human NACP . NACP has a much larger Stokes radius (34 A) but sedimented more slowly (s20,w = 1.7S) than globular proteins of similar molecular weight, indicating that the native protein is elongated . Circular dichroism (CD) and Fourier-transform infrared spectroscopy (FTIR) indicate the absence of significant amounts of secondary structure in NACP, while CD and ultraviolet spectroscopy suggest the lack of a hydrophobic core . The conformational properties of NACP were unchanged by boiling and were independent of concentration, pH, salt, and chemical denaturants . These features indicate that NACP exists as a mixture of rapidly equilibrating extended conformers and is representative of a class of "natively unfolded" proteins, many of which potentiate protein-protein interactions.

Mutat Res, 1996 Oct 28, 358(1), 113 - 22
Influence of the uvr-dependent nucleotide excision repair on DNA adducts formation and mutagenic spectrum of a potent genotoxic agent: 7-methoxy-2-nitronaphtho{2,1-b}furan (R7000); Quillardet P et al.; The influence of the uvr-dependent excision repair system on the lethal action, mutagenic specificity, SOS induction and DNA adducts formation of 7-methoxy-2-nitronaphtho{2,1-b}furan (R7000), a potent genotoxic nitrofuran, were examined in Escherichia coli . Binding measurements of 3H-labelled R7000 to DNA indicated that R7000-DNA adducts can be removed by excision repair soon after the action of the chemical: 50% of the DNA adducts were removed within 10 min of treatment . After 1 h of incubation the level of excision reached 70% . This result was confirmed using the postlabelling technique . We found that R7000 yielded at least 10 different DNA adducts . Each of the adducts detected could be removed by excision repair . The rates of excision appeared different from one to the other . In addition, using a lacZ reversion system that is able to detect each type of base substitution mutations {1}, we found that in uvrA bacteria deficient in excision repair, R7000 can induce 5 out of the 6 possible mutational events: GC-->TA, AT-->TA, GC-->CG, AT-->CG and GC-->AT . The transition AT-->GC was not observed . Only 3 transversions: GC-->TA, AT-->TA and GC-->CG could be detected in repair proficient uvr+ bacteria . The differences between the mutagenic spectra obtained in either uvr+ bacteria or uvrA mutants indicate that some potentially mutagenic DNA adducts induced by R7000 can be removed by excision repair, thus lowering the mutagenic potency of the chemical and modifying the mutagenic spectrum detected.

Mol Gen Genet, 1996 Oct 28, 252(6), 755 - 60
The grpD55 locus of Escherichia coli appears to be an allele of dnaB; Bull HJ et al.; Attempts to characterize the grpD55 mutation of Escherichia coli have led us to conclude that the gene had been assigned an incorrect map position . The mutation was found to cotransduce with malF3089:: Tn10 (at approximately 91.5 min) and a dnaB-expressing plasmid was able to complement fully the grpD55 defect in lambda replication . These studies strongly suggest that grpD55 is an allele of dnaB and is localized near 92 min on the E . coli linkage map.

Mol Gen Genet, 1996 Oct 28, 252(6), 723 - 32
The role of multiple binding sites in the activation of zein gene expression by Opaque-2; Muth JR et al.; Opaque-2 (O2) encodes a transcriptional activator of the basic domain-leucine zipper (bZIP) class, which controls the expression level in maize endosperm of the 22kD alpha-zeins and a number of non-storage proteins . The interaction of the O2 protein at three clustered binding sites on an isolated 22 kD zein gene promoter has been investigated . O2 is shown to transactivate transcription from these sites in tobacco mesophyll protoplasts as well as in maize endosperm cells transformed by particle bombardment . The binding sites have been mutated by base exchanges, singly or in different combinations, to determine their contribution to transactivation in vivo in both the leaf protoplast and the maize endosperm system . The effect of these mutations on binding of O2 in vitro was determined by electrophoretic mobility shift assays (EMSA), using O2 protein expressed in E . coli . Two of the sites seemed to be equally effective in responding to Opaque-2 in vivo in both cell types, although one of them does not contain an ACGT core sequence, and has a lower affinity for O2 in vitro than the ACGT-containing binding site . A third site, which has the lowest affinity of all three, confers no detectable O2-dependent promoter activation alone, but significantly increases activation in combination with either one of the other sites . Hence, weaker O2 binding sites can still mediate major O2-dependent effects when present in target promoters in vivo.

Mol Gen Genet, 1996 Oct 28, 252(6), 717 - 22
Genetic analysis of functional connectivity between substrate recognition domains of Escherichia coli glutaminyl-tRNA synthetase; Kitabatake M et al.; It has previously been shown that the single mutation E222K in glutaminyl-tRNA synthetase (GlnRS) confers a temperature-sensitive phenotype on Escherichia coli . Here we report the isolation of a pseudorevertant of this mutation, E222K/C171G, which was subsequently employed to investigate the role of these residues in substrate discrimination . The three-dimensional structure of the tRNA(Gln): GlnRS: ATP ternary complex revealed that both E222 and C171 are close to regions of the protein involved in interactions with both the acceptor stem and the 3' end of tRNA(Gln) . The potential involvement of E222 and C171 in these interactions was confirmed by the observation that GlnRS-E222K was able to mischarge supF tRNA(Tyr) considerably more efficiently than the wild-type enzyme, whereas GlnRS-E222K/C171G could not . These differences in substrate specificity also extended to anticodon recognition, with the double mutant able to distinguish supE tRNA(CUA)(Gln) from tRNA2(Gln) considerably more efficiently than GlnRS E222K . Furthermore, GlnRS-E222K was found to have a 15-fold higher K(m) for glutamine than the wild-type enzyme, whereas the double mutant only showed a 7-fold increase . These results indicate that the C171G mutation improves both substrate discrimination and recognition at three domains in GlnRS-E222K, confirming recent proposals that there are extensive interactions between the active site and regions of the enzyme involved in tRNA binding.

Mol Gen Genet, 1996 Oct 28, 252(6), 695 - 9
Cloning and sequence comparison of AvaI and BsoBI restriction-modification systems; Ruan H et al.; AvaI and BsoBI restriction endonucleases are isoschizomers which recognize the symmetric sequence 5'CYCGRG3' and cleave between the first C and second Y to generate a four-base 5' extension . The AvaI restriction endonuclease gene (avaIR) and methylase gene (avaIM) were cloned into Escherichia coli by the methylase selection method . The BsoBI restriction endonuclease gene (bsoBIR) and part of the BsoBI methylase gene (bsoBIM) were cloned by the "endo-blue" method (SOS induction assay), and the remainder of bsoBIM was cloned by inverse PCR . The nucleotide sequences of the two restriction-modification (RM) systems were determined . Comparisons of the predicted amino acid sequences indicated that AvaI and BsoBI endonucleases share 55% identity, whereas the two methylases share 41% identity . Although the two systems show similarity in protein sequence, their gene organization differs . The avaIM gene precedes avaIR in the AvaI RM system, while the bsoBI R gene is located upstream of bsoBI M in the BsoBI RM system . Both AvaI and BsoBI methylases contain motifs conserved among the N4 cytosine methylases.

J Mol Biol, 1996 Oct 25, 263(2), 369 - 82
Contributions to conformational entropy arising from bond vector fluctuations measured from NMR-derived order parameters: application to protein folding; Yang D et al.; The relation between order parameters derived from NMR spin relaxation experiments and the contribution to conformational entropy from ns-ps timescale bond vector dynamics is investigated by considering a number of simple models describing bond vector motion . In a few cases both classical and quantum mechanical derivations are included to establish the validity of obtaining order parameter-entropy relations using classical mechanics only . For these cases it is found that classical and quantum mechanical derivations give very similar results so long as the square of the order parameter of the bond vector is less than approximately 0.95 . For a given change in order parameter, the change in conformational entropy is sensitive to the model employed, with the absolute value of the entropy change increasing with the number of degrees of freedom in the model . The entropy-order parameter profile calculated from a 1.12 ns molecular dynamics trajectory of fully hydrated Escherichia coli ribonuclease HI is well fit using a simple expression based on a model assuming bond vector diffusion in a cone, suggesting that it may well be possible to extract meaningful entropy changes reflecting changes in ps-ns time scale motions from changes in NMR-derived order parameters . Contributions to the conformational entropy change associated with a folding-unfolding transition of an SH3 domain and calculated from changes in rapid N-HN backbone dynamics are presented.

J Mol Biol, 1996 Oct 25, 263(2), 359 - 68
Rational molecular design and genetic engineering of herbicide resistant crops by structure modeling and site-directed mutagenesis of acetohydroxyacid synthase; Ott KH et al.; Plants with specific resistance to a single class of herbicides have been genetically engineered by introduction of genes encoding rationally designed mutant acetohydroxyacid synthase (AHAS) enzymes . Suitable substitution mutations were identified from a three-dimensional model of an AHAS-inhibitor complex . The structural model was generated based on homology to pyruvate oxidase and an imidazolinone inhibitor was positioned in the proposed binding site using structure-activity data for this class of herbicide . Biochemical analysis of the mutant proteins expressed in Escherichia coli enabled iterative optimization of the mutant genes . Expression of recombinant proteins in tobacco plants conferred resistance in vivo . The novel approach coupling molecular modeling and molecular biology has many advantages over traditional random mutagenesis and selection methods and will be crucial to the future development for environmentally safe and sustainable agricultural systems.

J Mol Biol, 1996 Oct 25, 263(2), 149 - 62
Systematic mutational analysis revealing the functional domain organization of Escherichia coli nucleoid protein H-NS; Ueguchi C et al.; The Escherichia coli H-NS protein is one of the major constituents of the nucleoid structure . This protein has been implicated not only in the compact organization of the nucleoid structure, but also in the global regulation of gene expression . H-NS negatively regulates the transcription of a number of apparently unlinked genes on the chromosome, suggesting that it functions as a global transcriptional repressor . In this study, on systematic mutational analysis of hns, three distinct functional domains were found in H-NS, which appear to be responsible for DNA-binding, transcriptional repression and protein-protein interaction (dimerization and/or oligomerization), respectively . We first isolated a number of hns mutations which resulted in derepression of the proVWX operon . These included 20 independent missence mutations each resulting in a single amino acid substitution, and six nonsense mutations each giving a C-terminally truncated form of H-NS . The substituted amino acids were revealed to be located non-randomly in the primary sequence of H-NS . This set of hns mutants was examined extensively in terms of phenotypes and biochemical properties . Based on the in vivo and in vitro results, together with the locations of the altered amino acids, three distinct functional domains were identified in H-NS . Mutations in the C-terminal domain resulted in a loss of its DNA-binding ability, suggesting that this domain is directly involved in its binding to DNA . The N-terminal domain was suggested to be involved in the ability to repress transcription . Mutations in this region abolished its ability to repress the transcription of proV, in vivo and in vitro, without loss of its DNA-binding activity . None of the mutants examined was impaired in the formation of a dimer and/or oligomers, suggesting that the central region of H-NS is involved in oligomerization . These results are discussed with special reference to the molecular mechanism underlying the function of H-NS as a transcriptional repressor . In addition, expression of the bgl operon was found to be affected by only a subset of hns mutations in a highly allele-specific manner . This finding is also addressed with regard to a unique regulatory mechanism (i.e . silencing) for the bgl operon, which is partly mediated by H-NS.

J Mol Biol, 1996 Oct 25, 263(2), 140 - 8
Enhanced ribosome frameshifting in stationary phase cells; Barak Z et al.; We have examined the effect of growth phase in Escherichia coli on the translation of a plasmid-borne lacZ gene in which active enzyme synthesis requires a leftward frameshift . During the log phase of growth, the differential rate of enzyme synthesis is very low . It increases by about two orders of magnitude during the small amount of protein synthesis which occurs at the end of log phase and the early part of stationary phase . The increase is sufficient to increase the enzyme's specific activity in crude extracts to 30 times more than it would be if the log-phase differential rate continued unchanged . No such large increase is observed with a zero-frame lacZ+ control gene on the same plasmid under the control of the same promoter; a significant but much smaller increase is observed with a zero-frame control containing an in-frame terminator triplet in the region of the required frameshift . Protein sequence analysis of the enzyme made from the frameshift reporter in stationary cells shows that the increased enzyme synthesis is due to frameshifting, and not due to termination and reinitiation . The frameshift occurs at or right after the sequence U UUC AAG, an intrinsically shifty site.

J Mol Biol, 1996 Oct 25, 263(2), 126 - 39
Regulation of HU alpha and HU beta by CRP and FIS in Escherichia coli; Claret L et al.; The dimeric histone-like protein HU, one of the most abundant DNA binding proteins of Escherichia coli, is encoded by two closely related but unlinked genes, hupA and hupB . Overproduction of one or the other of the subunits has been shown to induce the SOS response and mucoidy . To understand how the synthesis of this protein is coordinated, we studied the transcription control of the two hup genes . We show here that CRP stimulated the transcription of both genes . In contrast, the FIS protein, one of the major positive regulators of the stable RNA operons, stimulated the transcription of the hupA gene, whereas it repressed that of the hupB gene . Moreover, stringent control, which like FIS also regulates the transcription of the stable RNA operons, affected the hupB transcription while it had no effect on hupA . This opposite regulation of the transcription of the two HU genes is reflected at the protein level signifying that changes in the composition of HU occur upon changes in the environment . It is rather unexpected that such divergent transcriptional regulation controls the two genes encoding a dimeric protein.

J Biol Chem, 1996 Oct 25, 271(43), 26855 - 62
Disulfide bond engineering to monitor conformational opening of apolipophorin III during lipid binding; Narayanaswami V et al.; Apolipophorin III (apoLp-III) from the Sphinx moth, Manduca sexta, is an exchangeable, amphipathic apolipoprotein that alternately exists in water-soluble and lipid-bound forms . It is organized as a five-helix bundle in solution, which has been postulated to open at putative hinge domains to expose the hydrophobic interior, thereby facilitating interaction with the lipoprotein surface (Breiter, D . R . , Kanost, M . R., Benning, M . M., Wesenberg, G., Law, J . H., Wells, M . A., Rayment, I., and Holden, H . M . (1991) Biochemistry 30, 603-608) . To test this hypothesis, we engineered two cysteine residues in apoLp-III, which otherwise lacks cysteine, by site-directed mutagenesis at Asn-40 and Leu-90 . Under oxidizing conditions the two cysteines spontaneously form a disulfide bond, which should tether the helix bundle and thereby prevent opening and concomitant lipid interaction . N40C/L90C apoLp-III was overexpressed in Escherichia coli and characterized for disulfide bond formation, secondary structure content, and stability, under both oxidizing and reducing conditions . Functional characterization was carried out by comparing the abilities of the oxidized and reduced protein to associate with modified lipoproteins in vitro . While the reduced form behaved like wild type apoLp-III, the oxidized form was unable to associate with lipoproteins . These results suggest that opening of the helix bundle is required for interaction with lipoproteins and provide a molecular basis for the dual existence of water-soluble and lipid-bound forms of apoLp-III . However, in phospholipid bilayer association assays, wild type, reduced, and oxidized N40C/L90C apoLp-III exhibited similar abilities to transform dimyristoylphosphatidylcholine multilamellar vesicles to disc-like complexes, as judged by electron microscopy . These data emphasize that underlying differences exist in initiating or maintaining a stable interaction of apoLp-III with phospholipid disc complexes versus spherical lipoprotein surfaces.

J Biol Chem, 1996 Oct 25, 271(43), 26835 - 42
Functional requirements of the active site position 185 in the human enzyme galactose-1-phosphate uridylyltransferase; Quimby BB et al.; The active site of galactose-1-phosphate uridylyltransferase (GALT) includes a HPH sequence that has been conserved in all species examined from Escherichia coli to humans . The crystal structure of the E . coli enzyme suggests that this proline is important in positioning the active site histidine (His-166) near the substrate . To examine the role of this proline in the homologous human sequence, we have performed saturating mutagenesis at Pro-185 within human GALT and characterized each resultant mutant enzyme using a yeast expression system . Activity analyses in crude lysates indicated that only proline at position 185 produced wild-type levels of activity, although five other amino acids, Ala, Gly, Ser, Gln, and Glu, all produced partially active enzymes . Western blot analyses of the GALT proteins in these lysates demonstrated that abundance varied from 9-118% of wild-type and was independent of activity . All five active mutant proteins were purified and characterized with regard to specific activity, apparent Km for both substrates, and temperature-dependence of activity . Finally, modeling of these mutations onto the conserved E . coli active site structure was performed . Together, these results provide functional evidence demonstrating the critical role of Pro-185 in facilitating the transferase reaction.

J Biol Chem, 1996 Oct 25, 271(43), 26684 - 9
Novel type of receptor-like protein kinase from a higher plant (Catharanthus roseus) . cDNA, gene, intramolecular autophosphorylation, and identification of a threonine important for auto- and substrate phosphorylation; Schulze-Muth P et al.; We characterize CrRLK1, a novel type of receptor-like kinase (RLK), from the plant Catharanthus roseus (Madagascar periwinkle) . The protein (90.2 kDa) deduced from the complete genomic and cDNA sequences is a RLK by predicting a N-terminal signal peptide, a large extracytoplasmic domain, a membrane-spanning hydrophobic region followed by a transfer-stop signal, and a C-terminal cytoplasmic protein kinase with all 11 conserved subdomains . It is a novel RLK type because the predicted extracytoplasmic region shares no similarity with other RLKs . The autophosphorylation was investigated with affinity-purified proteins expressed in Escherichia coli . The activity was higher with Mn2+ than with Mg2+ and achieved half-maximal rates at 2-2.5 microM ATP . The phosphorylation was predominantly on Thr, less on Ser, and not on Tyr . In contrast to other plant RLK, the kinase used an intra- rather than an intermolecular phosphorylation mechanism . After protein cleavage with formic acid, most of the radioactivity was in a 14.1-kDa peptide located at the end of the kinase domain . Mutagenesis of the four Thr residues in this peptide identified Thr-720 in the subdomain XI as important for autophosphorylation and for phosphorylation of beta-casein . This Thr is conserved in other related kinases, suggesting a subfamily sharing common autophosphorylation mechanisms.

J Biol Chem, 1996 Oct 25, 271(43), 26637 - 45
Molecular cloning, overexpression in Escherichia coli, structural and functional characterization of house fly cytochrome b5; Guzov VM et al.; A microsomal cytochrome b5 cDNA from the house fly, Musca domestica, was cloned and sequenced . The deduced amino acid sequence of the full-length house fly cytochrome b5 (134 residues) is 48% identical to that of rat microsomal cytochrome b5 . The house fly cytochrome b5 protein was overexpressed in Escherichia coli, purified, and characterized . Absorption and EPR spectroscopy reveal properties very similar to cytochromes b5 from vertebrates . NMR spectra indicate that the orientation of the heme in the protein relative to its alpha,gamma meso axis is about 1:1 . A redox potential of -26 mV versus standard hydrogen electrode was measured by cyclic voltammetry on a modified gold electrode in the presence of hexamminechromium(III) chloride . The cytochrome b5 is reduced by house fly cytochrome P450 reductase in a reconstituted system at a high rate (5.5 s-1), and it stimulates heptachlor epoxidation when reconstituted with house fly cytochrome P450 reductase, cytochrome P450 6A1, phospholipid, and detergent . Cytochrome b5 decreases the apparent Km for P450 reductase and increases the Vmax for heptachlor epoxidation at constant cytochrome P450 6A1 concentrations . The results indicate that cytochrome b5 stimulates a step following the first electron transfer during cytochrome P450 6A1 turnover.

J Biol Chem, 1996 Oct 25, 271(43), 26630 - 6
Signal detection by the PhoQ sensor-transmitter . Characterization of the sensor domain and a response-impaired mutant that identifies ligand-binding determinants; Waldburger CD et al.; The PhoP-PhoQ two-component system is required for virulence and/or regulatory stress responses in enteric bacteria . The PhoQ protein responds to low concentrations of extracellular divalent cations by activating PhoP-mediated transcription of a set of genes . PhoQ is a member of a family of transmembrane proteins that contain a periplasmic sensor domain coupled to a cytoplasmic transmitter domain . Here, we describe the cloning, purification, and properties of a fragment of Escherichia coli PhoQ corresponding to the sensor domain . This fragment is monomeric in solution and has a circular dichroism spectrum indicative of a mixture of alphahelix and beta-sheet . Divalent cations do not affect the oligomeric state, circular dichroism spectrum, or fluorescence spectrum of the sensor domain but do stabilize this domain to denaturation in a fashion expected for a direct binding model . We have also constructed a mutant in which a cluster of acidic amino acids (EDDDDAE) in the sensor domain is replaced with conservative, uncharged residues (QNNNNAQ) . The mutant sensor domain is indistinguishable from wild type in terms of oligomeric form and spectral properties but differs in being substantially more stable to urea denaturation, showing no additional stabilization in the presence of divalent cations, and showing little activation of PhoP-mediated transcription in response to divalent-cation starvation in vivo . These data are consistent with a model in which divalent cations bind to the acidic cluster of the wild-type sensor domain and stabilize a conformation that is inactive in signaling . Substituting uncharged residues for the acidic cluster appears to mimic the effect of divalent-cation binding by stabilizing the inactive conformation.

J Biol Chem, 1996 Oct 25, 271(43), 26622 - 9
Carboxyl-terminal fragments of phospholipase C-beta1 with intrinsic Gq GTPase-activating protein (GAP) activity; Paulssen RH et al.; Fragments of the approximately 50 kDa COOH-terminal region of phospholipase C-beta1 (PLC-beta1(1)), ranging in size from 14 to 38 kDa, were expressed in Escherichia coli, purified, and tested for their regulatory activities . As expected, none of the fragments had phospholipase activity . Several fragments, referred to as PLC tails, displayed GTPase-activating protein (GAP) activity for Gq, the G protein class that stimulates the PLC-betas in response to receptors . Gq GAP activity is characteristic of intact PLC-betas . In reconstituted phospholipid vesicles that contained purified Gq and m1 muscarinic cholinergic receptors, the most active tails increased agonist-stimulated, steady-state GTPase activity over 4-fold . Stimulation of steady-state GTPase by the tails depended on receptors for facilitation of GDP-GTP exchange, suggesting that the tails act by accelerating hydrolysis of bound GTP . In addition to intrinsic GAP activity, one tail with high GAP activity and others with low or minimal activity potentiated the GAP activity of intact PLC-beta1 . Other tails inhibited PLC-beta1s GAP effect . Both intrinsic GAP activity and potentiation of the PLC-beta1 GAP effect were often biphasic, with maxima as low as 100 nM tail and declining activities at higher concentrations . Several tails inhibited either the phospholipase activity of PLC-beta1, its stimulation by Gq, or both . The tails thus define the region of PLC-beta1 that has Gq GAP activity and suggest a mechanism of action in which the COOH terminus of PLC-betas can interact with Gq and with other PLC-beta1 molecules.

J Biol Chem, 1996 Oct 25, 271(43), 26536 - 42
Long term correction of bilirubin-UDP-glucuronosyltransferase deficiency in Gunn rats by administration of a recombinant adenovirus during the neonatal period; Takahashi M et al.; Injection of a recombinant adenovirus expressing human bilirubin-UGT1 (Ad-hBUGT1) (3 x 10(9) plaque-forming units (pfu) intravenously) in adult bilirubin-UDP-glucuronosyltransferase-1 (BUGT1)-deficient Gunn rats resulted in biliary excretion of bilirubin glucuronides and a 70% reduction of serum bilirubin levels . However, the effect was transient, and host humoral and cellular immune response prevented transgene expression after subsequent injections . To determine whether injection during the neonatal period would tolerize the host to the recombinant virus, we injected 1 x 10(8) pfu of Ad-hBUGT1 or Ad-LacZ (a recombinant adenovirus expressing Escherichia coli beta-galactosidase) into 1-3-day-old Gunn rats . Two subsequent injections (3 x 10(9) pfu) were given 56 and 112 days after the initial injection . Injection of Ad-BUGT1, but not Ad-LacZ, reduced serum bilirubin by 70-76% of the levels in untreated pups (9 +/- 1.3 mg/dl), followed by a gradual increase to 3.25 +/- 0.3 mg/dl in 56 days; similar or greater reductions occurred after the second and third injection . Serum neutralizing antibody titer and cytotoxic lymphocyte activity against adenovirus-infected hepatocytes were low or undetectable . Thus, tolerization by injection of the virus during the neonatal period permits long term gene therapy by repeated injection of the virus.

J Biol Chem, 1996 Oct 25, 271(43), 26522 - 8
Characterization of mutations in the beta subunit of the mitochondrial F1-ATPase that produce defects in enzyme catalysis and assembly; Liang Y et al.; The ATP2 gene, coding for the beta subunit of the mitochondrial F1-ATPase, was cloned from nine independent isolates of chemically mutagenized yeast . Seven different mutant alleles were identified . In one case the mutation occurs in the mitochondrial targeting sequence (M1I) . The remaining six mutations map to the mature part of the beta subunit protein and alter amino acids that are conserved in the bovine heart mitochondrial and Escherichia coli beta subunit proteins . Biochemical analysis of the yeast atp2 mutants identified two different phenotypes . The G133D, P179L, and G227D mutations correlate with an assembly-defective phenotype that is characterized by the accumulation of the F1 alpha and beta subunits in large protein aggregates . Strains harboring the A192V, E222K, or R293K mutations assemble an F1 of normal size that is none-the-less catalytically inactive . The effect of the atp2 mutations was also analyzed in diploids formed by crossing the mutants to wild type yeast . Hybrid enzymes formed with beta subunits containing either the G133D, E222K, or R293K mutations are compromised for steady-state ATPase activity . The display of partial dominance confirms the importance of Gly133 for structural stability and of Glu222 and Arg293 for catalytic cooperativity.

J Biol Chem, 1996 Oct 25, 271(43), 26499 - 507
Leucine-responsive regulatory protein-DNA interactions in the leader region of the ilvGMEDA operon of Escherichia coli; Rhee KY et al.; The leucine-responsive regulatory protein (Lrp) regulates the expression of many operons in Escherichia coli including several involved in the metabolism of the branched-chain amino acids, L-isoleucine, L-valine, and L-leucine . The ilvGMEDA operon contains the genes for four of the five enzymes of the common pathway for the biosynthesis of these amino acids . A high affinity, consensus-like Lrp-DNA binding site has been identified at an unusual position in the leader region of this operon 226 base pairs downstream of the transcriptional initiation site between the attenuator and the ilvG gene . Binding to this site facilitates the cooperative binding of a second Lrp protomer to an adjacent, upstream, secondary site . At higher Lrp concentrations, binding to a third site is observed . Chemical, enzymatic, and alkylation protection and interference footprinting experiments demonstrate that the Lrp homodimer contacts the DNA helix at symmetrical half-sites present in adjacent major grooves and that the primary and secondary binding sites are separated by one helical turn and aligned along the same face of the DNA helix . In vivo, Lrp represses transcription through the leader-attenuator region of the ilvGMEDA operon . Lrp-dependent production of attenuated RNA transcripts is also observed in vitro . No transcriptional effects are observed, in vivo or in vitro, in the absence of an intact Lrp primary binding site . A possible physiological role for Lrp in the regulation of ilvGMEDA operon expression is discussed.

Mutat Res, 1996 Oct 25, 357(1-2), 231 - 6
The rate of adaptive mutagenesis in Escherichia coli is enhanced by oxygen (superoxide); Benov L et al.; Adaptive mutagenesis is that which occurs in non-dividing cells and which allows growth under the selective conditions imposed . We now report that reversion of amino acid auxotrophies in E . coli fits that definition and is enhanced under conditions conducive to oxidative damage to DNA . Thus adaptive mutagenesis was approximately 4-fold more frequent in a sodA sodB strain than in the superoxide dismutase-replete parental strain and this mutagenesis was suppressed under anaerobic conditions . Moreover, a cell permeant manganic porphyrin, capable of catalyzing the dismutation of O2-, diminished the rate of occurrence of these mutations . Repair of oxidative damage to DNA, in the non-dividing cells, appears to provide the opportunity for adaptive mutagenesis.

Mutat Res, 1996 Oct 25, 357(1-2), 199 - 208
The mutational specificity of furazolidone in the lacI gene of Escherichia coli; Bertenyi KK et al.; The mutational specificity of the 5-nitrofuran derivative furazolidone was determined in the lacI gene of Escherichia coli . E . coli strain TC3960 (delta uvrB, pKM101) was treated with 10 microM furazolidone, yielding an induced mutation frequency of 30 times over the spontaneous frequency . Mutations from 88 furazolidone-induced mutants were analyzed by DNA sequencing: 74 were base substitutions, 7 were frameshift mutations, 3 were tandem base substitutions, 3 were complex mutations and 1 deletion was detected . The specificity of mutation was compared to that of furylfuramide (AF2) . Differences were observed in both the site specificity and the mutagenic specificity of the two 5-nitrofuran derivatives . (1) Furazolidone-induced point mutations were observed at both G:C and A:T base pairs; 93% of AF2-induced point mutations were targeted to G:C sites . (2) At G:C sites approximately equal numbers of G:C-->T:A transversions and G:C-->A:T transversions and G:C-->A:T transitions were induced by furazolidone; AF2-induced G:C-->T:A transversions outnumbered G:C-->A:T transitions 76:49 . (3) There was no observable preference for particular sequences of furazolidone-induced mutations; the prominent hotspots for AF2-induced G:C-->T:A transversions, G:C-->A:T transitions and -(G:C) frameshifts were at 5'-TGC-3' sequences in the lacI gene . (4) Furazolidone-induced frameshifts occurred at homopolymeric sequences suggesting that the mutations arose through a strand slippage mechanism; AF2-induced frameshifts occurred at a nonreiterated G:C base pair and could be templated, through formation of a palindrome, by a sequence 110 base pairs upstream from the site of mutation . The significant differences that we observe between the two spectra do not support the notion that structurally different 5-nitrofuran derivatives might react in a similar manner with DNA to produce premutational lesions with similar characteristics.

Mutat Res, 1996 Oct 25, 357(1-2), 1 - 15
Base analog N6-hydroxylaminopurine mutagenesis in Escherichia coli: genetic control and molecular specificity; Pavlov YI et al.; We have studied the molecular specificity of the base analog N6-hydroxylaminopurine (HAP) in the E . coli lacI gene, as well as the effects of mutations in DNA repair and replication genes on HAP mutagenesis . HAP induced base substitutions of the two transition types (A . T-->G . C and G . C-->A . T) at equal frequency . This bi-directional transition specificity is consistent with in vitro primer extension experiments with the Klenow fragment of DNA polymerase I in which we observed that either dTTP or dCTP were incorporated opposite HAP in an oligonucleotide template . The spectrum of HAP-induced transitions was different from the spontaneous transitions in either a wild-type or a mismatch-repair-defective (mutL) strain . Mutations in genes controlling excision repair, exonucleolytic proofreading, mismatch correction, error-prone (SOS) repair and 8-oxo-guanine repair did not affect HAP-induced mutagenesis substantially . However, an extensive deletion of several genes in the uvrB-bio region conferred supersensitivity to the lethal and mutagenic effects of HAP, perhaps due to an effect on HAP metabolism . dnaE antimutator alleles reduced HAP-forward mutagenicity in allele-specific manner: dnaE911 reduced it several fold, while dnaE915 abolished it almost completely . The results obtained are consistent with the idea that HAP is mutagenic in E . coli via a pathway generating replication errors.

Gene, 1996 Oct 24, 177(1-2), 267 - 8
Escherichia coli strain for thermoinducible T7 RNA polymerase-driven expression; Fedorova ND et al.; An Escherichia coli strain for thermoinducible T7Pol-driven transcription has been constructed . The strain was obtained by site-specific integration of the T7 gene 1 coding for T7Pol into the attB site of phage lambda in the E . coli chromosome . The expression of the inserted gene is regulated by cIts857 and major early promoter-operator regions of phage lambda.

Gene, 1996 Oct 24, 177(1-2), 223 - 8
Degradation products of the cytochrome c3 mRNA are similar in Desulfovibrio vulgaris Hildenborough and Escherichia coli; Cruz AA et al.; The transcription and mRNA degradation pattern of a cloned Desulfovibrio vulgaris (Dv) Hildenborough cytochrome c3-encoding gene (cyc) was analyzed in detail, both in Escherichia coli and its native species . Transcription in Dv seems to be controlled by the same promoter elements as in E . coli; the transcription start point (tsp) of this Dv gene has been mapped in both species and found to be identical . A major putative transcription terminator was mapped and it was found to be the same in both organisms . Furthermore, the intermediates in cyc mRNA degradation are similar in both bacterial species.

Gene, 1996 Oct 24, 177(1-2), 209 - 16
Molecular mechanisms of intramolecular recombination-dependent recircularization of linearized plasmid DNA in Escherichia coli: requirements for the ruvA, ruvB, recG, recF and recR gene products; McFarlane RJ et al.; Intramolecular recombinogenic recircularization (IRR) of linearized plasmid DNA was used to study mechanistic relationships between recombination functions in Escherichia coli in vivo . Homology requirement for IRR ranges from 1 to 11 bp, and does not exhibit any notable strain to strain variability, with recombination occurring at a large number of possible sites within the plasmid molecule . We show that recF- and recR-deficient strains exhibit greatly reduced IRR efficiency, although neither gene product is totally essential . Mutation of recF and recR does not alter the distribution of recombination sites nor the range of molecules produced during IRR . A recO-deficient strain did not exhibit dramatic reduction in efficiency of IRR, implying that RecF and RecR proteins maintain function during this mechanism in the absence of functional RecO . The main IRR mechanism is ruvA-, ruvB- and recG-dependent and there is a lower efficiency second IRR mechanism operating in ruvA, ruvB and recG mutants . Some evidence suggests that this second mechanism involves functions associated with the replisome.

Gene, 1996 Oct 24, 177(1-2), 173 - 7
Production of hepatitis B virus preS polypeptide in Escherichia coli by mutation of the 5'-end coding sequence and its purification and characterization; Kim HS et al.; The preS1 and preS2 antigens (preS Ag) of hepatitis B virus (HBV) elicit virus-neutralizing and protective antibodies which can overcome nonresponsiveness to the currently available vaccine for HBV and also carry the attachment site to HBV hepatocyte receptor . Therefore, in order to study the development of more effective vaccine and the receptor-ligand interaction, it will be helpful to obtain high-level production of the preS Ag from bacteria . We have found that the native preS region gene was not expressed under the control of commonly used promoters in Escherichia coli . By site-directed mutagenesis of some nucleotides at the 5'-end of the preS1 region gene, we have generated a mutant gene which is highly expressed in soluble form in E . coli . The produced polypeptide could be efficiently purified by 20% ammonium sulfate precipitation and a gel permeation chromatography and the purified polypeptide was demonstrated to exhibit the antigenicity and the immunogenicity of the preS1 and preS2 Ag, suggesting that it is functional.

Gene, 1996 Oct 24, 177(1-2), 155 - 62
Periplasmic expression of part of the major rotavirus capsid protein VP7 containing all the three antigenic regions in Escherichia coli; Wang L et al.; Part of the porcine rotavirus outer capsid protein VP7 containing all the three antigenic regions was expressed as a chimeric protein with bacterial alkaline phosphatase (AP) in E . coli . The construct contains an ompF promoter, the DNA encoding the signal sequence and the first 12 amino acids of mature OmpF, part of vp7 and the DNA encoding mature AP . The chimeric protein is stable, retains the biological property of AP and ability to react with polyclonal antiserum against the virus, and can be exported through the bacterial inner membrane into the periplasm.

Gene, 1996 Oct 24, 177(1-2), 133 - 6
A set of compatible tac promoter expression vectors; Dykxhoorn DM et al.; A series of expression vectors have been developed which all contain an identical expression cassette comprised of the lacIq gene, the tac promoter, a multiple cloning site (MCS) and a downstream transcriptional terminator . This cassette has been inserted into four distinct plasmid backbones, each of which is from a separate incompatibility group and carries a different drug resistance gene . Therefore, different combinations of these expression plasmids can be stably maintained together.

Gene, 1996 Oct 24, 177(1-2), 115 - 21
A mgl-like operon in Treponema pallidum, the syphilis spirochete; Porcella SF et al.; A 38-kDa lipoprotein of Treponema pallidum subsp . pallidum (T . pallidum), the syphilis spirochete, previously was identified as a putative homolog of E . coli MglB {Becker et al . (1994) Infect . Immun . 62, 1381-1391} . In the present study, genome walking in regions adjacent to the T . pallidum 38-kDa lipoprotein gene has identified three contiguous genes (tp-mglB {formerly tpp38}, tp-mglA, and tp-mglC) which appear to comprise a mgl-like operon in T . pallidum . A prominent transcript corresponding to tp-mglB, the first gene of the operon which encodes the carbohydrate receptor, is synthesized by T . pallidum along with lesser abundant transcript(s) corresponding to the entire T . pallidum mgl operon . An active promoter 135 bp upstream of tp-mglB is believed to direct mRNA synthesis for the operon . This is the first membrane protein-encoding operon of T . pallidum for which a putative function (glucose import) has been assigned . Furthermore, by analogy with E . coli MglB which interacts with the sensory transducer Trg to induce a chemotactic response, it is possible that T . pallidum also contains a homolog of E . coli Trg or other methyl-accepting chemotaxis proteins . The existence of a mgl operon in T . pallidum thus may have important implications with respect to T . pallidum survival, tissue dissemination, and sensory transduction during virulence expression.

Gene, 1996 Oct 24, 177(1-2), 55 - 8
Gene cloning and production of active recombinant Brugia malayi microfilarial chitinase; Southworth MW et al.; Canlas and coworkers {Canlas et al . (1984) Am . J . Trop . Med . Hyg . 33, 420-424} isolated a monoclonal antibody (MF1) which, upon passive transfer, led to the clearance of Brugia malayi (Bm) microfilariae (mf) from infected jirds . The target of MF1 is a developmentally regulated mf chitinase (Cht) (Fuhrman et al . (1992) Proc . Natl . Acad . Sci . USA 89, 1548-1552) . This paper describes the production of enzymatically active Bm Cht in Escherichia coli . Standard expression conditions resulted in production of an insoluble maltose-binding protein (MBP)::Cht fusion protein, but by optimizing expression conditions, the amount of soluble MBP::Cht was increased 25-fold . The specific activity of the soluble MBP::Cht isolated from the E . coli cytoplasm was low . Exporting MBP::Cht into the E . coli periplasmic space increased the specific activity by 12-fold . This suggests that secretion through the membrane and/or the environment of the periplasmic space results in improved folding of recombinant Bm Cht.

Biochim Biophys Acta, 1996 Oct 24, 1291(2), 122 - 30
Dynamics and morphology of the in vitro polymeric form of elongation factor Tu from Escherichia coli; Helms MK et al.; Elongation factor Tu from Escherichia coli is known to polymerize at slightly acidic pH and low ionic strength . The structure and dynamics of these aggregates have been examined using imaging and spectroscopic methodologies . Electron microscopy provides evidence for two-dimensional sheets and bundled filaments of EF-Tu, whereas fluorescence microscopy of EF-Tu covalently labeled with tetramethylrhodamine isothiocyanate showed highly branched polymers of EF-Tu several microns in diameter . These polymers were studied using quasi-elastic light scattering to determine the evolution of the translational diffusion coefficient during the polymerization process . The rotational dynamics of the aggregate were investigated using phosphorescence anisotropy of EF-Tu covalently labeled with erythrosin isothiocyanate . A high infinite-time anisotropy was observed, suggesting a lack of motion or entanglement of EF-Tu polymers . A sub-microsecond motion which was slowed in the presence of glycerol may be due to local flexibility of the polymers . The possible relevance of polymeric EF-Tu to its function in vivo is discussed.

Nature, 1996 Oct 24, 383(6602), 735 - 8
A new class of uracil-DNA glycosylases related to human thymine-DNA glycosylase; Gallinari P et al.; Mispairs in DNA of guanine with uracil and thymine can arise as a result of deamination of cytosine and 5-methylcytosine, respectively . In humans such mispairs are removed by thymine-DNA glycosylase (TDG) . By deleting the carboxy and amino termini of this enzyme we have identified a core region capable of processing G/U but not G/T mispairs . We have further identified two bacterial proteins with strong sequence homology to this core and shown that the homologue from Escherichia coli (dsUDG) can remove uracil from G/U mispairs . This enzyme is likely to act as a back-up to the highly efficient and abundant enzyme uracil-DNA glycosylase (UDG) which is found in most organisms . Pupating insects have been reported to lack UDG activity, but we have identified an enzyme similar to dsUDG in cell lines from three different insect species . These data imply the existence of a family of double-strand-specific uracil-DNA glycosylases which, although they are subservient to UDG in most organisms, may constitute the first line of defence against the mutagenic effects of cytosine deamination in insects.

Biochem Biophys Res Commun, 1996 Oct 23, 227(3), 909 - 14
M-glycogenin, the protein moiety of Neurospora crassa proteoglycogen, is an auto- and transglucosylating enzyme; Goldraij A et al.; Neurospora crassa proteoglycogen was purified and its protein moiety, M-glycogenin, was released by amylolytic treatment . The released protein was capable of autoglucosylation from UDP-glucose forming glucosyl-alpha 1,4-glucosyl linkage . The kinetics of autoglucosylation suggested an intramolecular mechanism of reaction . M-glycogenin was also able to glucosylate dodecyl-beta-maltoside and autoglucosylate, simultaneously and independently . Both auto- and transglucosylation reactions were dependent on Mn2+ . Thus, M-glycogenin, which has also been described as the constituent of Escherichia coli proteoglycogen (A . Goldraij and J . A . Curtino . 1993, Biochem . Mol . Biol . Int . 30, 453-458), is a glucosyltransferase that bears similar catalytic properties with mammalian glycogenin . This is the first report on the enzymatic character of the protein constituent of proteoglycogen in primitive organisms, which suggest that the mechanism for the de novo biosynthesis of glycogen was conserved over a very long period of evolution.

Biochemistry, 1996 Oct 22, 35(42), 13627 - 35
Bis-methionine ligation to heme iron in mutants of cytochrome b562 . 2 . Characterization by NMR of heme-ligand interactions; Barker PD et al.; Previous work has shown that, in variants of cytochrome b562 containing the H102M mutation, methionine residues provide both axial ligands to the heme iron . NMR spectroscopic studies of such bis-methionine-coordinated cytochrome have not previously been feasible, since the only other cytochrome with such a ligand arrangement, bacterioferritin, is too large to be studied by current NMR methods . The present work provides the first NMR characterization of 6-coordinate, bis-methionine-ligated heme centers in both ferrous and ferric oxidation states . We have used one and two dimensional, homonuclear NMR spectroscopy to assign the proton resonances of the heme group and ligand side chains in the reduced, cytochrome b562 variants, H102M and covR98C/H102M . The latter protein has heme covalently attached to the protein, and our results prove that the covalent linkage is a c-type thioether bond formed between the cysteine at residue 98 and the heme 2-vinyl group . Spectra of the ferrous H102M variant are consistent with the presence of two species differing in the orientation of the heme in the protein . We have interpreted results from NOESY experiments on the ferrous covR98C/H102M protein in terms of the conformation of the two methionine side chains, and we present a model for the structure of the heme ligand arrangement . The Met7 side chain adopts an extended conformation almost identical to that observed in the wild type protein with R stereochemistry at the chiral sulfur ligand . The Met102 side chain has a different, buckled side chain conformation and has S stereochemistry at the chiral center . Our NMR derived model is consistent with the spectroscopic data presented in the previous paper . Studies on the ferric forms of these proteins confirm that the double variant at low pH has a "stable" bis-methionine ligation arrangement, but that it is a thermal mixture of species with differing spin states . No hyperfine coupled proton resonances can be identified in spectra of the high-spin forms of either of these proteins.

Biochemistry, 1996 Oct 22, 35(42), 13618 - 26
Bis-methionine ligation to heme iron in mutants of cytochrome b562 . 1 . Spectroscopic and electrochemical characterization of the electronic properties; Barker PD et al.; We have generated mutants of cytochrome b562 in which the histidine ligand to the heme iron (His102) has been replaced by a methionine . The resulting proteins can have bis-methionine coordination to the heme iron, but the stability of this arrangement is dependent on oxidation state and solution pH . We have used optical, MCD, and EPR spectroscopies to study the nature of the heme coordination environment under a variety of conditions . Optical spectra of the reduced state of the single variant, H102M, are consistent with bis-methionine ligation . In its oxidized state, this protein is high-spin under all conditions studied, and the spectroscopic properties are consistent with only one of the methionine ligands being coordinated . We cannot identify what, if anything, provides the other axial ligand . A double variant, R98C/H102M (in which the heme is covalently attached to the protein through a c-type thioether linkage), is also bis-methionine coordinated in the ferrous state, but has significantly different properties in the oxidized state . With a pKa of 7.1 at 20 degrees C, the protein converts from a low-spin, 6-coordinate heme protein at low pH, to a high-spin species, similar to the high-spin species observed for the single variant . Our spectroscopic data prove that the low-spin species is bis-methionine coordinated . The reduction potential of this bis-methionine species has been measured using direct electrochemical techniques and is +440 mV at pH 4.8 . The electrochemistry of these proteins is complicated by coupled coordination-state changes . Proof that the ferrous state is bis-methionine coordinated is provided by NMR results presented in the following paper.

Biochemistry, 1996 Oct 22, 35(42), 13579 - 85
Furilisin: a variant of subtilisin BPN' engineered for cleaving tribasic substrates; Ballinger MD et al.; The serine protease, subtilisin BPN', was engineered to cleave proteins after tribasic sequences in a manner that resembles the substrate specificity of furin, one of the mammalian subtilisin homologs that processes prohormones . As a starting point we used a double mutant of subtilisin BPN' (N62D/ G166D) that showed substantial preference for cleaving after sequences having consecutive dibasic residues (namely, at the P1 and P2 substrate positions) {Ballinger et al . (1995) Biochemistry 34, 13312-13319} . Additional specificity for basic residues was engineered at the P4 position by introducing subtilisin-to-furin substitutions at three hydrophobic residues that composed the S4 subsite (Y104, I107, and L126) . Initial attempts to incorporate a Y104D or I107E mutation or the Y104D/I107E double mutation into the dibasic specific enzyme failed to generate the processed enzyme . The problem was traced to the inability of the mutant prosubtilisins to process themselves and fold correctly . Replacing the natural processing site sequence (AHAY) with a good furin substrate sequence (RHKR) resulted in expression of the triple subtilisin mutant (N62D/Y104D/G166D) we call "furilisin" . Furilisin hydrolyzes synthetic tribasic substrates (succinyl-RAKR-pNA or succinyl-KAKR-pNA) with high catalytic efficiency (kcat/K(m) > 3 x 10(5) M-1 s-1) and discriminates in favor of Arg versus Ala at the P4 position by a factor of 360 . The overall specificity change versus the wild-type enzyme was dramatic . For example, succinyl-RAKR-pNA was cleaved approximately 60000 times faster than succinyl-AAPF-pNA, a good substrate for wild-type subtilisin . Similarly, furilisin was inhibited (K1* = 29 nM) by a variant of the turkey ovomucoid third domain inhibitor that contained an engineered furin substrate site (RCKR decreases) {Lu et al . (1993) J . Biol . Chem . 268, 14583-14585} and not by one having a good wild-type subtilisin substrate sequence (ACTL decreases) . Interestingly, the extreme changes in substrate specificity resulted from substantial synergy between the engineered subsites . These studies provide a basic example of how to manipulate substrate specificity in a modular fashion, thereby creating an engineered-enzyme that may be useful as a protein processing tool.

J Theor Biol, 1996 Oct 21, 182(4), 459 - 62
Circumstantial evidence for cytochrome b1 involvement in the functioning of lac-permease in respiring Escherichia coli; Yariv J; The structure of the haem-binding site of cytochrome b1 and particularly the fact that the two protein ligands of the haem are methionines could explain a correlation found between loss of lac-permease activity and replacement of methionine by norleucine in the protein of aerobically respiring E . coli . If cytochrome b1 is essential for lac-permease mediated transport in whole bacteria as this correlation suggests, translocation of substrate by this permease must be coupled to electron transport . Such a dependence would invalidate the chemiosmotic interpretation of lactose transport in E . coli in its present form and would be in variance with the coupling-by-energy theories of lactose transport that exempted translocation from dependence on energy yielding processes.

FEBS Lett, 1996 Oct 21, 395(2-3), 286 - 92
Classification of tyrosine kinases from Dictyostelium discoideum with two distinct, complete or incomplete catalytic domains; Adler K et al.; Two new kinases of Dictyostelium discoideum were identified by screening of a (lambda)gt11 expression library with a phosphotyrosine specific antibody . Amino-acid sequences derived from cDNA and genomic clones indicate that DPYK3 is a protein of 150 kDa and DPYK4, a protein of 75 kDa . The C-terminal fragments of each protein were produced in Escherichia coli and shown to be autocatalytically phosphorylated at tyrosine residues . A common feature of these kinases is the presence of two different sequence stretches in tandem that are related to kinase catalytic domains . The sequence relationships of DPYK3 and 4 to other protein kinases, and the positions of their catalytic domain sequences within the phylogenetic tree of protein kinases were analysed . Domains I of both kinases and domain II of DPYK3 constitute, together with the catalytic domains of two previously described tyrosine kinases of D . discoideum, a branch of their own, separate from the tyrosine kinase domains in sensu strictu . Domain II in DPYK4 is found on a different branch close to serine/threonine kinases.

FEBS Lett, 1996 Oct 21, 395(2-3), 283 - 5
Modified nucleotides as substrates and inhibitors of adenylate kinase from different sources; Skoblov JS et al.; The substrate and inhibitory properties of modified nucleotides with respect to adenylate kinase from rabbit muscles, human placenta and Escherichia coli were studied . A number of 5'-hydrogenphosphonates and 5'-fluorophosphates of modified nucleotides were shown to inhibit the phosphorylation reaction catalyzed by these enzymes . A clear difference between phosphonates of 3'-deoxyribonucleotides and the corresponding ribo- and 2',3'-dideoxyribonucleotides was found . 3'-Azido-2',3'-dideoxythymidine and its phosphorus derivatives did not inhibit the adenylate kinase reaction.

FEBS Lett, 1996 Oct 21, 395(2-3), 235 - 40
Identification of residues in the putative 5th helical region of human interleukin-6, important for activation of the IL-6 signal transducer, gp130; Brakenhoff JP et al.; We have previously shown that L58 in the putative 5th helical region of human interleukin-6 (IL-6) is important for activation of the IL-6 signal transducer gp130 {de Hon et al . (1995) FEBS Lett . 369, 187-191} . To further explore the importance of individual residues in this region for gp130 activation we have now combined Ala substitutions of residues E52, S53, S54, K55, E56, L58 and E60 with other substitutions in IL-6, known to affect gp130 activation (Q160E and T163P) . The combination mutant protein with L58A completely lost the capacity to induce the proliferation of XG-1 myeloma cells, and could effectively antagonize wild type IL-6 activity on these cells . Moreover, the data suggest that besides L58, S54 particularly, but also E52, S53, K55 and E56 contribute to gp130 activation.

FEBS Lett, 1996 Oct 21, 395(2-3), 225 - 7
Ser644 is important for catalytic activity but is not involved in cAMP-dependent phosphorylation of yeast 6-phosphofructo-2-kinase; Kessler R et al.; To identify the target amino acid for the cAMP-dependent phosphorylation of yeast 6-phosphofructo-2-kinase Ser644 was mutated to Ala . The plasmid-encoded wild-type and mutant enzymes were overexpressed in E . coli TG2 cells and in the yeast strain DFY658 . Like the wild-type enzyme, the Ser644-->Ala mutant was phosphorylated in vivo after addition of glucose to yeast cells and in vitro by the catalytic subunit of protein kinase A . The specific activity of the mutant enzyme was 6-fold lower than that of the wild-type yeast 6-phosphofructo-2-kinase, but both enzymes were activated in response to the addition of glucose to yeast cells.

FEBS Lett, 1996 Oct 21, 395(2-3), 160 - 4
Structure of a methionine-rich segment of Escherichia coli Ffh protein; Oh DB et al.; The methionine-rich segments of the Ffh protein of Escherichia coli and its eukaryotic counterpart SRP54 are thought to bind signal sequences of secretory proteins . The structure of a chemically synthesized 25-residue-long peptide corresponding to one of the proposed methionine-rich amphiphilic helices of Ffh was determined in water and in aqueous trifluroethanol (TFE) solution using CD and NMR . An appreciable alpha-helix conformation exists even in water and this peptide assumes a stable alpha-helix along most of its length in aqueous TFE solution . It is clear that this segment of Ffh protein has a very strong propensity to form alpha-helical structure.

FEBS Lett, 1996 Oct 21, 395(2-3), 95 - 8
Expression of the gene for tear lipocalin/von Ebner's gland protein in human prostate; Holzfeind P et al.; Northern analysis of human multiple tissue blots containing poly A+ RNA from spleen, thymus, prostate, testis, ovary, small intestine, colon and peripheral blood leukocytes revealed that a prostate specific transcript hybridizes to a tear lipocalin/von Ebner's gland protein (TL/VEGP) gene probe . To characterize this transcript, the corresponding cDNA was amplified by reverse transcription (RT)-PCR . Cloning and sequence analysis showed that it was identical to the tear lipocalin cDNA isolated from human lachrymal glands . Immunohistochemical analysis on thin layer sections of human prostate using a tear lipocalin specific antiserum confirmed the expression of this cDNA in prostate . Thus, our results clearly argue against a unique function of TL/VEGP in human tear fluid or saliva . The human cDNA was expressed in E . coli using the pQE system yielding a recombinant protein which shows biochemical properties identical to the native TL/VEGP.

Hum Gene Ther, 1996 Oct 20, 7(16), 2015 - 24
Prolonged in vivo gene expression driven by a tyrosine hydroxylase promoter in a defective herpes simplex virus amplicon vector; Jin BK et al.; A 9.0-kb fragment of the tyrosine hydroxylase (TH) promoter, previously shown to direct tissue-specific expression in transgenic mice, was fused to an Escherichia coli LacZ reporter gene in a defective herpes simplex virus type-1 (HSV-1) amplicon vector (THlac) . The HSV immediate early (IE) 4/5 promoter (HSVlac) was used as a control . LacZ gene expression was visualized by X-Gal histochemical and TH immunocytochemical analysis . Two days and 10 weeks after THlac injection into rat caudate nucleus (CN), X-Gal-stained cells were observed in the substantia nigra (SN) and locus ceruleus (LC) ipsilateral to the injection site . These blue cells were TH-positive neurons as evidenced by double labeling with immunocytochemistry . Moreover, the number of X-Gal+, TH+ (double-positive) neurons in the SN increased at 10 weeks as compared to that seen 2 days after THlac injection . In marked contrast, few double-positive nigral neurons were observed either 2 days or 10 weeks after direct injection of THlac into SN . However, neither nigral nor striatal injection of HSVlac resulted in prolonged gene expression . These results suggest that a neuronal, but not a viral, promoter in an HSV vector can produce cell-type-specific, prolonged, and stable gene expression following retrograde transport . In addition, THlac produced infrequent gene expression in TH-negative cells (CN and dorsal to SN) after THlac injection into CN and SN, respectively . Overall, these results suggest that in some in vivo contexts cell-type-preferred expression can be achieved by a cellular promoter in an amplicon vector . Moreover, they underscore the need for the careful and systematic study of neuronal promoters in HSV vectors.

Hum Gene Ther, 1996 Oct 20, 7(16), 2003 - 13
A novel 'piggyback' packaging system for herpes simplex virus amplicon vectors; Pechan PA et al.; Recombinant and amplicon vectors derived from herpes simplex virus type 1 (HSV-1) have proven to be an efficient means of gene delivery to cells in culture and in vivo . In this study, a system was developed to make propagation of the amplicon vector and helper virus mutually dependent on each other, in a "piggyback' fashion . This combined system supports maintenance and enrichment of the amplicon vector when propagating stocks, while allowing the helper virus to serve as a recombinant vector in its own right . Amplicons bearing a gene essential for HSV-1 replication, IE3, as well as the Escherichia coli lacZ marker gene, were propagated using a mutant virus (d120) deleted in the same essential gene . Vector stocks could be propagated in Vero cells and other cultured cells not transfected with the IE3 gene with markedly delayed cytopathic effects, as compared to wild-type virus . Relatively high titers of amplicon vectors (6 x 10(7) infectious units/ml) were achieved with this piggyback system in Vero cells, with an apparent ratio of amplicon vector: helper virus of up of 5:1 under some conditions; however, recombinant wild-type virus was also generated . Injection of these stocks into experimental gliomas in rodent brain revealed gene delivery to tumor cells mediated by both amplicon vectors (lacZ) and helper virus (HSV-thymidine kinase), with no apparent neuropathology of normal brain . This basic piggyback vector model is amenable to modifications to promote conditional propagation of vectors in vivo and to allow incorporation of multiple transgene elements into both the amplicon and recombinant helper virus vectors.

Cell, 1996 Oct 18, 87(2), 241 - 51
The chaperonin ATPase cycle: mechanism of allosteric switching and movements of substrate-binding domains in GroEL; Roseman AM et al.; Chaperonin-assisted protein folding proceeds through cycles of ATP binding and hydrolysis by the large chaperonin GroEL, which undergoes major allosteric rearrangements . Interaction between the two back-to-back seven-membered rings of GroEL plays an important role in regulating binding and release of folding substrates and of the small chaperonin GroES . Using cryo-electron microscopy, we have obtained three-dimensional reconstructions to 30 A resolution for GroEL and GroEL-GroES complexes in the presence of ADP, ATP, and the nonhydrolyzable ATP analog, AMP-PNP . Nucleotide binding to the equatorial domains of GroEL causes large rotations of the apical domains, containing the GroES and substrate protein-binding sites . We propose a mechanism for allosteric switching and describe conformational changes that may be involved in critical steps of folding for substrates encapsulated by GroES.

Science, 1996 Oct 18, 274(5286), 425 - 6
Attractant signaling by an aspartate chemoreceptor dimer with a single cytoplasmic domain; Gardina PJ et al.; Signal transduction across cell membranes often involves interactions among identical receptor subunits, but the contribution of individual subunits is not well understood . The chemoreceptors of enteric bacteria mediate attractant responses by interrupting a phosphotransfer circuit initiated at receptor complexes with the protein kinase CheA . The aspartate receptor (Tar) is a homodimer, and oligomerized cytoplasmic domains stimulate CheA activity much more than monomers do in vitro . Intragenic complementation was used to show in Escherichia coli that heterodimers containing one full-length and one truncated Tar subunit mediated responses to aspartate in the presence of full-length Tar homodimers that could not bind aspartate . Thus, a Tar dimer containing only one cytoplasmic domain can initiate an attractant (inhibitory) signal, although it may not be able to stimulate kinase activity of CheA.

Science, 1996 Oct 18, 274(5286), 423 - 5
Signaling by the Escherichia coli aspartate chemoreceptor Tar with a single cytoplasmic domain per dimer; Tatsuno I et al.; Many transmembrane receptors are oligomeric proteins . Binding of a ligand may alter the oligomeric state of the receptor, induce structural changes within the oligomer, or both . The bacterial aspartate chemoreceptor Tar forms a homodimer in the presence or absence of ligands . Tar mediates attractant and repellent responses by modulating the activity of the cytoplasmic kinase CheA . In vivo intersubunit suppression was used to show that certain combinations of full-length and truncated mutant Tar proteins complemented each other to restore attractant responses to aspartate . These results suggest that heterodimers with only one intact cytoplasmic domain are functional . The signaling mechanism may require interactions between dimers or conformational changes within a single cytoplasmic domain.

Science, 1996 Oct 18, 274(5286), 415 - 21
Crystal structure of DNA recombination protein RuvA and a model for its binding to the Holliday junction; Rafferty JB et al.; The Escherichia coli DNA binding protein RuvA acts in concert with the helicase RuvB to drive branch migration of Holliday intermediates during recombination and DNA repair . The atomic structure of RuvA was determined at a resolution of 1.9 angstroms . Four monomers of RuvA are related by fourfold symmetry in a manner reminiscent of a four-petaled flower . The four DNA duplex arms of a Holliday junction can be modeled in a square planar configuration and docked into grooves on the concave surface of the protein around a central pin that may facilitate strand separation during the migration reaction . The model presented reveals how a RuvAB-junction complex may also accommodate the resolvase RuvC.

J Biol Chem, 1996 Oct 18, 271(42), 26430 - 4
Primary structure and tissue-specific expression of human beta-hydroxyisobutyryl-coenzyme A hydrolase; Hawes JW et al.; beta-Hydroxyisobutyryl-CoA (HIBYL-CoA) hydrolase is responsible for the specific hydrolysis of HIBYL-CoA, a saline catabolite, as well as the hydrolysis of beta-hydroxypropionyl-CoA, an intermediate in a minor pathway of propionate metabolism . We have obtained the amino acid sequences of several tryptic peptides derived from purified rat liver HIBYL-CoA hydrolase, and the NH2-terminal peptize sequence was matched to the translated sequence of a human expressed sequence tag present in the data base of the IMAGE Consortium (Lawrence Livermore National Laboratory, Livermore, CA) . The complete nucleotide sequence and the deduced amino acid sequence showed no similarity to the sequences of well known thioesterases but showed significant homology to the enoyl-CoA hydratase/isomerase enzyme family . The cDNA fragment corresponding to the mature (processed) protein was expressed in Escherichia coli . The purified recombinant enzyme displayed substrate specificity very similar to that of the rat enzyme and was specifically bound by polyclonal antibodies raised against purified rat liver HIBYL-CoA hydrolase . Northern and Western blot analyses with various human tissues indicated predominant expression in liver, heart, and kidney, with discrepancies occurring in the amounts of HIBYL-CoA hydrolase mRNA compared to stably expressed protein in several tissues.

J Biol Chem, 1996 Oct 18, 271(42), 26424 - 9
DNA strand cleavage is required for replication fork arrest by a frozen topoisomerase-quinolone-DNA ternary complex; Hiasa H et al.; The formation of a topoisomerase-quinolone-DNA ternary complex leads to cell death . We show here that an active strand breakage and reunion activity is required for formation of a norfloxacin-topoisomerase IV-DNA ternary complex that can arrest the progression of replication forks in vitro . Mutant topoisomerases containing either an active site mutation, a quinolone resistance-conferring mutation, or both, could all bind DNA as well as the wild-type, but unlike the wild-type, could not halt replication fork progression . The collision between the replication fork and the frozen topoisomerase converted the cleavable complex to a nonreversible form but did not generate a double-stranded break . Thus, the cytotoxicity of this class of topoisomerase inhibitors likely results from a two-step process: (i) conversion of the frozen topoisomerase-quinolone-DNA ternary complex to an unreversible form; and (ii) generation of a double-strand break by subsequent denaturation of the topoisomerase, perhaps by an aborted repair attempt.

J Biol Chem, 1996 Oct 18, 271(42), 26410 - 7
A model for the quaternary structure of the proteasome activator PA28; Song X et al.; PA28 is a protein activator of the 20S proteasome . It has a native molecular weight of approximately 200,000 and is composed of six 28,000-dalton subunits arranged in a ring-shaped complex . Purified preparations of PA28 contain two polypeptides, alpha and beta, which are about 50% identical in primary structure . It has been unclear whether native PA28 consists of two distinct homohexameric proteins or of a single protein containing both alpha and beta subunits . To distinguish between these possibilities, we prepared antibodies that reacted specifically with either the alpha or beta subunit and used these subunit-specific antibodies in two types of experiments designed to elucidate PA28 quaternary structure . In the first experiment, the alpha and beta subunits were completely co-immunoprecipitated by each subunit-specific antibody, indicating that both subunits were part of a single protein complex . In the second experiment, PA28 was chemically cross-linked using bis(sulfosuccinimidyl)suberate . When the cross-linked products were immunoblotted after SDS-polyacrylamide gel electrophoresis, indistinguishable patterns were obtained with each subunit-specific antibody . These results confirm that the alpha and beta subunits were part of the same protein complex . The pattern of cross-linked products also provided insight as to the relative abundance and arrangement of the subunits within the PA28 complex and indicated that the ring-shaped PA28 hexamer may be composed of alternating alpha and beta subunits with a stoichiometry of (alphabeta)3 . PA28 was inactivated by treatment with carboxypeptidase Y, which cleaved Tyr and Ile residues from the carboxyl terminus of the alpha subunit but had very little effect on the beta subunit . This selective and limited proteolysis prevented binding of both alpha and beta subunits to the proteasome and therefore provides additional evidence of the heterodimeric nature of PA28 . These results indicate that a short carboxyl-terminal sequence of the alpha subunit is critical for binding of native PA28 to the proteasome . To learn about the relative functions of the alpha and beta subunits, PA28alpha was expressed in Escherichia coli and purified to homogeneity . Purified PA28alpha stimulated proteasome activity but required 5-10-fold greater concentrations than the heterodimeric PA28 to achieve a given level of activity . These results suggest that the heterodimeric structure of PA28 is required for maximal proteasome activation.

J Biol Chem, 1996 Oct 18, 271(42), 26362 - 8
The Caenorhabditis elegans p21-activated kinase (CePAK) colocalizes with CeRac1 and CDC42Ce at hypodermal cell boundaries during embryo elongation; Chen W et al.; The p21-activated kinase (PAK) is a downstream target of Rac and CDC42, members of the Ras-related Rho subfamily, that mediates signaling pathway leading to cytoskeletal reorganization . To investigate its function in Caenorhabditis elegans development, we have isolated the cDNA coding for the p21-activated kinase homologue (CePAK) from a C . elegans embryonic cDNA library . This 2.35-kilobase pair cDNA encodes a polypeptide of 572 amino acid residues, with the highly conserved N-terminal p21-binding and the C-terminal kinase domains . Similar to its mammalian and Drosophila counterparts, the CePAK protein expressed in E . coli exhibits binding activity toward GTP-bound CeRac1 and CDC42Ce . Polyclonal antibodies raised against the recombinant CePAK recognize a specific 70-kDa protein from embryonic extracts that displays CeRac1/CDC42Ce-binding and kinase activities . Immunofluorescence analysis indicates that CePAK is specifically expressed at the hypodermal cell boundaries during embryonic body elongation, which involves dramatic cytoskeletal reorganization . Interestingly, CeRac1 and CDC42Ce are found at the same location, which might point to their common involvement in hypodermal cell fusion, a crucial morphogenetic event for nematode development.

J Biol Chem, 1996 Oct 18, 271(42), 26349 - 55
The NH2-terminal extension of protein phosphatase PPZ1 has an essential functional role; Clotet J et al.; Deletion of the yeast Ser/Thr protein phosphatase PPZ1 results in increased tolerance to sodium and lithium . PPZ1 is also important for cell integrity, as ppz1Delta cells undergo lysis under caffeine stress and PPZ1 overexpression overrides the lytic defect of mutants in the protein kinase C/mitogen-activated protein (MAP) kinase pathway . The PPZ1 protein can be dissected in two halves . The COOH-terminal half is related to type 1 phosphatases, whereas the NH2-terminal half is unrelated to phosphatases and contains a consensus site for N-myristoylation . Several mutated versions of PPZ1 have been constructed and tested for complementation of ppz1Delta mutants . We show that PPZ1 can be myristoylated in vivo and that change of Gly-2 to Ala results in lack of myristoylation and loss of complementation of salt tolerance . Removal of the entire NH2-terminal half results in complete loss of function, although it does not abolish the phosphatase activity of the protein expressed in Escherichia coli . The deletion of a large region of the NH2-terminal half (residues 17-193) does not affect the ability to complement the salt tolerance phenotype but abolish complementation of caffeine sensitivity, whereas the opposite behavior is observed upon removal of residues from 241 to 318 . Mutation of Arg-451 to Leu results in both complete loss of function and of phosphatase activity . These results indicates that the NH2-terminal half of the protein contains structural determinants that are specific for certain functions and that the phosphatase activity is required but not sufficient for full PPZ1 function.

J Biol Chem, 1996 Oct 18, 271(42), 26251 - 60
Biochemical and biophysical properties of the core-binding factor alpha2 (AML1) DNA-binding domain; Crute BE et al.; The Runt domain is the DNA-binding domain defining a small family of transcription factors that are involved in important developmental processes . Developmental pathways controlled by Runt domain proteins include sex determination, neurogenesis, segmentation, and eye development in Drosophila and hematopoiesis in mammals . In addition to binding DNA, the Runt domain also mediates heterodimerization with another subunit called the core-binding factor beta (CBFbeta) subunit . In this study we overexpress the Runt domain from the mouse CBFalpha2 (AML1) protein in Escherichia coli, and purify it from the insoluble fraction . We determine the equilibrium constants for Runt domain binding to two different DNA sequences by surface plasmon resonance technology . Circular dichroism spectroscopy demonstrates that the Runt domain is a folded beta-domain with essentially no alpha-helical content . The single tryptophan residue in the CBFalpha2 Runt domain at amino acid 79 is shown by tryptophan fluorescence spectroscopy to reside in a polar environment . Finally, we demonstrate that ATP can be UV cross-linked to the Runt domain and that ATP binding is sensitive to an amino acid substitution in the putative Kinase-1a motif (P-loop).

J Biol Chem, 1996 Oct 18, 271(42), 26105 - 9
Analysis of substrate specificity of the RuvC holliday junction resolvase with synthetic Holliday junctions; Shida T et al.; The Escherichia coli RuvC protein endonucleolytically resolves Holliday junctions, which are formed as intermediates during genetic recombination and recombination repair . Previous studies using model Holliday junctions suggested that a certain size of central core of homology and a specific sequence in the junction were required for efficient cleavage by RuvC, although not for binding . To determine the minimum length of sequence homology required for RuvC cleavage, we made a series of synthetic Holliday junctions with various lengths of homologous sequence in the core region . It was demonstrated that a monomobile junction possessing only 2 base pairs of the homology core was efficiently cleaved by RuvC . To study the sequence specificity for cleavage, we made 16 bimobile junctions, which differed only in the homologous core sequence . Among them, 6 junctions were efficiently cleaved . Cleavage occurred by introduction of nicks symmetrically at the 3'-side of thymine in all cases . However, the nucleotide bases at the 3'-side of the thymines were not always identical between the two strands nicked . These results suggest that RuvC recognizes mainly topological symmetry of the Holliday junction but not the sequence symmetry per se, that the thymine residue at the cleavage site plays an important role for RuvC-mediated resolution, and that a long homologous core sequence is not essential for cleavage.

J Biol Chem, 1996 Oct 18, 271(42), 25912 - 5
Membrane topology of Kch, a putative K+ channel from Escherichia coli; Johansson M et al.; We have mapped the topology of the C-terminal half of the putative potassium channel protein Kch in the inner membrane of Escherichia coli using PhoA fusions . Our results are consistent with the widely assumed six-transmembrane helix model for eukaryotic voltage-gated ion channels and place both ends of the proposed channel-lining P-segment on the periplasmic side of the inner membrane . The rather hydrophobic P-segment is found to translocate only slowly across the inner membrane and seems to be near a threshold for stop-transfer function.

J Biol Chem, 1996 Oct 18, 271(42), 25898 - 905
Regulation of UDP-3-O-{R-3-hydroxymyristoyl}-N-acetylglucosamine deacetylase in Escherichia coli . The second enzymatic step of lipid a biosynthesis; Sorensen PG et al.; The first enzyme of lipid A assembly in Escherichia coli is an acyltransferase that attaches an R-3-hydroxymyristoyl moiety to UDP-GlcNAc at the GlcNAc 3-OH . This reaction is reversible and thermodynamically unfavorable . The subsequent deacetylation of the product, UDP-3-O-{R-3-hydroxymyristoyl}-GlcNAc, is therefore the first committed step of lipid A biosynthesis . We now demonstrate that inhibition of either the acyltransferase or the deacetylase in living cells results in a 5-10-fold increase in the specific activity of the deacetylase in extracts prepared from such cells . Five other enzymes of the lipid A pathway are not affected . The elevated specific activity of deacetylase observed in extracts of lipid A-depleted cells is not accompanied by a significant change in the Km for the substrate, but is mainly an effect on Vmax . Western blots demonstrate that more deacetylase protein is indeed made . However, deacetylase messenger RNA levels are not significantly altered . Inhibition of lipid A biosynthesis must either stimulate the translation of available mRNA or slow the turnover of pre-existing deacetylase . In contrast, inhibition of 3-deoxy-D-manno-octulosonic acid (Kdo) biosynthesis has no effect on deacetylase specific activity . The underacylated lipid A-like disaccharide precursors that accumulate during inhibition of Kdo formation may be sufficient to exert normal feedback control.

J Biol Chem, 1996 Oct 18, 271(42), 25864 - 72
Secobarbital-mediated inactivation of cytochrome P450 2B1 and its active site mutants . Partitioning between heme and protein alkylation and epoxidation; He K et al.; Secobarbital (SB) is a relatively selective mechanism-based inactivator of cytochrome P450 2B1, that partitions between epoxidation and heme and protein modification during its enzyme inactivation . The SB-2B1 heme adduct formed in situ in a functionally reconstituted system has been spectrally documented and structurally characterized as N-(5-(2-hydroxypropyl)-5-(1-methylbutyl)barbituric acid)protoporphyrin IX . The SB-protein modification has been localized to 2B1 peptide 277-323 corresponding to the active site helix I of cytochrome P450 101 . The targeting of heme and this active site peptide suggests that the 2B1 active site topology could influence the course of its inactivation . To explore this possibility, the individual SB epoxidation, heme and protein modification, and corresponding molar partition ratios of the wild type and seven structural 2B1 mutants, site-directed at specific substrate recognition sites, and known to influence 2B1 catalysis were examined after Escherichia coli expression . These studies reveal that Thr-302 is critical for SB-mediated heme N-alkylation, whereas Val-367 is a critical determinant of 2B1 protein modification, and Val-363 is important for SB epoxidation . SB docking into a refined 2B1 homology model coupled with molecular dynamics analyses provide a logical rationale for these findings.

J Biol Chem, 1996 Oct 18, 271(42), 25850 - 8
Studies with flavin analogs provide evidence that a protonated reduced FMN is the substrate-induced transient intermediate in the reaction of Escherichia coli chorismate synthase; Macheroux P et al.; Chorismate synthase catalyzes the 1,4-elimination of phosphate and the C-(6-pro-R) hydrogen from 5-enolpyruvylshikimate 3-phosphate (EPSP) to generate chorismate . Although this reaction does not involve an overall change in redox state, the enzyme requires reduced FMN . To investigate the role of the flavin in catalysis we have employed chemically modified flavins: 1- and 5-deaza-, 2- and 4-thio-, 6-hydroxy-, 8-nor-6-methyl-, 8-methyl-sulfonyl-, 8-chloro-, 8-fluoro-, 8-nor-methyl-, 8-S-methyl-, 8-methoxy, 8-mercapto- and 8-amino-FMN . Photoreduction of 4-thio-FMN in the presence of chorismate synthase at pH 7.5 produced a reduced flavin species with an absorbance maximum at lambda = 410 nm indicative of monoanionic, reduced 4-thio-FMN . Binding of 8-mercapto- and 6-hydroxy-FMN to chorismate synthase in the presence of EPSP or (6R)-6-fluoro-EPSP resulted in an increase of the flavin analogs' pKa values by 4 and 1 pH units, respectively . On the basis of these findings it is concluded that chorismate synthase preferentially binds neutral flavin species, including the protonated reduced form, rather than anionic flavin species in the presence of EPSP or the 6-fluoro-substrate analog . Further support for this conclusion was obtained using 5-deaza- and 4-thio-FMN . Addition of EPSP to enzyme-bound, reduced 5-deaza-FMN produced spectral changes consistent with protonation of the flavin . Photoreduction of 4-thio-FMN in the presence of enzyme and the (6R)-6-fluoro-EPSP generated a reduced flavin species with absorbance properties of a neutral, reduced 4-thio-flavin . These results and their implications for the nature and kinetic properties of an observed flavin intermediate are discussed in the context of a possible role of reduced flavin as an electron donor to bound EPSP.

Gene, 1996 Oct 17, 176(1-2), 249 - 55
A multifunctional vector system for heterologous expression of proteins in Escherichia coli . Expression of native and hexahistidyl fusion proteins, rapid purification of the fusion proteins, and removal of fusion peptide by Kex2 protease; Ghosh S et al.; Vectors have been constructed for the general purpose of expressing foreign proteins in E . coli . These vectors allow the production in high yield of either native proteins or of fusion proteins which contain, at their amino terminus, the peptide Met Gly His6 Ser Gly Leu Phe Lys Arg/, where Leu Phe Lys Arg/ is the recognition site for Kex2 protease which cleaves at the site indicated by / . The His6 sequence is used as a ligand for the one-step affinity purification of the expressed proteins on columns containing Ni or Zn ions chelated to iminodiacetic acid-agarose . After affinity chromatography, the purification peptide is cleaved off with Kex2 protease from Saccharomyces cerevisiae . The vectors also allow site-directed mutagenesis and sequencing of the cloned gene to be expressed without any intermediate subcloning . For practical examples of over-expression, affinity purification, and removal of the purification peptide, we chose a high-molecular-weight protein, phospholipase C gamma 1 (PLC gamma 1, M(r) 148,000) and a low-molecular-weight protein, Hit-1 (M(r) 16,000) . Both were obtained pure and in high yield . PLC gamma 1 was fully active; the function of Hit-1 is not known . A set of companion vectors for co-expression of additional proteins has also been developed . These allow expression of proteins which enhance the production or activity of the protein of primary interest and of proteins which exhibit trans-interactions.

Gene, 1996 Oct 17, 176(1-2), 171 - 6
A Mycobacterium tuberculosis gene cluster encoding proteins of a phosphate transporter homologous to the Escherichia coli Pst system; Braibant M et al.; We report the cloning and sequencing of three M . tuberculosis genes encoding proteins homologous to E . coli PstA, PstC and PstB . They are tentatively called pstA-2, pstC-1 and pstB . They encode proteins of 302, 336 and 275 amino acids, respectively . In E . coli, PstB is the ATP binding component and PstA/PstC are the two hydrophobic subunits of a phosphate permease belonging to the family of ABC (ATP-binding cassette) transporters . In mycobacteria, PstS-1, the phosphate binding subunit (Andersen and Hansen, 1989), is encoded by a gene directly surrounded by pstB, pstC-1 and pstA-2 within a potential operon (pstB, pstS-1, pstC-1, pstA-2) . Phosphate uptake by whole, suspension grown, M . bovis BCG cells was measured and could be inhibited by a monoclonal antibody directed against the PstS-1 subunit, suggesting that these genes encode subunits of a functional mycobacterial phosphate permease.

Gene, 1996 Oct 17, 176(1-2), 163 - 9
Homologs of Escherichia coli recJ, gltX and of a putative 'early' gene of avian Chlamydia psittaci are located upstream of the 'late' omp2 locus of Chlamydia psittaci strain guinea pig inclusion conjunctivitis; Hsia RC et al.; The nucleotide sequence of nearly 6 kb of genomic DNA located immediately upstream of the omp3-omp2 operon of Chlamydia psittaci strain GPIC was obtained, revealing four significant open reading frames (ORFs), named ORF1, ORF2, ORF4 and ORF5 . Searches for homologous sequences in the GenBank/EMBL databases have revealed that: (a) the open-ended ORF1 putatively encodes an homolog of RecJ of Escherichia coli, thought to be required for RecBCD-independent and conjugational recombination, and for UV repair; (b) the predicted translation product of ORF4 is highly homologous to the putative product of EUO, a previously described ORF of avian C . psittaci strain 6BC which is preferentially transcribed early during the life cycle; and (c) ORF5 putatively encodes an homolog of bacterial glutamyl-tRNA synthetases . This analysis establishes the genetic linkage of late (omp3-omp2) and of a proposed early (EUO) genes in Chlamydia.

Biochim Biophys Acta, 1996 Oct 17, 1297(2), 228 - 34
Regulation of the Escherichia coli biosynthetic ornithine decarboxylase activity by phosphorylation and nucleotides; Anagnostopoulos CG et al.; A protein kinase which phosphorylates in vitro the biosynthetic ornithine decarboxylase (ODC) was partially purified from Escherichia coli . In vivo phosphorylation of ODC occurs after incubation of E . coli with {32P}orthophosphate . When the recombinant ODC was incubated with calf intestine alkaline phosphatase it was inactivated and this inactive ODC could be reversibly activated allosterically only by guanyl or uracyl phosphate analogues at a concentration of 10(-4) or 10(-3) M . The pH optimum of the {8-3H}GTP binding was determined and it was shown that the GTP binding is proportional to the amount of ODC . The {8-3H}GTP binds specifically to ODC as was proved by the addition of cold GTP or ATP . High concentration of GTP can dissociate the ODC-antizyme complex and either reactivate or liberate the ODC . Therefore, a working hypothesis is suggested describing the regulation of ODC by phosphorylation-dephosphorylation reaction or by antizyme and nucleotide analogues interaction.

Biochim Biophys Acta, 1996 Oct 17, 1297(2), 127 - 30
Primary structure of the plant serpin BSZ7 having the capacity of chymotrypsin inhibition; Rasmussen SK et al.; The primary structure of barley grain serpin BSZ7 was deduced from a cDNA encoding 397 amino-acid residues . More than 70% of the residues were confirmed by sequencing peptide fragments . The N-terminus was identified as an acetylated Ala by using mass spectrometry coupled with amino-acid analysis . None of the four putative N-glycosylation sites were found to be glycosylated . The positional identity of BSZ7 with plant and mammalian serpins is 69-72% and 25-32%, respectively.

Nature, 1996 Oct 17, 383(6601), 630 - 3
Nuclear import of the homeodomain protein extradenticle in response to Wg and Dpp signalling; Mann RS et al.; In Drosophila, Decapentaplegic (Dpp) and Wingless (Wg) are two secreted signalling proteins of the transforming growth factor (TGF)-beta and Wnt families, respectively . Although both are often required during development, only a few downstream components of these signalling pathways have been described . Here we present evidence that in the embryonic midgut both signalling pathways control the subcellular localization of the homeodomain protein encoded by the extradenticle (exd) gene . Exd protein is predominantly nuclear in endoderm cells close to Dpp-and Wg-secreting cells of the visceral mesoderm, but is in the cytoplasm in more distant endoderm cells . Both dpp and wg are required for the nuclear localization of Exd in the endoderm, whereas ectopic expression of dpp and wg expands the domain of nuclear Exd . Furthermore, the nuclear import of Exd correlates with the transcription of an exd-dependent reporter gene in the endoderm . Thus one mechanism by which extracellular signals might control pattern is by directing the graded nuclear localization of homeodomain proteins such as Exd that directly control the expression of target genes.

Nature, 1996 Oct 17, 383(6601), 598 - 603
Structure of a replication-terminator protein complexed with DNA; Kamada K et al.; The crystal structure of the Escherichia coli replication-terminator protein (Tus) bound to terminus-site (Ter) DNA has been determined at 2.7 A resolution . The Tus protein folds into a previously undescribed architecture divided into two domains by a central basic cleft . This cleft accommodates locally deformed B-form Ter DNA and makes extensive contacts with the major groove, mainly through two interdomain beta-strands . The unusual structural features of this complex may explain how the replication fork is halted in only one direction.

Mol Gen Genet, 1996 Oct 16, 252(5), 580 - 6
Allele specificity of the Escherichia coli dnaA gene function in the replication of plasmids derived from phage lambda; Wegrzyn G et al.; We demonstrate a variation in the effects of seven alleles of the Escherichia coli dnaA gene, which cause temperature sensitivity of initiation of chromosomal replication, on the replication of lambda phage-derived plasmids at 30 degrees C . These mutants showed no allele specificity of dnaA function in replication of either of two lambda pi plasmids studied . On the other hand, the inability of the lambda P+ plasmid to replicate in dnaA508, 46 and 204 cells, in dnaB (groP A15) or in cells that are temperature sensitive for the chaperone genes dnaK756, dnaJ259 and grpE280 at 30 degrees C was suppressible by a single pi mutatation . This suggests that it is a common property of the pi protein, probably its weaker interaction with DnaB helicase, that is responsible for the suppression . One can also conclude that the DnaA-regulated transcriptional activation of ori lambda acts at the step, in which all these gene products cooperate, i.e . during preprimosome loading and chaperone-mediated release of DnaB from P protein inhibition.

J Immunol Methods, 1996 Oct 16, 197(1-2), 39 - 49
Engineering of a recombinant colorimetric fusion protein for immunodiagnosis of insulin; Chanussot C et al.; A synthetic DNA encoding human proinsulin was inserted in frame in the bacterial alkaline phosphatase gene . A homogeneous recombinant human proinsulin-alkaline phosphatase conjugate was obtained directly from the periplasm of Escherichia coli transformed with a plasmid carrying the hybrid gene . The recombinant conjugate was stable and could be produced in the bacteria . The immunological properties of the recombinant conjugate and those of the human insulin and human proinsulin were compared using a panel of six different human insulin-specific monoclonal antibodies . Three immunological groups were thus distinguished and one of them indiscriminately recognized all of the insulin-like molecules . One monoclonal antibody from this group was used in combination with the recombinant conjugate to develop successfully a competitive immunoenzymatic assay for detecting insulin.

Structure, 1996 Oct 15, 4(10), 1141 - 51
Helix unwinding in the effector region of elongation factor EF-Tu-GDP; Polekhina G et al.; BACKGROUND: Elongation factor Tu (EF-Tu) in its GTP conformation is a carrier of aminoacylated tRNAs (aa-tRNAs) to the ribosomal A site during protein biosynthesis . The ribosome triggers GTP hydrolysis, resulting in the dissociation of EF-Tu-GDP from the ribosome . The affinity of EF-Tu for other molecules involved in this process, some of which are unknown, is regulated by two regions (Switch I and Switch II) that have different conformations in the GTP and GDP forms . The structure of the GDP form of EF-Tu is known only as a trypsin-modified fragment, which lacks the Switch I, or effector, domain . The aim of this work was to establish the overall structure of intact EF-Tu-GDP, in particular the structure of the effector domain . RESULTS: The crystal structures of intact EF-Tu-GDP from Thermus aquaticus and Escherichia coli have been determined at resolutions of 2.7 A and 3.8 A, respectively . The structures confirm the domain orientation previously found in the structure of partially trypsin-digested EF-Tu-GDP . The structures of the effector region in T . aquaticus and E . coli EF-Tu-GDP are very similar . The C-terminal part of the effector region of EF-Tu-GDP is a beta hairpin; in EF-Tu-GTP, this region forms an alpha helix . This conformational change is not a consequence of crystal packing . CONCLUSIONS: EF-Tu undergoes major conformational changes upon GTP hydrolysis . Unlike other GTP-binding proteins, EF-Tu exhibits a dramatic conformational change in the effector region, involving an unwinding of a small helix and the formation of a beta hairpin structure . This change is presumably involved in triggering the release of tRNA, and EF-Tu, from the ribosome.

Anal Biochem, 1996 Oct 15, 241(2), 186 - 9
Identification and eradication of a denatured DNA isolated during alkaline lysis-based plasmid purification procedures; Sayers JR et al.; Many plasmid isolation procedures use strongly alkaline conditions in the initial stages to facilitate lysis of the host bacteria . We demonstrate that such procedures can give rise to a minor but significantly altered form of plasmid . After electrophoresis and uv transillumination of ethidium bromide-stained agarose gels we and others have noticed a faint band migrating near to the major fluorescent product, covalently closed circular plasmid DNA . This faint band is resistant to cleavage by restriction endonucleases which have recognition sites in the parent plasmid . We were able to show that the contaminating band is able to transform competent Escherichia coli cells and that normal double-stranded plasmids were isolated from such transformants . We were able to selectively hydrolyze the contaminating band using T5 exonuclease which is a 5'-nuclease with a single-strand specific endonuclease activity . Plasmid preparations carried out under nonalkaline conditions failed to produce the contaminant band . We suggest methods for purifying plasmid DNA which remove the denatured band and could improve cloning efficiencies where the largest recombinant libraries are required.

Anal Biochem, 1996 Oct 15, 241(2), 173 - 9
Epitope mapping using histidine-tagged protein fragments: application to Escherichia coli RNA polymerase sigma 70; Rao L et al.; We present a simple, rapid, and inexpensive method for mapping epitopes of monoclonal antibodies using His-tagged protein fragments . In essence, four steps are involved: (i) purify overproduced His-tagged protein from inclusion body; (ii) fragment the protein by partial chemical or enzymatic hydrolysis and isolate the His-tagged peptides with a Ni(2+)-chelate column to generate a "ladder" containing a distribution of peptide fragment sizes; (iii) fractionate the isolated peptides by SDS-polyacrylamide gel electrophoresis and probe the ladder by immunoblot analysis; (iv) determine the size of the fragments that do and do not bind the monoclonal antibody using size markers specific to the His-tagged protein being studied . We have applied this method successfully to map the epitope positions of known and new monoclonal antibodies to Escherichia coli sigma 70 . We believe this method will find broad application.

Nucleic Acids Res, 1996 Oct 15, 24(20), 4071 - 7
Genomic structure and expression of the murine Hmgi-c gene; Zhou X et al.; The murine Hmgi-c gene, a member of the Hmgi gene family, contains five exons encompassing >110 kb of genomic DNA at the pygmy locus on mouse chromosome 10 . Northern analysis identified a 4.1 kb transcript which contains a 324 bp open reading frame encoding a 12 kDa HMGI-C protein . Further analysis defined both the 5' and 3' untranslated regions of the Hmgi-c mRNA species as 658 and 2967 bp respectively . The HMGI-C protein has three consecutive AT hook DNA binding domains and an acidic domain, each of which are encoded by individual exons; such an organization is conserved among the HMGI gene family members from insects to mammals . Similar to the HMGI/Y proteins, the HMGI-C protein does not function as a typical transcriptional activator . Developmental studies revealed that the Hmgi-c gene is expressed predominantly during mouse embryogenesis . Since the human homolog is disrupted in a number of tumors, HMGI-C could play an important role in cell proliferation and differentiation during mammalian development.

Nucleic Acids Res, 1996 Oct 15, 24(20), 3996 - 4002
Effects of antisense DNA against the alpha-sarcin stem-loop structure of the ribosomal 23S rRNA; Meyer HA et al.; Antisense DNAs complementary against various sequences of the alpha-sarcin domain (C2646-G2674) of 23S rRNA from Escherichia coli were hybridized to naked 23S rRNA as well as to 70S ribosomes . Saturation levels of up to 0.4 per 70S ribosome were found, the identical fraction was susceptible to the attack of the RNase alpha-sarcin . The hybridization was specific as demonstrated with RNase H digestion, sequencing the resulting fragments and blockage of the action of alpha-sarcin . The RNase alpha-sarcin seems to approach its cleavage site from the 3' half of the loop of the alpha-sarcin domain . Hybridization is efficiently achieved at 37 degrees C and can extend at least into the 3' strand of the stem of the alpha-sarcin domain . However, the inhibition of alpha-sarcin activity is observed at 30 degrees C but not at 37 degrees C . For a significant inhibition of poly(Phe) synthesis the temperature had to be lowered to 25 degrees C . The results imply that the alpha-sarcin domain changes its conformation during protein synthesis and that the conformational changes may include a melting of the stem of the alpha-sarcin domain.

Nucleic Acids Res, 1996 Oct 15, 24(20), 3942 - 6
Transcriptional regulation of the Drosophila CycA gene by the DNA replication-related element (DRE) and DRE binding factor (DREF); Ohno K et al.; The Drosophila gene for cyclin A is expressed in dividing cells throughout development . This expression pattern is similar to those of genes related to DNA replication, suggesting involvement of some common control mechanism(s) . In the upstream region (-71 to -64 with respect to the transcription initiation site) of the CycA gene, we found a sequence identical to the DNA replication-related element (DRE; 5'-TATCGATA), which is important for high level expression of replication-related genes such as those encoding DNA polymerase alpha and proliferating cell nuclear antigen . Transient expression assays with chloramphenicol acetyltransferase (CAT) were carried out to examine the function of the DRE sequence of the CycA gene . Deletion or base substitution mutations resulted in an extensive reduction in CAT expression . Furthermore, monoclonal antibodies against DRE binding factor (DREF) diminished or supershifted the complex of the DREF and DRE-containing fragment . The results indicate that the Drosophila CycA gene is under the control of a DRE/DREF system, as are DNA replication-related genes.

Nucleic Acids Res, 1996 Oct 15, 24(20), 3903 - 10
Interaction of the Escherichia coli fdhF mRNA hairpin promoting selenocysteine incorporation with the ribosome; Huttenhofer A et al.; The codon UGA located 5' adjacent to an mRNA hairpin within fdhF mRNA promotes the incorporation of the amino acid selenocysteine into formate dehydrogenase H of Escherichia coli . The loop region of this mRNA hairpin has been shown to bind to the special elongation factor SELB, which also forms a complex with selenocysteinyl-tRNA(Sec) and GTP . We designed seven different mRNA constructs derived from the fdhF mRNA which contain a translation initiation region including an AUG initiation codon followed by no, one, two, three, four, five or six UUC phenylalanine codon(s) and the UGA selenocysteine codon 5' adjacent to the fdhF mRNA hairpin . By binding these different mRNA constructs to 30S ribosomal subunits in vitro we attempted to mimic intermediate steps of elongation of a structured mRNA approaching the ribosome by one codon at a time . Toeprint analysis of the mRNA-ribosome complexes showed that the presence of the fdhF mRNA hairpin strongly interferes with binding of the fdhF mRNA to 30S ribosomal subunits as soon as the hairpin is placed closer than 16 bases to the ribosomal P-site . Binding is reduced up to 25-fold compared with mRNA constructs where the hairpin is located outside the ribosomal mRNA track . Surprisingly, no toeprint signals were observed in any of our mRNA constructs when tRNA(Sec) was used instead of tRNA(fMet) . Lack of binding of selenocysteinyl-tRNA(Sec) to the UGA codon was attributed to steric hindrance by the fdhF mRNA hairpin . By chemical probing of the shortest mRNA construct (AUG-UGA-fdhF hairpin) bound to 30S ribosomal subunits we demonstrate that the hairpin structure is not unfolded in the presence of ribosomes in vitro; also, this mRNA is not translated in vivo when fused in-frame 5' of the lacZ gene . Therefore, our data indicate that the fdhF mRNA hairpin has to be unfolded during elongation prior to entering the ribosomal mRNA track and we propose that the SELB binding domain within the fdhF mRNA is located outside the ribosomal mRNA track during decoding of the UGA selenocysteine codon by the SELB-selenocysteinyl-tRNA(Sec)-GTP complex.

Eur J Biochem, 1996 Oct 15, 241(2), 664 - 74
Expression, characterisation and mutagenesis of the aspartic proteinase from equine infectious anaemia virus; Powell DJ et al.; The gene encoding the proteinase from equine infectious anaemia virus (EIAV) was cloned and expressed in Escherichia coli . The recombinant EIAV proteinase was purified to homogeneity and shown to have the ability to process polyprotein and synthetic peptide substrates of human immunodeficiency virus (HIV) origin with an efficiency that can approach that exhibited by HIV proteinase . EIAV proteinase, however, was not susceptible to inhibition by a wide variety of inhibitors of HIV-1 proteinase, including those which have been licenced as anti-AIDS drugs . In this respect, EIAV proteinase behaves like an extreme case of a drug-resistant mutant of HIV-1 proteinase that has arisen under selective drug pressure . Only one potent inhibitor (HBY-793) of HIV-1 proteinase showed comparable efficiency against the EIAV enzyme; the compounds A-77003 and A-76889, which differ only in their stereochemistry and which are otherwise structurally identical to HBY-793 from residues P2 to P2' {nomenclature of Schechter, I . & Berger, A . (1967) Biochem . Biophys . Res . Commun . 27, 157-162}, were not effective inhibitors of EIAV proteinase . Mutant forms of EIAV proteinase (Thr30-->Asp and Ile54-->Gly) were generated and their ability to interact with substrates and inhibitors was characterised . HBY-793 inhibited {Gly54}proteinase as effectively as the wild-type proteinase but was tenfold less potent against {Asp30}proteinase . Data interpretations are presented, based on the structure solved for the complex between HBY-793 and EIAV {Gly54}proteinase {Gustchina A., Kervinen, J., Powell, D . J., Zdanov, A., Kay, J . & Wlodawer, A . (1996) Protein Sci . 5, 1453-1465}.

Eur J Biochem, 1996 Oct 15, 241(2), 611 - 8
The role of conserved histidine residues in the pyridine nucleotide transhydrogenase of Escherichia coli; Bragg PD et al.; The pyridine nucleotide transhydrogenase of Escherichia coli catalyzes the reversible transfer of hydride ion equivalents between NAD+ and NADP+, coupled to translocation of protons across the cytoplasmic membrane . The role of histidine residues in catalysis was investigated by chemical modification with diethylpyrocarbonate and by site-directed mutagenesis . Diethylpyrocarbonate inhibited both hydride ion transfer and coupled proton translocation . Histidine residues were modified as shown spectroscopically and by the ability of hydroxylamine to cause reversal of inhibition . Complete inhibition of hydride ion transfer occurred following modification of 10 residues/enzyme molecule . Site-directed mutagenesis of single conserved histidine residues or the presence of substrates did not provide resistance to inhibition by diethylpyrocarbonate . It is concluded that diethylpyrocarbonate inhibition was a consequence of the structural changes brought about by modification of many histidine residues . With the exception of beta-subunit residue His91 (beta His91), in which mutation can result in specific loss of proton translocation activity {Glavas, N . A., Hou, C . & Bragg, P . D . (1995) Biochemistry 34, 7694-7702}, site-directed mutation of the remaining conserved residues alpha His450, beta His161, beta His345 and beta His354 did not demonstrate a direct role for these residues in catalysis . Mutation of beta His161 had relatively little effect on the properties of the enzyme . By contrast, mutation of alpha His450, beta His345 and beta His354 caused major loss of enzyme activities which was probably due to alterations in the structure of the enzyme . These alterations were reflected in changes in the K(m) values for transhydrogenation.

Eur J Biochem, 1996 Oct 15, 241(2), 557 - 63
Structural and functional characterization of vitronectin-derived RGD-containing peptides from human hemofiltrate; Standker L et al.; Bioactive peptides derived from the adhesive plasma protein vitronectin are present at submicromolar concentrations in human hemofiltrate of patients with renal diseases and were isolated by a combination of high-efficiency chromatographic steps . The structural and functional properties of these peptides were characterized . Sequencing and mass spectrometry revealed the existence of peptide isoforms (5-6 kDa) which corresponded to the N-terminus (residues 1 to 44-50) of vitronectin . The isolated peptides bound directly to plasminogen-activator inhibitor-1 (PAI-1) and were effective competitors of the interaction of PAI-1 with isolated intact vitronectin or extracellular matrix . These functional properties were indistinguishable from the binding properties of a recombinant fusion protein containing residues 1-52 of vitronectin linked to a portion of glutathione S-transferase, expressed in Escherichia coli . Peptides containing the RGD sequence of vitronectin competed for vitronectin binding to the alpha v beta 3 integrin . No indication for direct growth-factor binding was noted, whereas natural peptides were found associated with PAI-1 as the major binding protein in plasma . These data demonstrate that functionally active vitronectin-derived peptides are released by unknown protease(s) from the mature protein and that these peptides are identical, in terms of activity, to recombinant vitronectin fragments . These natural peptides may interact with active PAI-1 in plasma or at extravascular sites and thereby interfere with established biological functions of intact vitronectin.

Eur J Biochem, 1996 Oct 15, 241(2), 516 - 24
The role of a trans-proline in the folding mechanism of ribonuclease T1; Schindler T et al.; Protein folding is often retarded by the cis reversible trans isomerizations of prolyl peptide bonds both in vitro and in vivo . An important role for the folding mechanism is well established for the prolyl peptide bonds that are cis in the native protein, but not for those that are trans . Here we investigated the role of trans-Pro73 for the folding of ribonuclease T1 (which additionally contains two cis-prolines) by comparing the wild-type protein with the Pro73-->Val variant . The Pro-->Val substitution led to a destabilization of the folded protein by 8.5 kJ/mol, which is explained by the strong, 25-fold increase in the rate of unfolding . In contrast, the rates and amplitudes of the fast and slow refolding reactions were virtually unchanged . trans-Proline residues remain largely trans after unfolding, and therefore their contributions to the observed folding kinetics should indeed be insignificant for proteins which also contain one or more cis prolines . The cis-proline residues dominate the kinetics of refolding, because almost all slow-folding molecules contain the respective incorrect (trans) isomers, and because trans-->cis isomerizations are slower than cis-->trans isomerizations . The inability to detect contributions from a trans-proline to the kinetics of folding does not imply that this proline is non-essential for folding in the sense that its cis reversible trans isomerization is energetically uncoupled from conformational folding.

Eur J Biochem, 1996 Oct 15, 241(2), 489 - 97
Two-dimensional 1H-NMR of transmembrane peptides from Escherichia coli phosphatidylglycerophosphate synthase in micelles; Morein S et al.; Two 28-residue peptides, PTLLTLFRVILIPFFVLVFYKKKGKKKG {Pgs-(6-25)-peptidyl-KKKGKKKG; Pgs peptide A} and VEYAGIALFFVAAVLTLWSMLQYLSAAR {Pgs-(149-176)-peptide, Pgs peptide E}, were synthesized and studied by CD and two-dimensional 1H-NMR spectroscopy . The first 20 amino acid residues of Pgs peptide A are identical to one predicted transmembrane segment (Pro6-Tyr25) of the integral membrane protein phosphatidylglycerophosphate synthase (Pgs) of Escherichia coli . Pgs peptide E is identical to another predicted transmembrane segment (Val149-Arg176), which is located in the C-terminal end of this lipid synthase . Pgs peptides A and E were dissolved in methanol or trifluoroethanol or were incorporated into solvent-free micelles of fully deuterated SDS . In all these systems, CD spectra of both peptides indicated an alpha-helical secondary structure . However, peptides that were solubilized in micelles exhibited the highest content of alpha-helix as judged from comparison of the CD spectra . Thermodynamically stable isotropic solutions at high peptide concentrations (1-3 mM) could only be obtained with the peptide incorporated in micelles; in organic solvents, significant peptide aggregation occurred . Relatively sharp peaks were obtained with 1H-NMR spectroscopy of the peptides in SDS micelles, which indicates rapid tumbling of the peptides in the micellar environment . Translational-diffusion coefficients of the micelles with and without peptide, determined by pulsed-field-gradient NMR, showed that the micellar size was unaffected by the solubilized peptide . The radius of the hydrated micelles was estimated to be about 2.7 nm (i.e . the mass of the aggregate is almost 30 kDa) . Two-dimensional NMR spectroscopy of both peptides solubilized in the micelles indicated an alpha-helical conformation . This observation is strengthened by an investigation of the hydrogen exchange of the peptide amide protons, where significantly less exchange of the amide protons was observed in the middle of the peptides compared with the ends.

Eur J Biochem, 1996 Oct 15, 241(2), 484 - 8
Determination of a binding site for a non-substrate ligand in mammalian cytosolic glutathione S-transferases by means of fluorescence-resonance energy transfer; Sluis-Cremer N et al.; To determine the location of the non-substrate-ligand-binding region in mammalian glutathione S-transferases, fluorescence-resonance energy transfer was used to calculate distances between tryptophan residues and protein-bound 8-anilinonaphthalene 1-sulphonate (an anionic ligand) in the human class-alpha glutathione S-transferase, and in a human Trp28-->Phe mutant class-pi glutathione S-transferase . Distance values of 2.21 nm and 1.82 nm were calculated for the class-alpha and class-pi enzymes, respectively . Since glutathione S-transferases bind one non-substrate ligand/protein dimer, the ligand-binding region, according to the calculated distances, is found to be located in the dimer interface near the twofold axis . This region is the same as that in which the parasitic helminth Schistosoma japonicum glutathione S-transferase binds praziquantel, a non-substrate drug used to treat schistosomiasis {McTigue, M . A., Williams, D . R . & Tainer, J . A . (1995) J . Mol . Biol . 246, 21-27} . Since the overall folding topology is conserved and certain features at the dimer interface are similar throughout the superfamily, it is reasonable to expect that all cytosolic glutathione S-transferases bind non-substrate ligands in the amphipathic groove at the dimer interface.

Eur J Biochem, 1996 Oct 15, 241(2), 453 - 61
X-ray structure of native full-length human fibroblast-growth factor at 0.25-nm resolution; Romero A et al.; Acidic fibroblast-growth factor (aFGF) is one of the typical members of a group of nine polypeptides of relatively similar amino acid sequence known as the fibroblast-growth-factor family of proteins . Widely distributed throughout the organism, fibroblast-growth factors seem to be involved in numerous physiological processes ranging from control of cell proliferation and differentiation to modulation of animal behaviour and arterial blood pressure . This wide assortment of biological activities explains their involvement in numerous pathologies . Instability and low yields of the purified protein have precluded high-resolution structural studies of the physiological form of aFGF . Nevertheless, modifications introduced recently into the synthesis and purification procedures of this protein have allowed preparations of samples that, as shown here, are reliable substrates to obtain crystals suitable for X-ray-diffraction studies . These analyses have allowed us to elucidate the three-dimensional structure of the physiological form of human aFGF by molecular-replacement methods, from the previously reported structure of a shortened form of bovine aFGF that was stabilized by point-directed mutagenesis . The structure was refined at a resolution of 0.25 nm to an R factor of 20.4% for 13,109 reflections between 0.6 nm and 0.25 nm, with rmsd of 1.1 pm and 1.9 degrees from ideal bond lengths and bond angles, respectively . Human aFGF folds according to a beta-trefoil topology . This fold consist of six beta-strand pairs . Three of them form a six-stranded beta-barrel structure that is capped at one end by the other three pairs arranged in a triangular array . The N-terminus of aFGF up to residue Pro19 appears flexible in the structure and does not specifically interact with the rest of the molecule.

Eur J Biochem, 1996 Oct 15, 241(2), 432 - 9
Mössbauer, electron-paramagnetic-resonance and X-ray-absorption fine-structure studies of the iron environment in recombinant human tyrosine hydroxylase; Meyer-Klaucke W et al.; Isoforms (1-4) of human tyrosine hydroxylase (TH) have been expressed in Escherichia coli and purified as apoenzymes (metal-free) . Apo-human TH binds 1.0 atom Fe(II)/enzyme subunit, and iron binding is associated with an immediate and dramatic (40-fold) increase in specific activity . For X-ray absorption fine structure (XAFS) and electron paramagnetic resonance (EPR) measurements the apoenzyme was reconstituted with 56Fe and for Mossbauer measurements with 57Fe . XAFS measurements at the Fe-K edge of human TH were performed on the native form {Fe(II)-human TH}, as well as after addition of stoichiometric amounts of the substrate tetrahydropterin, the inhibitor dopamine and of H2O2 . The addition of dopamine or H2O2 oxidizes the ferrous iron of the native human TH to the ferric state . In both redox states the iron is octahedrally coordinated by low-Z backscatterers, thus sulfur coordination can be excluded . From the multiple scattering analysis of the EXAFS region is was surmised that part of the iron coordination is due to (3 +/- 1) imidazols . Addition of tetrahydropterin does not significantly change the iron coordination of the Fe(II) enzyme . The Mossbauer results confirm the valence states and the octahedral coordination of iron as well as the exclusion of sulfur ligation . Both the EPR spectra and the Mossbauer magnetic hyperfine pattern of dopamine- and H2O2-treated native human TH, were analyzed with the spin-Hamiltonian formalism . This analysis provides significantly different features for the two forms of human TH: the ferric iron (S = 5/2) of the H2O2-treated form exhibits a rhombic environment while that of the dopamine-treated form exhibits near-axial symmetry . The specific spectroscopic signature of dopamine-treated human TH, including that of an earlier resonance-Raman study {Michaud-Soret, I., Andersson, K . K., Que, L . Jr & Haavik, J . (1995) Biochemistry 34, 5504-5510} is most likely due to the bidentate binding of dopamine to iron.

Eur J Biochem, 1996 Oct 15, 241(2), 425 - 31
Characterisation of the nucleic-acid-binding activity of KH domains . Different properties of different domains; Dejgaard K et al.; The KH module is a sequence motif recently identified in a number of diversified RNA-binding proteins and suggested to be the functional element responsible for RNA binding . So far, however, this hypothesis has not received direct experimental support . We have expressed the three KH-domains from heterogeneous nuclear ribonucleoprotein K (hnRNP-K), the poly(C)-binding proteins PCBP-1 and PCBP-2, the first three to four domains from the high-density binding protein HBP, the one and a half domain from the archaeon Halobacterium halobium ORF139 and one and a half domain of the fragile-X protein FMR1 in Escherichia coli and analysed their nucleic-acid-binding properties in vitro . The results showed that the in vitro poly(rC)-binding activity of hnRNP-K can be assigned to KH-domain 3, whereas both domains 1 and 3 in the PCBPs bind poly(rC) . In addition, all these domains exhibit binding activity towards other nucleic acids, albeit at a significantly lower level . The first KH domain from the FMR1 protein binds poly(rG) and single-stranded and double-stranded DNA . The N-terminal three or four domains from HBP bind poly(rG) and, at a much lower level, single-stranded and double-stranded DNA . Thus, single KH domains are discrete and independent nucleic-acid-binding units . Moreover, different KH domains bind different nucleic acids, suggesting that KH domains are composed of a conserved, weakly nucleic-acid-binding, structure that is fine tuned, by sequence variation, resulting in sequence-specific nucleic-acid-binding entities.

Eur J Biochem, 1996 Oct 15, 241(2), 411 - 6
Specific binding of DnaA protein to a DnaA box in the guaB gene of Escherichia coli K12; Tesfa-Selase F et al.; Expression of the guaBA operon of Escherichia coli is regulated by the DNA replication-initiating protein, DnaA . Two DnaA boxes, which are potential binding sites for DnaA, are present in the gua operon . One box (with 8/9 match to the DnaA box consensus sequence) is at the gua promoter; the other box, which has a consensus sequence, is on the non-transcribed strand within the guaB coding region approximately 200 bp downstream of the initiation codon . The binding in vitro of purified DnaA protein to these boxes was investigated by filter retention and gel retardation analysis, and by deoxyribonuclease I footprinting, using restriction fragments of gua operon DNA . DnaA protein was shown to bind specifically only to the fragment carrying the consensus sequence DnaA box, and to protect this box from deoxyribonuclease I . Transcription termination resulting from the binding of DnaA to this box within the guaB gene explains repression by DnaA of the gua operon in vivo.

Eur J Biochem, 1996 Oct 15, 241(2), 363 - 7
The Cys292-->Ala substitution in protein R1 of class I ribonucleotide reductase from Escherichia coli has a global effect on nucleotide binding at the specificity-determining allosteric site; Ormo M et al.; Ribonucleotide reductase from aerobically grown Escherichia coli is allosterically regulated, both with respect to general activity and substrate specificity . Protein R1, the homodimeric enzyme component which harbours binding sites for allosteric effectors (nucleoside triphosphates) as well as substrates (ribonucleoside diphosphates), has been engineered at Cys292 close to the dimer interaction area . This residue was earlier shown to be specifically photoaffinity labelled with the allosteric nucleotide dTTP . In this study the effect of the Cys292-->Ala substitution is shown to be an overall diminished nucleotide binding at the specificity site reflected in Kd values for dTTP, dGTP and dATP higher by more than one order of magnitude with respect to wild type . The mutant protein's interaction with other protein components of the ribonucleotide reductase system was unaffected by the mutation . These results show that Cys292 in protein R1 of class I ribonucleotide reductase from E . coli is located in the allosteric specificity site.

Eur J Biochem, 1996 Oct 15, 241(2), 309 - 14
Molecular characterization of a tissue-polypeptide-specific-antigen epitope and its relationship to human cytokeratin 18; Rydlander L et al.; Tissue-polypeptide-specific antigen (TPS) from the human colon adenocarcinoma cell line WiDr was purified using the monoclonal antibody M3 as a probe . Upon SDS/polyacrylamide gradient gel electrophoresis, several TPS-positive bands were detected (corresponding to 13 kDa, 22 kDa and a doublet at 42 kDa) . The 13-kDa moiety was purified about 30,000-fold by a 5-step protocol . The electro-phoretically homogeneous component was obtained in a 7% yield of the total TPS activity of the crude extract . N-terminal sequence analysis showed the presence of an N-terminally truncated molecule and identified the 13-kDa TPS component as a fragment of human cytokeratin 18, with a major from starting at position 284 of the parent molecule . Laser-desorption mass spectrometry showed the presence of one major component with a molecular mass corresponding to a C-terminal end close to position 396 (which gives 12776 Da for the form with non-truncated N-terminus) . The M3 antibody was also used to screen a human prostate cDNA lambda gt11 library . Four identical phage clones were detected, each producing a fusion protein with beta-galactosidase and the M3-positive component . PCR amplification showed the presence of an approximately 1200-bp insert, and sequence analysis revealed it to contain a 996-nucleotide fragment corresponding to residues 103-429 of human cytokeratin 18 (plus a non-coding human desmin artifact fragment) . Smaller fragments, engineered by PCR and expressed as fusion proteins using the pET3xc vector in Escherichia coli, showed that the M3 epitope is localized to cytokeratin 18, residues 322-340 . Two other TPS-active monoclonal antibodies were localized to cytokeratin 18 with similar techniques, ascribing an epitope (to M21) to residues 414-429 and another (to M24) to residues 139-297 . Combined, the results demonstrate that TPS reactivity is derived from specific epitopes of human cytokeratin 18.

Biochem J, 1996 Oct 15, 319 ( Pt 2), 435 - 40
Expression of the human spermidine/spermine N1-acetyltransferase in spermidine acetylation-deficient Escherichia coli; Ignatenko NA et al.; A cDNA encoding the human spermidine/spermine N1-acetyltransferase (N1SSAT) was conditionally expressed in a strain of Escherichia coli deficient in spermidine-acetylating activity . Conditional expression of this cDNA was performed under the control of the lac promoter, by addition of the non-hydrolysable lactose analogue isopropyl beta-D-thiogalactoside . Expression of the N1SSAT cDNA oriented in the sense direction resulted in the acetylation of spermidine at the N1 but not the N8 position and a decrease in endogenous spermidine contents and growth rates in these bacteria . When this cDNA was expressed in the antisense orientation, spermidine acetylation was not detected and endogenous spermidine contents and growth rates were unaffected . Increasing the endogenous N1-acetylspermidine concentration by addition of this amine to the culture medium did not suppress growth, and increasing endogenous spermidine pools by exogenous addition was not sufficient to restore optimal growth in cells expressing the human N1SSAT . Exogenous spermidine, but neither N1- nor N8-acetylspermidine, stimulated cell growth in strains unable to synthesize spermidine . These results suggest that one physiological consequence of spermidine acetylation in E . coli is growth inhibition . The mechanism of this inhibition seems to involve the formation of acetylspermidine, and is not simply due to a decrease in the intracellular concentration of non-acetylated spermidine.

Biochem J, 1996 Oct 15, 319 ( Pt 2), 351 - 9
The folding and activity of the extracellular lipase of Rhizopus oryzae are modulated by a prosequence; Beer HD et al.; The fungus Rhizopus oryzae synthesizes an extracellular lipase precursor bearing N-terminal pre- and pro-sequences . Our studies in Escherichia coli and using recombinant lipase in vitro indicate that the prosequence of 97 amino acids has at least two functions . First, it modulates the enzyme activity of the lipase so that this enzyme can initially be synthesized in a non-destructive form . Direct synthesis of the mature form of the lipase in the cell has toxic consequences, at least partly because of phospholipase activity that is suppressed in the proprotein . Secondly, it supports folding of the lipase via a pathway influenced by a single cysteine residue at position - 68 . Mutational analysis of the prosequence demonstrates not only the key role of this cysteine residue but also the importance of the neighbouring amino acids . In particular, Arg-69 probably enhances the leaving group character of Cys-68 . We propose a model in which Cys-68 acts as an intramolecular thiodisulphide reagent, playing a catalytic role in the folding of the enzyme . The prosequence is capable of performing the described functions both in cis and in trans.

Arch Biochem Biophys, 1996 Oct 15, 334(2), 268 - 76
Structure-function relationships in Escherichia coli transcription termination protein Rho revealed by radiation target analysis; Stitt BL et al.; High-energy electrons were used to measure the target sizes for inactivation of the RNA-dependent ATPase activity of Escherichia coli transcription termination factor Rho, for its ATP binding ability, and for its physical destruction . SDS-PAGE analysis of irradiated samples indicated that the target size for polypeptide destruction in the homohexameric enzyme is the dimer, indicating that energy transfer must occur from a hit subunit to one other subunit, although the subunits are not known to be linked by any covalent bonds . The ATP binding ability of Rho also inactivates as a dimer, a result that is consistent with the physical destruction target size . However, a single subunit as the ATP binding entity is not excluded . The RNA-dependent ATPase activity of Rho inactivates with the apparent target size of trimer to tetramer, indicating that interactions among the subunits of Rho are required for ATP hydrolysis . Rho hexamers are known to exchange subunits, although the identity of the exchanging unit is not known . Models in which this property of Rho is taken into account indicate that the closest fit to the experimental data is for an ATPase target size of a hexamer with dimers as the exchanging units, consistent with earlier chemical inactivation studies.

Arch Biochem Biophys, 1996 Oct 15, 334(2), 183 - 92
Studies of distant members of the P450 superfamily (P450scc and P450c27) by random chimeragenesis; Pikuleva IA et al.; "Random chimeragenesis" (Kim, J.-Y., and Devreotes, P . N . (1994) J . Biol . Chem . 269, 28724-28731; Levin, L . R., and Reed, R . R . (1995) J . Biol . Chem . 270, 7573-7579) has been used to generate chimeras between two distant P450s involved in catabolism of cholesterol in mammals, P450scc (product of CYP11A1 gene) and P450c27 (product of CYP27 gene) . Both are mitochondrial P450s which hydroxylate the side chain of cholesterol . Even though these P450s are only about 25% identical, we wondered whether their similar substrate specificity might permit mapping of the active sites by this technique . Four chimeric DNAs encoding three different proteins have been obtained testifying that short stretches of nucleotide identity (six nucleotides preceding the crossover point) are sufficient for homologous recombination in Escherichia coli, the basis of the random chimeragenesis technique . The N-terminal part of the chimeras was formed by P450scc and the C-terminus was from P450c27 . The chimeric P450s have been expressed in E . coli and partially purified . Though they displayed a peak at 453 nm in their CO-difference spectra, indicating that the proteins are properly folded and hemin is correctly incorporated, none of the chimeras had detectable catalytic activity with either cholesterol (substrate for P450scc) or 5beta-cholestane-3alpha,7alpha,12alpha-triol (substrate for P450c27) . To investigate whether the chimeras bind substrate, titrations with 22R-hydroxycholesterol have been carried out . Addition of 22R-hydroxycholesterol to recombinant P450scc which is in the low spin form leads to conversion to the high spin form . Titration of chimeras with 22R-hydroxycholesterol did not result in their conversion to the high spin form . However, a shift to 426-430 nm was observed in the difference spectrum of two of the chimeras with a minimum around 406 nm . Thus, addition of 22R-hydroxycholesterol to these chimeras results in binding of the steroid in the enzyme active site and conversion of one low spin form to a different low spin form . Possible explanations for the absence of enzymatic activity and the potential advantages of the random chimeragenesis technique to generate chimeras between different P450s are discussed.

EMBO J, 1996 Oct 15, 15(20), 5547 - 56
A novel type of protein kinase phosphorylates actin in the actin-fragmin complex; Eichinger L et al.; Actin-fragmin kinase (AFK) from Physarum polycephalum specifically phosphorylates actin in the EGTA-resistant 1:1 actin-fragmin complex . The cDNA deduced amino acid sequence reveals two major domains of approximately 35 kDa each that are separated by a hinge-like proline/serine-rich segment of 50 residues . Whereas the N-terminal domain does not show any significant similarity to protein sequences from databases, there are six complete kelch repeats in the protein that comprise almost the entire C-terminal half of the molecule . To prove the intrinsic phosphorylation activity of AFK, full-length or partial cDNA fragments were expressed both in a reticulocyte lysate and in Escherichia coli . In both expression systems, we obtained specific actin phosphorylation and located the catalytic domain in the N-terminal half . Interestingly, this region did not contain any of the known protein kinase consensus sequences . The only known sequence motif present that could have been involved in nucleotide binding was a nearly perfect phosphate binding loop (P-loop) . However, introduction of two different point mutations into this putative P-loop sequence did not alter the catalytic activity of the kinase, which indicates an as yet unknown mechanism for phosphate transfer . Our data suggest that AFK belongs to a new class of protein kinases and that this actin phosphorylation might be the first example of a widely distributed novel type of regulation of the actin cytoskeleton in non-muscle cells.

Proc Natl Acad Sci U S A, 1996 Oct 15, 93(21), 11652 - 7
Single residue substitutions that change the gating properties of a mechanosensitive channel in Escherichia coli; Blount P et al.; MscL is a channel that opens a large pore in the Escherichia coli cytoplasmic membrane in response to mechanical stress . Previously, we highly enriched the MscL protein by using patch clamp as a functional assay and cloned the corresponding gene . The predicted protein contains a largely hydrophobic core spanning two-thirds of the molecule and a more hydrophilic carboxyl terminal tail . Because MscL had no homology to characterized proteins, it was impossible to predict functional regions of the protein by simple inspection . Here, by mutagenesis, we have searched for functionally important regions of this molecule . We show that a short deletion from the amino terminus (3 amino acids), and a larger deletion of 27 amino acids from the carboxyl terminus of this protein, had little if any effect in channel properties . We have thus narrowed the search of the core mechanosensitive mechanism to 106 residues of this 136-amino acid protein . In contrast, single residue substitutions of a lysine in the putative first transmembrane domain or a glutamine in the periplasmic loop caused pronounced shifts in the mechano-sensitivity curves and/or large changes in the kinetics of channel gating, suggesting that the conformational structure in these regions is critical for normal mechanosensitive channel gating.

Proc Natl Acad Sci U S A, 1996 Oct 15, 93(21), 11580 - 5
Identification of the coding region for a second poly(A) polymerase in Escherichia coli; Cao GJ et al.; We had earlier identified the pcnB locus as the gene for the major Escherichia coli poly(A) polymerase (PAP I) . In this report, we describe the disruption and identification of a candidate gene for a second poly(A) polymerase (PAP II) by an experimental strategy which was based on the assumption that the viability of E . coli depends on the presence of either PAP I or PAP II . The coding region thus identified is the open reading frame f310, located at about 87 min on the E . coli chromosome . The following lines of evidence support f310 as the gene for PAP II: (i) the deduced peptide encoded by f310 has a molecular weight of 36,300, similar to the molecular weight of 35,000 estimated by gel filtration of PAP II; (ii) the deduced f310 product is a relatively hydrophobic polypeptide with a pI of 9.4, consistent with the properties of partially purified PAP II; (iii) overexpression of f310 leads to the formation of inclusion bodies whose solubilization and renaturation yields poly(A) polymerase activity that corresponds to a 35-kDa protein as shown by enzyme blotting; and (iv) expression of a f310 fusion construct with hexahistidine at the N-terminus of the coding region allowed purification of a poly(A) polymerase fraction whose major component is a 36-kDa protein . E . coli PAP II has no significant sequence homology either to PAP I or to the viral and eukaryotic poly(A) polymerases, suggesting that the bacterial poly(A) polymerases have evolved independently . An interesting feature of the PAP II sequence is the presence of sets of two paired cysteine and histidine residues that resemble the RNA binding motifs seen in some other proteins.

Proc Natl Acad Sci U S A, 1996 Oct 15, 93(21), 11563 - 8
Selective response of ternary complex factor Sap1a to different mitogen-activated protein kinase subgroups; Strahl T et al.; Mitogenic and stres signals results in the activation of extracellular signal-regulated kinases (ERKs) and stress-activated protein kinase/c-Jun N-terminal kinases (SAPK/JNKs), respectively, which are two subgroups of the mitogen-activated protein kinases . A nuclear target of mitogen-activated protein (MAP) kinases is the ternary complex factor Elk-1, which underlies its involvement in the regulation of c-fos gene expression by mitogenic and stress signals . A second ternary complex factor, Sap1a, is coexpressed with Elk-1 in several cell types and shares attributes of Elk-1, the significance of which is not clear . Here we show that Sap1a is phosphorylated efficiently by ERKs but not by SAPK/JNKs . Serum response factor-dependent ternary complex formation by Sap1a is stimulated by ERK phosphorylation but not by SAPK/JNKs . Moreover, Sap1a-mediated transcription is activated by mitogenic signals but not by cell stress . These results suggest that Sap1a and Elk-1 have distinct physiological functions.

Proc Natl Acad Sci U S A, 1996 Oct 15, 93(21), 11552 - 7
In vitro characterization of mutant yeast RNA polymerase II with reduced binding for elongation factor TFIIS; Wu J et al.; We have reported previously the isolation and genetic characterization of mutations in the gene encoding the largest subunit of yeast RNA polymerase II (RNAPII), which lead to 6-azauracil (6AU)-sensitive growth . It was suggested that these mutations affect the functional interaction between RNAPII and transcription-elongation factor TFIIS because the 6AU-sensitive phenotype of the mutant strains was similar to that of a strain defective in the production of TFIIS and can be suppressed by increasing the dosage of the yeast TFIIS-encoding gene, PPR2, RNAPIIs were purified and characterized from two independent 6AU-sensitive yeast mutants and from wild-type (wt) cells . In vitro, in the absence of TFIIS, the purified wt polymerase and the two mutant polymerases showed similar specific activity in polymerization, readthrough at intrinsic transcriptional arrest sites and nascent RNA cleavage . In contrast to the wt polymerase, both mutant polymerases were not stimulated by the addition of a 3-fold molar excess of TFIIS in assays of promoter-independent transcription, readthrough or cleavage . However, stimulation of the ability of the mutant RNAPIIs to cleave nascent RNA and to read through intrinsic arrest sites was observed at TFIIS:RNAPII molar ratios greater than 600:1 . Consistent with these findings, the binding affinity of the mutant polymerases for TFIIS was found to be reduced by more than 50-fold compared with that of the wt enzyme . These studies demonstrate that TFIIS has an important role in the regulation of transcription by yeast RNAPII and identify a possible binding site for TFIIS on RNAPII.

Proc Natl Acad Sci U S A, 1996 Oct 15, 93(21), 11528 - 33
Mutant LexA proteins with specific defects in autodigestion; Shepley DP et al.; In self-processing biochemical reactions, a protein or RNA molecule specifically modifies its own structure . Many such reactions are regulated in response to the needs of the cell by an interaction with another effector molecule . In the system we study here, specific cleavage of the Escherichia coli LexA repressor, LexA cleaves itself in vitro at a slow rate, but in vivo cleavage requires interaction with an activated form of RecA protein . RecA acts indirectly as a coprotease to stimulate LexA autodigestion . We describe here a new class of lexA mutants, lexA (Adg-; for autodigestion-defective) mutants, termed Adg- for brevity . Adg- mutants specifically interfered with the ability of LexA to autodigest but left intact its ability to undergo RecA-mediated cleavage . The data are consistent with a conformational model in which RecA favors a reactive conformation capable of undergoing cleavage . To our knowledge, this is the first example of a mutation in a regulated self-processing reaction that impairs the rate of self-processing without markedly affecting the stimulated reaction . Had wild-type lexA carried such a substitution, discovery of its self-processing would have been difficult; we suggest that, in other systems, a slow rate of self-processing has prevented recognition that a reaction is of this nature.

Proc Natl Acad Sci U S A, 1996 Oct 15, 93(21), 11504 - 9
Structural studies of p21Waf1/Cip1/Sdi1 in the free and Cdk2-bound state: conformational disorder mediates binding diversity; Kriwacki RW et al.; The cyclin-dependent kinase (Cdk) inhibitor p21Waf1/Cip1/Sdi1, important for p53-dependent cell cycle control, mediates G1/S arrest through inhibition of Cdks and possibly through inhibition of DNA replication . Cdk inhibition requires a sequence of approximately 60 amino acids within the p21 NH2 terminus . We show, using proteolytic mapping, circular dichroism spectropolarimetry, and nuclear magnetic resonance spectroscopy, that p21 and NH2-terminal fragments that are active as Cdk inhibitors lack stable secondary or tertiary structure in the free solution state . In sharp contrast to the disordered free state, however, the p21 NH2 terminus adopts an ordered stable conformation when bound to Cdk2, as shown directly by NMR spectroscopy . We have, thus, identified a striking disorder-order transition for p21 upon binding to one of its biological targets, Cdk2 . This structural transition has profound implications in light of the ability of p21 to bind and inhibit a diverse family of cyclin-Cdk complexes, including cyclin A-Cdk2, cyclin E-Cdk2, and cyclin D-Cdk4 . Our findings suggest that the flexibility, or disorder, of free p21 is associated with binding diversity and offer insights into the role for structural disorder in mediating binding specificity in biological systems . Further, these observations challenge the generally accepted view of proteins that stable secondary and tertiary structure are prerequisites for biological activity and suggest that a broader view of protein structure should be considered in the context of structure-activity relationships.

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