Escherichia coli

Biochemistry, 1993 Nov 16, 32(45), 12096 - 104
Preferential binding of the xeroderma pigmentosum group A complementing protein to damaged DNA; Jones CJ et al.; The xeroderma pigmentosum group A complementing protein (XPAC) is involved in an early step of nucleotide excision repair, the main process that removes UV damage and many chemical lesions from DNA . To explore the properties and function of XPAC, recombinant protein encoded by the human XPAC cDNA was expressed with an N-terminal polyhistidine tag in Escherichia coli and purified to homogeneity . The soluble fusion protein could correct the repair defect in vitro of XP-A cell extracts . XPAC protein bound to DNA with a preference for UV-irradiated over nonirradiated DNA, as determined by a gel electrophoresis mobility shift assay with a 258 base pair DNA fragment (the association constant was approximately 3 x 10(6) M-1 for the fragment irradiated with 6 kJ/m2 UV light) . Removal of cyclobutane pyrimidine dimers from UV-irradiated DNA by enzymatic photoreactivation did not significantly reduce binding of XPAC to the irradiated fragment, indicating that binding was mostly due to (6-4) photoproducts, with a preference for a (6-4) photoproduct over an undamaged base pair up to 300-fold . Undamaged single-stranded DNA competed about 4-fold more effectively than undamaged double-stranded DNA for binding of XPAC to a UV-irradiated fragment . In addition, XPAC bound to DNA treated with the chemotherapeutic agent cis-diamminedichloroplatinum(II) . The results suggest that XPAC functions as a key component in recognition of DNA damage during repair.

Biochemistry, 1993 Nov 16, 32(45), 12054 - 61
Boundary of the autoinhibitory region of smooth muscle myosin light-chain kinase; Yano K et al.; It has been proposed that myosin light-chain kinase (MLCK) activity is inhibited in the absence of Ca2+/calmodulin by a pseudosubstrate sequence {Kemp, B . E., Pearson, R . B., Guerriero, V . J., Bagchi, I., & Means, A . R . (1987) J . Biol . Chem . 262, 2542-2548} . To evaluate this hypothesis, the role of a cluster of basic residues, Arg797-Arg798-Lys799, which are essential for the pseudosubstrate sequence, in the inhibition of MLCK was studied . A full-length cDNA of chicken gizzard MLCK was obtained, and the recombinant MLCK which contains the entire amino acid sequence was expressed in Escherichia coli . The Ca2+/calmodulin-dependent activity of the recombinant MLCK was comparable to that of the naturally isolated MLCK . Two truncation mutants, MT799 and MT796, were produced, of which MT799 but not MT796 contained a cluster of basic residues . Neither MT799 nor MT796 bound calmodulin, and kinase activity was inhibited (similar to MLCK activity in the absence of Ca2+/calmodulin) . However, the kinase activity of the mutants was increased markedly by subsequent tryptic proteolysis . The tryptic digestion of the mutants initially produced a 64-kDa fragment then, subsequently, the 61-kDa fragment, and the increase in activity coincided with the appearance of the 61-kDa fragment . This was similar to the digestion profile of native MLCK, and it is known that the 61-kDa fragment is the constitutively active kinase {Ikebe, M., Stepinska, M., Kemp, B . E., Means, A . R., & Hartshorne, D . J . (1987) J . Biol . Chem . 262, 13828-13834}.(ABSTRACT TRUNCATED AT 250 WORDS)

Biochemistry, 1993 Nov 16, 32(45), 11953 - 6
The gateway to the active site of heme-copper oxidases; Lemon DD et al.; The spectroscopy and dynamics of CO binding were measured for wild-type and mutant cytochromes bo, members of the superfamily of heme-copper oxidases . The results suggest that access of ligands, including substrate O2, to the binuclear Fe-Cu active site is controlled at two levels . CO recombination to the wild-type ubiquinol oxidase exhibited saturation kinetics (kmax = 190 s-1, Km = 2.4 mM), indicative of the existence of an intermediate in the ligand-binding pathway . FTIR spectroscopy and TRIR spectroscopy were used to demonstrate conclusively that this intermediate was a CuB-CO complex . Two mutant oxidases (His333Leu, His334Leu) which lack CuB showed no evidence of saturation of CO rebinding, even up to 21 mM CO . Also, the absolute rates of CO binding to the mutant oxidases were much greater than for wild type, even at CO concentrations well below the apparent Km for wild-type enzyme . These results clearly indicate that the copper ion at the binuclear site acts as an obligatory way station, or gate, severely limiting the approach of ligands to the heme active site . Further, an analysis of the rate constants for CO binding to CuB suggests that the protein structure external to the binuclear site regulates ligand entry into this site . We propose that these control mechanisms for substrate binding are operative throughout this general class of enzymes.

Biochemistry, 1993 Nov 16, 32(45), 12203 - 8
Modification of the head-group selectivity of porcine pancreatic phospholipase A2 by protein engineering; Bhat MK et al.; On the basis of the three-dimensional structures of phospholipid and porcine pancreatic phospholipase A2 (pla2), it was predicted that the removal of a negative charge in the hydrophilic region of the phospholipid binding site would influence the head-group selectivity of porcine pancreatic pla2 . To test this prediction, glutamic acid 46 was changed to leucine by site-directed mutagenesis . The E46L mutant, expressed in Escherichia coli, was purified and characterized . The mutation did not affect the activity toward the mixed micellar substrate, but the activity of E46L toward DiC12-P, which has two negative charges on the head group, was three times higher than that of DiC12-PC, which carries no net charge in the head group . The native pla2 was inhibited by the product(s) released from DiC12-P but not the mutant enzyme . Kinetic analysis revealed that the E46L mutant and the native pla2 had comparable affinities (Km) toward monomeric and micellar phospholipids of zwitterionic type while the activity (kcat) of E46L, toward the same substrates, was approximately 50% lower compared to that of native pla2 . When micellar DiC12-P was used as a substrate, the Kmapp value for E46L was four times lower and the kcatapp/kmapp was 5-fold higher than those of native pla2 . However, the kinetic parameters of mutant and native pla2s remained unchanged for monomeric HEPG, with one negative charge in the head group . Thus, we have modified the head-group selectivity of porcine pancreatic pla2 by protein engineering.

Anal Biochem, 1993 Nov 15, 215(1), 118 - 28
Membrane protein topology determination by proteolysis of maltose binding protein fusions; Miller KW et al.; A method is presented for determining the topology of Escherichia coli inner membrane proteins that is based on proteolysis of fusion proteins between maltose binding protein (MBP) and the membrane protein of interest . Fusion proteins are constructed wherein the MBP domain is fused upstream of the membrane protein domain . A secreted MBP domain is attached to the protein if its N-terminus resides in the periplasm . A cytosolic MBP domain (MBP delta 2-26) is attached to the protein when its N-terminus resides in the cytoplasm . The method has been developed using a fusion protein in which a secreted MBP domain is attached to the pBR322 tetracycline resistance protein at its first periplasmic loop . Fusion proteins are subjected to partial proteolysis in membrane vesicles under conditions where digestion does not occur in MBP . The mixture of digestion products is analyzed by Western immunoblotting using anti-MBP antiserum to detect cleavage fragments . Sites of digestion in the membrane protein are identified by comparing the mobilities of digestion products to truncated fusion standards that terminate at defined locations within the membrane protein domain . The technique has the advantage that neither overexpression of the protein nor high-quality antiserum to it are required for detection of protease fragments . Furthermore, the method can be applied to screen membrane proteins for structure alterations that have been introduced deliberately into them.

Tijdschr Diergeneeskd, 1993 Nov 15, 118(22), 731 - 4
{Infectious diarrhea of calves: therapeutic and preventive possibilities based on etiological, pathophysiological and immunological data}; Vanopdenbosch E et al.; In this article the current knowledge on the development mechanisms of bacterial, viral and cryptosporidial neonatal calf diarrhoea are briefly reviewed as basis for an efficacious prophylaxis strategy . Hygienic measures will prevent the infection of the calf before colostrum intake and will diminish the infection pressure during the first weeks of life . Moreover, local intestinal immunity will be provided by early administration of immune colostrum for the prevention of E.coli diarrhoea and prolonged administration of colostrum or immune milk during the first weeks can protect against viral diarrhoea . To obtain a maximum level of E.coli antibodies in colostrum, the last vaccination has to be administered at least 8 weeks before parturition . Prolonged excretion of antiviral antibodies in the milk for several weeks, can be achieved by a 'priming' vaccination with an adjuvanted inactivated vaccine during pregnancy, followed by a booster vaccination within 24 hours after parturition . Cryptosporidium parvum diarrhoea can be prevented with halofuginone lactate.

Biochem Biophys Res Commun, 1993 Nov 15, 196(3), 1552 - 7
Sensitive detection of low levels of ribonuclease H activity by an improved renaturation gel assay; Frank P et al.; Renaturation gel assays are good tools to assign enzymatic activities to protein bands . First, proteins are separated by denaturating electrophoresis on substrate-containing gels . Then, following the elimination of the denaturing agent, polypeptides are allowed to renature, thus leading to the degradation of the embedded substrate at positions at which the corresponding activity has moved . Nevertheless, this in situ technique does not only reflect a certain amount of enzyme activity, it also depends upon the ability of an enzyme to renature . Here we present a renaturation gel assay procedure with an improved sensitivity and discuss the detection of E . coli and human ribonuclease H activities as an example.

Biochem Biophys Res Commun, 1993 Nov 15, 196(3), 1466 - 73
Cloning, sequencing and expression of the gene for alpha antigen from Mycobacterium intracellulare and use of PCR for the rapid identification of Mycobacterium intracellulare; Kitaura H et al.; The complete nucleotide sequence of alpha antigen secreted from Mycobacterium intracellulare (ATCC13950) was determined . The gene encoded 330 amino acids including 40 amino acids for signal peptide, followed by 290 amino acids for a mature protein with molecular mass 30,645 Da . The cloned gene was expressed in Escherichia coli by using an E . coli expression vector . Based on these results, the feasibility of rapid identification of M . intracellulare by two step polymerase chain reaction (PCR) was demonstrated.

Biochem Biophys Res Commun, 1993 Nov 15, 196(3), 1376 - 82
Binding site of annexin XI on the calcyclin molecule; Watanabe M et al.; We purified rabbit calcyclin of S100 family protein and a calcyclin associated protein which has proved to be a novel annexin, annexin XI . Using a co-precipitation assay of annexin XI with phospholipid, the binding site of annexin XI on calcyclin was examined . The peptide fragment of calcyclin, CNBr-3 (residues 1-57), digested with cyanogen bromide completely inhibited the interaction of native calcyclin with annexin XI, while CNBr-1 (residues 83-90) and CNBr-2 (residues 58-82) did not affect the binding . We then constructed and expressed recombinant cDNAs for wild type and four different deletion mutants lacking N-terminal portions . The wild type (wt) and mt1 mutant lacking three amino acids from N-terminal bound to annexin XI with phosphatidylserine and Ca2+, whereas mt2, mt3 and mt4 with seven, twelve and eighteen amino acids deleted, respectively, did not bind to annexin XI . Moreover, the truncated mutant from residues 4 to 7 (mt5) decreased the binding capacity . These observations suggest that four amino acids (residues 4-7) at the N-terminal portion of calcyclin play an important role in the interaction of calcyclin with annexin XI.

Biochem Biophys Res Commun, 1993 Nov 15, 196(3), 1343 - 8
Modulation of human endothelial cell tetrahydrobiopterin synthesis by activating and deactivating cytokines: new perspectives on endothelium-derived relaxing factor; Schoedon G et al.; Endothelial cells, through soluble mediators, play an important role in the regulation of the vascular tone . In the present paper we investigated whether endothelial tetrahydrobiopterin (BH4), an obligatory cofactor of nitric oxide (NO) synthase, could serve as such a regulatory mediator . By studying the human vascular endothelial hybrid cells EA hy 926, we found that 1) BH4 biosynthesis is highly regulated (70-fold) by activating and deactivating cytokines; that 2) up to 90% of the induced BH4 is released by activated endothelium, and 3) while intracellular BH4 could be related to cyclic GMP concentrations within the endothelial cells, the bulk of BH4 (up to 90 pmol/10(6) cells) appears not to serve endothelial cell requirements . Activation and deactivation of BH4 synthesis by cytokines was paralleled by other endothelial cell responses reflecting their activity . We propose that BH4 serves as an endothelial mediator augmenting the activity of cytokine-inducible NO synthase in vascular smooth muscle cells . BH4 could thereby account for endothelium-derived relaxing factor activity.

Biochem Biophys Res Commun, 1993 Nov 15, 196(3), 1255 - 60
Expression of the fungal cytochrome P-450nor cDNA in Escherichia coli; Obika K et al.; We succeeded in expressing the unique cytochrome P-450 (P-450), nitric oxide reductase (P-450nor), in Escherichia coli by utilizing P-450nor cDNA and the expression vector pKK233-2 or pUC19 . The expression was confirmed by Western blot analysis and by detecting the unique nitric oxide reductase activity . The expressed protein was recovered in the soluble fraction of transformed cells, supporting that P-450nor is the first soluble P-450 of eukaryote . The results also provided conclusive evidence that the enzymatic reaction depends solely on P-450nor without support of any other electron transferring components.

Biochem J, 1993 Nov 15, 296 ( Pt 1), 235 - 43
Mode of action, kinetic properties and physicochemical characterization of two different domains of a bifunctional (1-->4)-beta-D-xylanase from Ruminococcus flavefaciens expressed separately in Escherichia coli; Garcia-Campayo V et al.; Two catalytic domains, A and C, of xylanase A (XYLA) from Ruminococcus flavefaciens were expressed separately as truncated gene products from lacZ fusions in Escherichia coli . The fusion products, referred to respectively as XYLA-A1 and XYLA-C2, were purified to homogeneity by anion-exchange chromatography and chromatofocusing . XYLA-A1 was isoelectric at pH 5.0 and had a molecular mass of 30 kDa, whereas XYLA-C2 had a pI of 5.4 and a molecular mass of 44 kDa . The catalytic activity shown by both domains was optimal at 50 degrees C, but XYLA-A1 was more sensitive than XYLA-C2 to temperatures higher than the optimum . XYLA-A1 showed a higher sensitivity to pH than XYLA-C2 . The enzyme activity of both domains was completely inactivated in the presence of copper or silver ions and partially inactivated by iron or zinc ions . Neither domain was active on xylo-oligosaccharides shorter than xylopentaose: the rate of degradation of longer xylo-oligosaccharides (degree of polymerization 5-10) increased as the chain length increased . Analysis of the products of hydrolysis of xylo-oligosaccharides and xylan (arabinoxylan) polysaccharide showed that the two domains differed in their modes of action: xylobiose was the shortest product of the hydrolysis . With oat spelt xylan as substrate, XYLA-A1 activity was apparently restricted to regions where xylopyranosyl residues did not carry arabinofuranosyl substituents, whereas XYLA-C2 was able to release hetero-oligosaccharides carrying arabinofuranosyl residues . Neither domain was able to release arabinose from oat spelt xylan.

Biochem J, 1993 Nov 15, 296 ( Pt 1), 209 - 15
Expression of functional human retinol-binding protein in Escherichia coli using a secretion vector; Sivaprasadarao A et al.; In order to express human serum retinol-binding protein (sRBP) in Escherichia coli in a form that is structurally indistinguishable from the native protein, we placed the coding sequence of the RBP cDNA next to that of the outer membrane protein A (OmpA) signal sequence in the secretion vector, pIN-III-OmpA1 . However, this construct did not generate detectable expression of RBP in E . coli . When the DNA fragment consisting of the ribosome-binding site and the OmpA-RBP fusion sequence was subcloned downstream to the T7 promoter of pKS-Bluescript, however, the resultant construct (pOmp-RBP2) gave low but detectable secretion of RBP into the periplasm . Deletion of the 3' untranslated region of the RBP cDNA (pOmp-RBP3) further improved the expression (by approx . 20-fold) . After charging with retinol, the secreted RBP was purified from the periplasm on a transthyretin-affinity resin . The purified protein exhibited all the three molecular recognition properties characteristic of sRBP, i.e . it interacted with retinol, transthyretin and its cell-surface receptor . Comparison of the receptor binding properties of the recombinant RBP (rRBP) with those of the serum protein revealed that while the affinity of rRBP is similar to sRBP (50 +/- 20 nM), the Bmax of the rRBP is about 6-8-fold higher . This indicates that a major proportion of RBP, isolated from serum, is incapable of interacting with the receptor.

Biochem J, 1993 Nov 15, 296 ( Pt 1), 189 - 97
Reversible modification of rat liver glutathione S-transferase 3-3 with 1-chloro-2,4-dinitrobenzene: specific labelling of Tyr-115; Liu LF et al.; Rat liver glutathione S-transferase 3-3 (GST, EC 2.5.1.18), a triple mutant with all three cysteine residues replaced with serine (CallS) and a quadruple mutant with a Tyr-115 to phenylalanine substitution on CallS (CallSY115F) were overexpressed in Escherichia coli under the control of a phoA promoter . Using this system, we obtained over 35 mg of fully active pure protein/litre of cell medium . GST 3-3 and CallS mutant were modified with 1-chloro-2,4-dinitrobenzene (CDNB), a model substrate for the enzyme, in the absence of GSH . Dinitrophenol, but not S-methylglutathione, inhibits this process . The dinitrophenyl groups are readily removed from the enzyme with GSH, but much more slowly with dithiothreitol . Results from peptide mapping and amino acid sequence analyses indicate that CDNB modifies the cysteine residues and Tyr-115 on wild-type GST 3-3, but only Tyr-115 on CallS . In addition, CDNB cannot modify the CallSY115F mutant . We propose that Tyr-115 is located at or near the H-site of GST 3-3.

Proc Natl Acad Sci U S A, 1993 Nov 15, 90(22), 10861 - 5
Cell growth and lambda phage development controlled by the same essential Escherichia coli gene, ftsH/hflB; Herman C et al.; The lambda phage choice between lysis and lysogeny is influenced by certain host functions in Escherichia coli . We found that the frequency of lambda lysogenization is markedly increased in the ftsH1 temperature-sensitive mutant . The ftsH gene, previously shown to code for an essential inner membrane protein with putative ATPase activity, is identical to hflB, a gene involved in the stability of the phage cII activator protein . The lysogenic decision controlled by FtsH/HflB is independent of that controlled by the protease HflA . Overproduction of FtsH/HflB suppresses the high frequency of lysogenization in an hflA null mutant . The FtsH/HflB protein, which stimulates cII degradation, may be a component of an HflA-independent proteolytic pathway, or it may act as a chaperone, maintaining cII in a conformation subject to proteolysis via such a pathway . Suppressor mutations of ftsH1 temperature-sensitive lethality, located in the fur gene (coding for the ferric uptake regulator), did not restore FtsH/HflB activity with respect to lambda lysogenization.

Proc Natl Acad Sci U S A, 1993 Nov 15, 90(22), 10583 - 7
A second hepatitis C virus-encoded proteinase; Grakoui A et al.; Host and viral proteinases are believed to be required for the production of at least nine hepatitis C virus (HCV)-specific polyprotein cleavage products . Although several cleavages appear to be catalyzed by host signal peptidase or the HCV NS3 serine proteinase, the enzyme responsible for cleavage at the 2/3 site has not been identified . In this report, we have defined the 2/3 cleavage site and obtained evidence which suggests that this cleavage is mediated by a second HCV-encoded proteinase, located between aa 827 and 1207 . This region encompasses the C-terminal portion of the 23-kDa NS2 protein, the 2/3 cleavage site, and the serine proteinase domain of NS3 . Efficient processing at the 2/3 site was observed in mammalian cells, Escherichia coli, and in plant or animal cell-free translation systems in the absence of microsomal membranes . Cleavage at the 2/3 site was abolished by alanine substitutions for NS2 residues His-952 or Cys-993 but was unaffected by several other substitution mutations, including those that inactivate NS3 serine proteinase function . Mutations abolishing cleavage at the 2/3 site did not block cleavage at other sites in the HCV polyprotein . Cotransfection experiments indicate that the 2/3 site can be cleaved in trans, which should facilitate purification and further characterization of this enzyme.

Proc Natl Acad Sci U S A, 1993 Nov 15, 90(22), 10558 - 62
Mechanistic aspects of genome-wide demethylation in the preimplantation mouse embryo; Kafri T et al.; Gene-specific methylation patterns in mammals play a role in a variety of biological processes in the embryo and adult tissues . These patterns are established during embryo development by a process that involves genome-wide demethylation in the morula and de novo methylation in the pregastrula . To elucidate the mechanism of demethylation in the early mouse embryo, we have injected mouse zygotes with gene sequences that were methylated in vitro by Hpa II methylase and analyzed the methylation status of specific sites in blastocyst DNA . Because it had been propagated in Escherichia coli, the DNA used for these injections was also methylated at adenine residues in GATC sites . This allowed us to eliminate fully methylated, unintegrated DNA by Dpn I digestion and fully unmethylated, integrated DNA that underwent several rounds of replication by Mbo I digestion . The integrated, originally injected DNA strands were in a hemimethylated state and survived this treatment . The methylation status of Hpa II sites in these molecules was analyzed by Hpa II digestion of the genomic DNA isolated from blastocysts, followed by PCR amplification using appropriate primers . The results demonstrate that demethylation is achieved by an active mechanism and that specific sites in imprinted genes escape demethylation, maintaining a methylated state throughout preimplantation development.

Proc Natl Acad Sci U S A, 1993 Nov 15, 90(22), 10444 - 8
Production and fluorescence-activated cell sorting of Escherichia coli expressing a functional antibody fragment on the external surface; Francisco JA et al.; We have expressed a single chain Fv (scFv) antibody fragment, consisting of the variable heavy and variable light domains from two separate anti-digoxin monoclonal antibodies, on the external surface of Escherichia coli by fusing it to an Lpp-OmpA hybrid previously shown to direct heterologous proteins to the cell surface . This scFv fusion was expressed at a high level and was shown to bind the hapten with high affinity and specificity . Whole cell ELISAs, fluorescence microscopy, protease sensitivity, and flow cytometry all confirmed that the scFv was anchored on the outer membrane and was accessible on the surface . Utilizing fluorescence-activated cell sorting, we were able to specifically enrich scFv-producing cells from a 10(5)-fold excess of control cells in only two steps . The expression of antibody fragments on the surface of E . coli is being evaluated as an attractive method for the in vitro production and selection of useful antibody fragments.

Eur J Biochem, 1993 Nov 15, 218(1), 205 - 11
Recombinant coho salmon insulin-like growth factor I . Expression in Escherichia coli, purification and characterization; Moriyama S et al.; Recombinant coho salmon insulin-like growth factor I (rsIGF-I) was produced in Escherichia coli, purified and characterized . The rsIGF-I expression vector was constructed by polymerase chain reaction and cloning into a plasmid containing a phage T7 RNA polymerase promoter . The rsIGF-I was recovered from bacterial inclusion bodies, solubilized under reducing conditions, immediately refolded, then fractionated by a two-step ion-exchange chromatography on DEAE-52 and Mono-S columns . It was further purified by HPLC on a reverse-phase Asahi-Pak C4P-50 C4 column . Purification of rsIGF-I was monitored by SDS/PAGE and immunoblot with anti-{human somatomedin C (SM C)/IGF-I} serum . The rsIGF-I appeared as a single band with molecular mass of 7 kDa, the same size as recombinant human IGF-I (rhIGF-I) and cross-reacted with anti-(human SM C/IGF-I) serum . The amino acid sequence of rsIGF-I contained an NH2-terminal methionine residue followed by the sequence predicted for mature sIGF-I . At concentrations in the range 3.9-250 ng/ml, rsIGF-I significantly stimulated sulfate uptake by the cultured branchial cartilage of coho salmon . The stimulatory effect of rsIGF-I was concentration dependent and slightly more potent than that of rhIGF-I at the highest concentration tested.

Arch Biochem Biophys, 1993 Nov 15, 307(1), 46 - 51
Amino acid substitutions at position 73 in motif 2 of Escherichia coli alanyl-tRNA synthetase; Filley SJ et al.; Lysine73, located in the adenylate synthesis domain of Escherichia coli alanyl-tRNA synthetase (AlaRS), was previously indicated to be an important residue for the interaction of this enzyme with the acceptor stem of its cognate tRNA (tRNA(Ala)) . Replacement of this residue with glutamine produced a reduction in the catalytic efficiency of AlaRS in the aminoacylation assay, primarily through an increase in the apparent KM for tRNA(Ala) {Hill, K., and Schimmel, P . (1989) Biochemistry 28, 2577-2586} . Studies on the role of residue 73 in the interaction of AlaRS with its substrates have now been extended using the additional substitutions of asparagine, alanine, and glutamate . Analysis of each substituted enzyme in the ATP-PPi exchange and aminoacylation reactions reveals kinetic characteristics similar to those obtained with the glutamine substitution, except that the glutamate substitution causes a fivefold decrease in the affinity for alanine . These data verify that the positive charge on lysine 73, rather than its hydrophilic side chain, is of importance in the binding of the cognate tRNA, but do not support an ionic interaction of this residue with the RNA phosphate backbone . The collective data support the prediction that lysine73 is in motif 2 of AlaRS {Cusack, S., Hartlein, M., and Leberman, R . (1991) Nucleic Acids Res . 19, 3489-3498}, but question the predicted alignment of this motif with other enzymes in its class.

Arch Biochem Biophys, 1993 Nov 15, 307(1), 193 - 9
Overexpression, purification, and kinetic characterization of a carboxyl-terminal-truncated yeast squalene synthetase; LoGrasso PV et al.; Yeast squalene synthetase which has been truncated by 24 amino acids at the C-terminus has been overexpressed in Escherichia coli and constitutes approximately 20% of the total soluble cell protein . For the first time, milligram quantities of this essential enzyme in the cholesterol biosynthetic pathway have been purified to near homogeneity by ammonium sulfate precipitation and Mono Q anion-exchange chromatography so that the steady-state rate constants could be measured . A combination of 10% methanol, 10% glycerol, 30 mM octyl-beta-D-glucopyranoside, 0.4% Brij-58, and 1 mM dithiothreitol in 25 mM sodium phosphate, pH 7.4, was essential for the stability and maximal enzyme activity of the near homogeneous enzyme . Kinetic analysis indicated a Km for farnesyl pyrophosphate of 2.5 microM, suggesting fairly tight binding of farnesyl pyrophosphate to truncated yeast squalene synthetase . The turnover number, kcat, for the conversion of farnesyl pyrophosphate to squalene was 0.53 s-1, and the apparent second order rate constant, kcat/Km, was 2.1 x 10(5) M-1 s-1, indicating a relatively slow conversion of farnesyl pyrophosphate to squalene and a low specificity constant for this enzyme . In addition, Km for NADPH and NADH was 0.5 and 3.6 mM, respectively . Moreover, truncated yeast squalene synthetase shows a preference for NADPH over NADH as reflected in the sevenfold higher kcat/Km value for NADPH similar to that for the native enzyme.

Am J Epidemiol, 1993 Nov 15, 138(10), 849 - 69
Epidemiologic studies of Escherichia coli diarrheal infections in a low socioeconomic level peri-urban community in Santiago, Chile; Levine MM et al.; The incidence of diarrhea due to six categories of diarrheogenic Escherichia coli was determined in two pediatric cohorts in a low socioeconomic level community in Santiago, Chile, with access to chlorinated water . An age cross-sectional cohort of 340 children aged birth to 47 months was assembled . A newborn cohort was assembled by enrolling 10-12 newborns monthly for 12 months . Episodes of diarrhea were detected by twice weekly household visits . E . coli from stool cultures of cases and matched controls were hybridized with DNA probes specific for enterotoxigenic, enteroinvasive, enteropathogenic, enterohemorrhagic, enteroaggregative, and diffuse adherence E . coli . Overall, the incidence of diarrhea was low (2.1 episodes/infant/year) . Nevertheless, a putative E . coli enteropathogen was found in a large proportion of diarrheal episodes, particularly during the summer . In both cohorts, enterotoxigenic E . coli were important pathogens . Enteropathogenic E . coli were incriminated during the first year of life in the newborn cohort, where they were found significantly more often in cases (p = 0.021) than in controls; beyond this age, isolation rates were similar . In contrast, the relative risk of isolation of diffuse adherence E . coli increased with age in the age cross-sectional cohort, where, overall, the difference in rate of isolation between cases and controls was significant (p = 0.0024) . Enteroinvasive and enterohemorrhagic E . coli were isolated infrequently . Enteroaggregative E . coli were encountered equally in cases and controls . Facile transmission of E . coli enteropathogens is occurring in this community despite the availability of potable waterPIP: Researchers conducted an age cross sectional cohort analysis of 340 0-47 month old children and newborn cohort analysis of 144 newborns to determine the diarrheogenic Escherichia coli incidence in Santa Julia, a low socioeconomic community in Santiago, Chile . Children in the age cross sectional cohort had age, sex, and sector matched controls . The newborns had sex matched controls . A public health nurse or nurse auxiliary visited the household of each subject 2 times a week to detect diarrhea episodes . Between December 1986 and February 1990, the age cross sectional cohort had 1178 episodes of diarrhea and the newborn cohort had 674 episodes . The overall diarrhea incidence was only 2.1 episodes/child/year . An E . coli enteropathogen was isolated in many of these episodes, especially during the summer (e.g . enterotoxigenic E . coli {ETEC}, 2.2 cases/month in summer vs . 0.4 cases/month in winter; p = .00001) . Diffuse adherence E . coli (DAEC) and enteropathogenic E . coli (EPEC) infections also peaked in the summer . ETEC contributed greatly to diarrheal episodes in both cohorts . Among newborns, EPEC was isolated significantly more often in cases than controls during the 1st 12 months of life (6.7% vs . 2.5%; p = .021) . After 1 year, however, E . coli isolation rates were essentially the same . On the other hand, in the age cross sectional cohort, the relative risk of isolation of DAEC rose with age (e.g., 1.1 for 0.11 months, 1.4 for 36-47 months, and 2.1 for = or 48 months) . In the same cohort, DAEC infections were much more common in cases than controls (16.6% vs . 11.9%; p = .0024) . Enteroinvasive and enterohemorrhagic E . coli were the most rarely isolated E . coli types . No difference in the isolation rate of enteroaggregative E . coli existed between cases and controls . Since most households in Santa Julia have access to potable water (68%) and an indoor toilet (64%), food contamination were likely the vehicles of E . coli transmission because more than 50% of households do not have a refrigerator .

J Biol Chem, 1993 Nov 15, 268(32), 24498 - 505
Kallistatin: a novel human serine proteinase inhibitor . Molecular cloning, tissue distribution, and expression in Escherichia coli; Chai KX et al.; We have recently purified a novel human serine proteinase inhibitor (serpin), designated as kallistatin, which binds to tissue kallikrein and inhibits kallikrein's kininogenase and amidolytic activities . In the present studies, we have cloned a full-length cDNA encoding kallistatin from human liver RNA by the polymerase chain reaction . The cDNA is 1284 base pairs in length and encodes 427 amino acid residues, including a 26-residue signal peptide and a 401-residue mature peptide . The translated amino acid sequence of kallistatin matches with the protein sequence and shares 44-46% sequence identity with human alpha 1-antichymotrypsin, protein C inhibitor, corticosteroid-binding globulin, alpha 1-antitrypsin, thyroxin-binding globulin, and rat kallikrein-binding protein . Kallistatin is a new member of the serpin superfamily with a unique reactive site P1-P1' of Phe-Ser . Four potential glycosylation sites are found in the translated amino acid sequence of kallistatin . In a Southern blot analysis following reverse transcription and polymerase chain reaction, kallistatin was found to be expressed in human liver, stomach, pancreas, kidney, aorta, testes, prostate, artery, atrium, ventricle, lung, renal proximal tubular cell, and a colonic carcinoma cell line T84 . A genomic Southern blot using the full-length kallistatin cDNA probe revealed simple banding patterns suggesting the gene encoding kallistatin is single-copied . The kallistatin cDNA encoding the mature peptide was expressed in Escherichia coli . The recombinant kallistatin forms an SDS-stable complex with 125I-human tissue kallikrein and has a molecular mass of 40 kDa . The cloning of human kallistatin cDNA established the identity of the novel kallikrein inhibitor and its expression in a functional form in E . coli provides means for studying its structure-function relationship through protein engineering.

J Biol Chem, 1993 Nov 15, 268(32), 24491 - 7
Transcriptional regulation of puc operon expression in Rhodobacter sphaeroides . Involvement of an integration host factor-binding sequence; Lee JK et al.; The putative overlapping consensus sequences (-129 to -105) for binding of fumarate nitrate reductase regulator- and integration host factor (IHF)-like proteins to puc operon upstream DNA of Rhodobacter sphaeroides was protected from DNase I digestion by purified Escherichia coli IHF . The binding of E . coli IHF to the purported IHF-binding site in the puc upstream DNA is highly sequence-specific . The recorded binding affinity was significantly lower than that of E . coli IHF to the lambda attP site . Employing site-directed changes in the DNA sequence within the -129 to -105 region, a loss in IHF binding, as monitored through gel retardation analysis, was correlated with alterations in puc operon expression monitored through the use of puc::lacZ transcriptional fusions . These results suggest that the IHF-binding site is involved in repression of puc operon transcription by oxygen as well as modulation of puc operon transcription levels by incident light intensity . Mutations specific to the upstream half of the putative fumarate nitrate reductase regulator-binding site of the puc upstream DNA did not show any physiological effects under the experimental conditions employed . Taken together, these studies reveal that the DNA sequence between -129 to -105 may involve facilitation of the interaction between upstream and downstream cis-acting regulatory sequences involved in puc operon expression.

J Biol Chem, 1993 Nov 15, 268(32), 24361 - 6
Molecular cloning of fish Pit-1 cDNA and its functional binding to promoter of gene expressed in the pituitary; Yamada S et al.; Pit-1 is a pituitary-specific transcription factor responsible for activating growth hormone (GH) and prolactin genes . Here, we describe the isolation of a rainbow trout cDNA clone that contains the entire Pit-1 coding region . The deduced amino acid sequence contains 358 residues, encoding a 39-kDa protein . Comparison of the protein sequences of the rainbow trout and rat Pit-1 shows that the 160 residue POU domain at the C terminus is highly conserved (86% identical) . However, homology is much weaker in the N-terminal region (56% identical), and the rainbow trout Pit-1 contains segments of 29 and 33 amino acids that are not present in rat Pit-1 . The protein produced by expression of rainbow trout Pit-1 cDNA in Escherichia coli binds specifically to at least four sites in the rainbow trout GH gene promoter . Moreover, we demonstrate that the promoter region of salmon somatolactin gene, which belongs to the GH prolactin gene family and is also expressed specifically in the pituitary, has at least five rainbow trout Pit-1 binding sites . The consensus sequence of these binding sites closely matches the 9-base pair motif, (T/A)(T/A)TATNCAT, recognized by rat Pit-1 . Rainbow trout Pit-1 specifically activates rainbow trout GH promoter fusion gene expression, confirming the ability of Pit-1 to bind in a transcriptionally active conformation in GH gene promoter.

J Biol Chem, 1993 Nov 15, 268(32), 24190 - 6
The purification and characterization of recombinant yeast dolichyl-phosphate-mannose synthase . Site-directed mutagenesis of the putative dolichol recognition sequence; Schutzbach JS et al.; Yeast dolichyl-phosphate-mannose synthase was purified from cultures of Escherichia coli carrying the gene for this enzyme in a high expression vector . The synthase contains a highly conserved hydrophobic amino acid sequence proposed to be involved in the recognition of dolichols (Albright, C . F., Orlean, P., and Robbins, P . W . (1989) Proc . Natl . Acad . Sci . U . S . A . 86, 7366-7369) and amino acid residues in this sequence were altered by site-directed mutagenesis . Conservative substitutions had no effect on the affinity of the enzyme for dolichyl-P . The substitution of asparagine for isoleucine at position 253 resulted in higher values for the apparent Km for Dol-P when assayed in detergent solutions, but this substitution had no effect on Km when the enzyme was reconstituted with phosphatidylethanolamine . Enzyme containing a deletion of the entire putative dolichol recognition sequence retained catalytic activity . The apparent Km for Dol-P was increased when this enzyme was assayed in detergent solution but was the same as wild type enzyme when reconstituted in phosphatidylethanolamine . These results suggest that the amino acid composition and sequence of the conserved domain are not critically important for the recognition and binding of Dol-P when the synthase is present in a lipid matrix.

J Biol Chem, 1993 Nov 15, 268(32), 24156 - 62
Expression of active human DNA ligase I in Escherichia coli cells that harbor a full-length DNA ligase I cDNA construct; Teraoka H et al.; A recombinant plasmid for expression of full-length human DNA ligase I (phLig-I) was constructed in a plasmid/phage chimeric vector, pTD-T7N, which was derived from pUC118 by oligonucleotide-directed mutagenesis . The insert contained a 2757-base pair coding sequence for a whole human DNA ligase I and an extra ACC codon adjacent to the ATG initiation codon . This ACC codon was required for achieving high levels of expression of full-length DNA ligase I in Escherichia coli strain BL21 . The recombinant plasmid, which was designed to exploit the T7 late promoter and the ATG initiation codon for beta-galactosidase was transfected into E . coli BL21 cells that express T7 RNA polymerase . The recombinant clone produced relatively high levels of DNA ligase I with a molecular mass of 130 kDa, as estimated by SDS-polyacrylamide gel electrophoresis . The DNA ligase was purified to near-homogeneity by the two-step column chromatographic procedure from BLphLig-I cells that had been induced with isopropyl beta-D-thiogalactoside . The specific activity, chromatographic behavior, kinetic properties, molecular mass, and antigenicity of the recombinant human DNA ligase I were indistinguishable from those of purified mammalian DNA ligase I . Metabolically labeling experiments with 32P(i) indicate that the recombinant DNA ligase I was present as an enzyme-AMP reaction intermediate, but not as a phosphoprotein, in the E . coli cells.

J Biol Chem, 1993 Nov 15, 268(32), 24005 - 11
Cytosine deaminase . The roles of divalent metal ions in catalysis; Porter DJ et al.; Cytosine deaminase (CDase, EC 3.5.4.1) isolated from Escherichia coli contains a catalytically essential divalent metal ion . Fe2+ was efficiently removed from the enzyme with o-phenanthroline to yield an apoenzyme with less than 5% of the catalytic activity of native enzyme . The time courses for inactivation and for removal of Fe2+ from the enzyme by o-phenanthroline were similar . Apoenzyme reconstituted with Fe2+, Mn2+, Co2+, or Zn2+ (M2+CDase) had kcat values of 185, 88, 50, and 32 s-1, respectively . The Km values of these M2+CDases for cytosine were similar (0.22-0.39 mM) . Cytosine potently inhibited reconstitution of the apoenzyme with Fe2+ . Fe2+CDase was rapidly inactivated by 1 mM H2O2 (t1/2 < 1 s), whereas Mn2+CDase, Co2+CDase, and Zn2+CDase were not inactivated by H2O2 . CDase was also inhibited by excess divalent cations . Cu2+ and Zn2+ reversibly inhibited Fe2+CDase activity with inhibition constants of 1.8 and 5.8 microM, respectively . Cu2+ dissociated slowly from the secondary binding on CDase with a rate constant of 2 x 10(-3) s-1.

J Biol Chem, 1993 Nov 15, 268(32), 23986 - 90
Addition of an endoplasmic reticulum retrieval sequence to ricin A chain significantly increases its cytotoxicity to mammalian cells; Wales R et al.; An Escherichia coli expression system was used to produce recombinant ricin A chain (RTA) and RTA modified either by the addition of a carboxyl-terminal endoplasmic reticulum retrieval sequence Lys-Asp-Glu-Leu (RTAKDEL) or a nonfunctional analogue Lys-Asp-Glu-Ala (RTAKDEA) . These RTA molecules can enter mammalian cells by fluid phase endocytosis . RTAKDEL was significantly more cytotoxic than either RTA or RTAKDEA to both Vero cells and HeLa cells (250- and 10-fold, respectively), despite the fact that all these RTA molecules had comparable enzymatic activities . This difference did not result from KDEL-mediated binding of RTAKDEL to the cell surface . Enhanced cytotoxicity could be correlated with an increased level of ribosome inactivation, measured as the RTA-catalyzed depurination of 28 S ribosomal RNA . These results indicate that the added KDEL sequence facilitated RTA entry into the cytosol . We propose that interaction with the intracellular KDEL receptor promotes retrograde transport of the toxin to the endoplasmic reticulum, where translocation of RTA into the cytosol occurs.

J Biol Chem, 1993 Nov 15, 268(32), 23972 - 80
Phosphorylation/dephosphorylation of the receiver module at the conserved aspartate residue controls transphosphorylation activity of histidine kinase in sensor protein ArcB of Escherichia coli; Iuchi S; A membrane sensor protein, ArcB, recognizes anaerobic environments and signals the information to the cognate regulator, ArcA . This in vitro study presents the process and control of the signal-transduction phosphorylation . In the presence of ATP, the ArcB transmitter module undergoes autophosphorylation and then transfers the phosphoryl group to its own receiver module as well as to the ArcA receiver module . Results suggest that the phosphoryl group of the ArcB receiver module is released by an intrinsic phosphatase activity . D-Lactate inhibits the phosphatase activity that removes phosphoaspartate groups from the receiver module in ArcB, and the associated increase in phosphorylation of this module leads to an activation of transphosphorylation of subsequently added phosphohistidine groups on ArcB to the receiver module of ArcA . A similar effect was also observed in the presence of pyruvate, acetate, or NADH . Conversely, the non-phosphorylated ArcB receiver module completely inhibits the intermolecular transphosphorylation . Thus, the phosphorylation state of the ArcB receiver module controls signal transduction from ArcB to ArcA . Since the intrinsic phosphatase activity is inhibited by cellular metabolites that increasingly accumulate by anaerobiosis, the enzyme portion of ArcB may be involved in sensing anaerobic environments through cellular metabolites in vivo.

J Biol Chem, 1993 Nov 15, 268(32), 23964 - 71
Alternative splicing in a novel tyrosine phosphatase gene (DPTP4E) of Drosophila melanogaster generates two large receptor-like proteins which differ in their carboxyl termini; Oon SH et al.; A novel Drosophila receptor-like protein tyrosine phosphatase gene, DPTP4E, was isolated and characterized . DPTP4E, located at cytological position 4E1-2, is comprised of 10 exons; its RNA products are widely expressed during embryonic development, including the developing central nervous system . DPTP4E produces three major developmentally regulated transcripts of 6.5, 7.0, and 7.5 kilobases . The two major embryonic transcripts arise as the result of the alternative splicing of exon IX; as a consequence, two proteins (200 and 183 kDa) are produced which differ in their carboxyl-terminal sequences . The deduced extracellular domain, which lies between two putative hydrophobic transmembrane segments, contains 11 fibronectin type III-like repeats and 25 putative N-glycosylation sites . A single conserved protein tyrosine phosphatase (PTPase) catalytic domain, which shows a high level of amino acid identity to the Drosophila PTPase DPTP10D and human HPTP beta, is found in the predicted intracellular domain; this PTPase domain, when expressed as a fusion protein in Escherichia coli, exhibits PTPase activity . The possible implications of these findings are discussed.

J Biol Chem, 1993 Nov 15, 268(32), 23876 - 80
Inactivation of rabbit muscle glycogen synthase by glycogen synthase kinase-3 . Dominant role of the phosphorylation of Ser-640 (site-3a); Wang Y et al.; Rabbit skeletal muscle glycogen synthase, a rate-limiting enzyme for glycogen biosynthesis, is regulated by multisite phosphorylation . The protein kinase glycogen synthase kinase 3 (GSK-3) phosphorylates 4 Ser residues (Ser-640, Ser-644, Ser-648, and Ser-652; also known as sites 3a, 3b, 3c, and 4, respectively) at the COOH terminus of the subunit . Phosphorylation of these sites by GSK-3 is sequential, from COOH- to NH2-terminal, and is wholly dependent on prior phosphorylation by casein kinase II at Ser-656 (site 5) . Expression in Escherichia coli was used to generate mutant forms of glycogen synthase, S640A, S644A, and S648A, in which site 3a, site 3b, or site 3c was changed to Ala, respectively . The purified enzymes had -/+ glucose-6-P activity ratios in the range of 0.8-0.9 . Phosphorylation by casein kinase II and GSK-3 gave results consistent with the model of obligate sequential action of GSK-3 . Phosphorylation at site 5, sites 4 + 5, or sites 3c + 4 + 5 had no measurable effect on activity . When sites 3b + 3c + 4 + 5 were phosphorylated, modest inactivation resulted . Additional phosphorylation at site 3a, however, was potently inactivating, reducing the -/+ glucose-6-P activity ratio to 0.1 and increasing the glucose-6-P concentration needed for half-maximal activation by an order of magnitude . Introduction of each additional phosphate, in the order site 4, 3c, 3b, and 3a, caused an incremental reduction in the mobility of the subunit when analyzed by polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate . The results of this study demonstrate that GSK-3 phosphorylation of site 3a (Ser-640), and to a lesser extent, site 3b, correlates with inactivation of glycogen synthase by GSK-3 . Evidence is also presented for an allosteric mechanism of inactivation whereby modification of one subunit influences the activity state of adjacent subunits.

J Biol Chem, 1993 Nov 15, 268(32), 23837 - 42
Role of the conserved Lys-X-Gly-Gly sequence at the ADP-glucose-binding site in Escherichia coli glycogen synthase; Furukawa K et al.; Although bacterial and mammalian glycogen synthases differ in the primary structure and specificity for glucosyl donor, lysyl residues identified at their substrate-binding sites by affinity labeling are present in a conserved tetrapeptide sequence, Lys-X-Gly-Gly, where X is a residue not conserved (Tagaya, M., Nakano, K., and Fukui, T . (1985) J . Biol . Chem . 260, 6670-6676; Furukawa, K., Tagaya, M., Inouye, M., Preiss, J., and Fukui, T . (1990) J . Biol . Chem . 265, 2086-2090) . To elucidate the functional role of this conserved sequence, Lys-15, Gly-17, and Gly-18 in Escherichia coli glycogen synthase have been replaced by other amino acid residues via site-directed mutagenesis . Kinetic analyses of the Lys-15 mutant enzymes showed that the epsilon-amino group of Lys-15 is mainly involved in binding of the phosphate moiety adjacent to the glycosidic linkage in the substrate ADP-glucose, presumably through an ionic interaction . The mutant enzyme in which Ala was substituted for Gly-17 had a catalytic rate constant 3 orders of magnitude smaller than that of the wild-type enzyme with a slightly increased Michaelis constant for ADP-glucose, whereas the Gly-18-->Ala mutant showed a rate constant only 3.2-fold smaller . In addition, mutations of Gly-17 and Gly-18 resulted in marked changes in the reactivity of Lys-15 with affinity labeling reagents . These results suggest that the 2 glycyl residues in the conserved Lys-X-Gly-Gly sequence, in particular the one closer to the ADP-glucose-binding lysyl residue, participate in catalysis by assisting conformational change(s) of the active site or stabilizing the transition state.

J Biol Chem, 1993 Nov 15, 268(32), 23824 - 9
Cloning and characterization of a unique elastolytic metalloproteinase produced by human alveolar macrophages; Shapiro SD et al.; Human alveolar macrophages have the capacity to degrade elastin . As an approach to define proteinases responsible for this activity, we recently cloned a murine macrophage elastase cDNA and demonstrated that it is a member of the matrix metalloproteinase gene family (Shapiro, S . D., Griffin, G . L., Gilbert, D . J., Jenkins, N . A., Copeland, N . G., Welgus, H . G., Senior, R . M., and Ley, T . J . (1992) J . Biol . Chem . 267, 4664-4671) . We now report that there is a human orthologue of murine macrophage metalloelastase that we call human macrophage metalloelastase (HME) . The full-length HME cDNA spans 1.8 kilobases and contains an open reading frame of 1410 base pairs; the predicted molecular mass of the HME proenzyme is 54 kDa . HME mRNA and protein were detected in human alveolar macrophages . Similar to murine macrophage metalloelastase, HME readily undergoes NH2- and COOH-terminal processing to a mature 22-kDa form . Both recombinant HME expressed in Escherichia coli and native HME derived from human alveolar macrophage-conditioned media degraded insoluble elastin . HME is a unique human metalloproteinase that possesses elastolytic activity and is expressed in alveolar macrophages; it is therefore a candidate molecule for the causation of diseases characterized by damage to the extracellular matrix.

Gene, 1993 Nov 15, 133(2), 291 - 4
Production of human plasma retinol-binding protein in Escherichia coli; Wang TT et al.; We designed a polymerase chain reaction (PCR) primer pair which allowed us to clone the cDNA coding for the human plasma retinol-binding protein (hRBP) into an Escherichia coli expression vector . Production of hRBP was confirmed by probing Western blots with antisera against plasma hRBP . Purification and characterization of the E . coli-produced plasma hRBP are also described . The availability of this expression system makes it possible to obtain large quantities of hRBP to facilitate our continuing studies of retinol and RBP metabolism.

Gene, 1993 Nov 15, 133(2), 223 - 5
Translational properties of the human papillomavirus type-6 L1-coding mRNA; Tomita Y et al.; A cDNA encoding a bicistronic mRNA, E1E4L1, which was generated by double-splicing of the E1, E4 and L1 genes, of the type-6 human papillomavirus (HPV-6), was cloned . The E1E4 and L1 open reading frames (ORFs) in this cDNA were expressed in COS-1 or CV-1 cells as fusion proteins with Escherichia coli beta-galactosidase (beta Gal), and the products were analyzed by immunoprecipitation and enzyme assay . The results showed that the translational efficiency of the L1 ORF was about 9-15-fold less efficient than that of the E1E4 ORF . Substitution of the ATG of the E1E4 ORF with AAG increased translation of the L1 ORF about 30-fold . Lengthening of the intercistronic sequence to 31 bp, equivalent in length to the bicistronic HPV-1 mRNA, showed little translational effect relative to the wild-type 12-bp intercistronic sequence . (Carets {} represent splicing of RNA.)

Gene, 1993 Nov 15, 133(2), 213 - 7
One-step purification and characterization of a lignin-specific O-methyltransferase from poplar; Van Doorsselaere J et al.; O-Methyltransferases (OMT; EC 2.1.1.6) play an important role in the synthesis of lignin precursors by catalyzing the O-methylation of o-diphenolic substrates such as caffeic acid (CA) and 5-hydroxyferulic acid (5OH) . Here, we report on the purification of a lignin-specific OMT (38 kDa) from poplar (Populus trichocarpa x P . deltoides) . The OMT was purified from xylem by a single affinity chromatography step on adenosine agarose . The enzyme uses both CA and 5OH as substrates . We previously have reported the cloning of a corresponding OMT cDNA {Dumas et al., Plant Physiol . 98 (1992) 796-797} . Expression of this OMT cDNA in Escherichia coli further confirmed the identity of the clone . Genomic hybridization demonstrates the presence of one or two OMT genes per haploid poplar genome . RNA gel blot hybridization shows high levels of steady-state OMT mRNA in the xylem of young poplar trees, as compared to the levels in leaves.

FEBS Lett, 1993 Nov 15, 334(2), 165 - 9
The antirepressor of phage P1 . Isolation and interaction with the C1 repressor of P1 and P7; Riedel HD et al.; Two antirepressor proteins, Ant1 and Ant2, of molecular weight 42 and 32 kDa, respectively, are encoded by P1 as a single open reading frame, with the smaller protein initiating at an in-frame start codon . Another open reading frame, icd, 5' upstream of and overlapping ant1 is required for ant1 expression . Using appropriate ant gene-carrying plasmids we have overproduced and purified Ant1/2 in the form of a protein complex and Ant2 as a single protein . Sequence analysis confirmed the N-terminal amino acids predicted from the DNA sequence of ant1/ant2, except that the N-terminal methionine is missing in the Ant2 protein . Under appropriate conditions the C1 repressors of phages P1 and P7 specifically co-precipitate with the Ant1/2 complex but not with Ant2 protein alone . The results suggest that the antirepressor may exert its C1-inactivating function by a direct protein-protein interaction.

Gene, 1993 Nov 15, 133(2), 237 - 42
Isolation of the chicken NF-kappa B p65 subunit-encoding cDNA and characterization of its products; Ikeda T et al.; NF-kappa B is a heterodimeric transcription factor consisting of subunits of 50 kDa (p50) and 65 kDa (p65) . cDNA clones encoding the chicken NF-kappa B p65 subunit were isolated . Sequence analysis showed that chicken p65 is approximately 55% identical to the mouse and human p65 proteins, and contains the Rel homology domain (RHD) in its N-terminal 286 amino acids (aa) and the putative transactivation domain in its C-terminal region . The RHD is particularly highly conserved between the chicken and mammalian p65 proteins . Northern blot hybridization analysis detected the expression of a 2.6-kb transcript of p65 in various organs, with the highest level in spleen . A fusion protein containing the RHD of chicken p65 was found to bind to a consensus kappa B-site in an electrophoretic mobility shift assay (EMSA) . This binding was specifically inhibited by the presence of fusion proteins containing the C-terminal ankyrin repeats domain (ARD) of chicken p105, the precursor protein for the p50 subunit . Immunoprecipitation analysis showed that p65 formed a complex(es) with multiple cellular proteins, including p50, p105 and c-Rel in chicken spleen cells.

Arch Biochem Biophys, 1993 Nov 15, 307(1), 165 - 74
Threonine synthase of Escherichia coli: inhibition by classical and slow-binding analogues of homoserine phosphate; Farrington GK et al.; L-threo-3-Hydroxyhomoserine phosphate, derived from the antimetabolites L-threo-3-hydroxyaspartate and L-threo-3-hydroxyhomoserine {Shames, S . L., Ash, D . E., Wedler, F . C., and Villafranca, J . J . (1984) J . Biol . Chem . 258, 15331-15339}, is a classical competitive inhibitor of threonine synthase (Ki = 6 microM) with structural elements of both substrate and product . L-2-Amino-5-phosphonovaleric acid also inhibits the enzyme competitively with a Ki (31 microM), comparable to Km for L-homoserine phosphate . In contrast, a structural analogue of Hse-P, L-2-amino-3-{(phosphonomethyl)thio}propionic acid exhibits a Ki = 0.11 microM (ca . 100-fold less than Km for L-Hse-P), along with "slow, tight" inhibition kinetics . Nuclear magnetic resonance was used with these inhibitors to probe for pyridoxal phosphate-catalyzed hydrogen-deuterium exchange reactions characteristic of substrates . With L-threo-3-hydroxy-homoserine phosphate, H-D exchange occurs only at the C-alpha position, but for homoserine in the presence of phosphate and for L-2-amino-5-phosphonovaleric acid and L-amino-3{(phosphonomethyl)thio}propionic acid (APMTP), H-D exchange occurs at C-alpha and stereospecifically at C-beta . For L-homoserine plus phosphate and L-2-amino-5-phosphonovaleric acid, the rate of H-D exchange at C-alpha is 8-45 times faster than at C-beta . For L-2-amino-3-{(phosphonomethyl)thio}propionic acid, the C-alpha to C-beta exchange rate ratio is near unity, due to a 700-fold decrease in the C-alpha rate for the analogue . Taken with information from molecular modeling, these data can be interpreted in terms of the current working hypothesis for the catalytic mechanism . Specifically, the slow, tight inhibition by APMTP results from its being carried further into the catalytic cycle than other analogues prior to forming an intermediate that is blocked from further catalysis.

J Biol Chem, 1993 Nov 15, 268(32), 24149 - 55
Evidence for a catalytic role of glutamic acid 129 in the NAD-glycohydrolase activity of the pertussis toxin S1 subunit; Antoine R et al.; The S1 subunit of pertussis toxin is an ADP-ribosyl-transferase capable of transferring the ADP-ribose moiety of NAD+ to nucleotide-binding signal-transducing proteins of the Gi/G(o) family . In the absence of G proteins, the enzyme also catalyzes the hydrolysis of NAD+ . Glu-129 was previously shown to be critical for both enzymatic activities . In this study, site-directed mutagenesis was used to make the conservative substitution of aspartate for Glu-129 . The recombinant wild type and mutant proteins were purified to near homogeneity and used for enzymatic analyses . Kinetic experiments showed that the kcat of the mutant protein was about 200 times less than that of the wild type enzyme, whereas the Km for NAD+ of the two proteins were very similar, suggesting that Glu-129 is a catalytic residue for the NAD-glycohydrolase reaction of S1 . This hypothesis was confirmed by a less than 2-fold change in Kd as measured by fluorescence quenching studies, indicating that the binding of NAD+ is not affected in the mutant protein in any important way . In another experiment, the replacement of Glu-129 by cysteine resulted in a disulfide bridge between Cys-129 and Cys-41 in rS1d-E129C, suggesting that the folding of the polypeptide chain is such that the catalytic Glu-129 residue is close to the amino-terminal NAD-binding site of S1 . These findings imply that Glu-129 plays a key role in catalysis of the NAD-glycohydrolase reaction, possibly by electrostatically stabilizing a cationic transition state intermediate, or by serving as a general base to deprotonate the ADP-ribosyl acceptor substrates.

J Biol Chem, 1993 Nov 15, 268(32), 24074 - 7
Specificity of the Escherichia coli chaperone DnaK (70-kDa heat shock protein) for hydrophobic amino acids; Richarme G et al.; Molecular chaperones form a class of proteins that bind selectively to nascent, unfolded, misfolded, or aggregated polypeptides . This property is the basis of their implication in many cellular processes such as protein folding, protein targeting to membranes, or protein renaturation after stress . It has been suggested that the recognition of non-native proteins by chaperones is mediated by their binding to exposed hydrophobic areas, to the polypeptide backbone, or to specific secondary structures . We show in the present study that DnaK, the 70-kDa chaperone of Escherichia coli specifically recognizes hydrophobic amino acids . The peptide-dependent ATPase activity of DnaK is specifically stimulated by Ile, Phe, Leu, and Val in a manner which is consistent with an interaction of these amino acids with the polypeptide binding site of DnaK . Two classes of amino acid binding site can be distinguished, one being specific for the aliphatic amino acids and the other for the aromatic amino acids . Since the hydrophobic amino acids are buried inside the hydrophobic core of native proteins and are exposed in non-native forms, their interaction with DnaK could be the basis of the specific interaction of the chaperone with non-native proteins in protein folding, protein targeting to membranes, or protein renaturation.

J Biol Chem, 1993 Nov 15, 268(32), 24330 - 8
The cystic fibrosis transmembrane conductance regulator . Overexpression, purification, and characterization of wild type and delta F508 mutant forms of the first nucleotide binding fold in fusion with the maltose-binding protein; Ko YH et al.; The first nucleotide binding fold (NBF1) of the cystic fibrosis transmembrane conductance regulator (CFTR) and its disease-causing mutant form (delta F508,NBF1) were overexpressed in high yield in Escherichia coli in fusion with the maltose-binding protein (MBP) . The rationale for producing the chimerae was to aid in domain purification, solubilization, and crystallization and to examine the effect of protein-protein interactions on the properties of the mutant NBF1 . Both the purified wild type and delta F508 mutant fusion proteins fold into functional nucleotide binding domains as determined by using the fluorescent nucleotide analog TNP-ATP (2'-(3')-O-(2,4,6-trinitrophenyl)adenosine-5'-triphosphate) . Moreover, the prominent secondary structural features of the two proteins as assessed by ultraviolet circular dichroism spectropolarimetry are very similar, as is the higher order structure evident in three separate protease digestion patterns . Finally, the stability of the nucleotide binding function of the two proteins is similar as assessed by sensitivity to urea . Gel filtration chromatography and electron and confocal microscopy reveal that both fusion proteins, but not MBP alone, form organized fibers, suggesting that NBF1 self-associates, thus raising the possibility that CFTR may be oligomeric in the plasma membrane . Significantly, in the presence of high salt, these fusion proteins also have a propensity to form microcrystals . Finally, the two separate domains (NBF1 and MBP) constituting the fusion proteins appear to interact quite strongly as both proteins remain associated even after cleavage of their fusion junction . The possible relevance of these novel findings to those approaches that might be taken to elucidate the three-dimensional structural differences between the wild type and delta F508 mutant forms of CFTR, as well as to ameliorate the severity of cystic fibrosis, is discussed.

Biochem Biophys Res Commun, 1993 Nov 15, 196(3), 1214 - 20
Characterization of cDNA for a dehydration-inducible gene that encodes a CLP A, B-like protein in Arabidopsis thaliana L; Kiyosue T et al.; Sequence was obtained from a cDNA clone, designated ERD1, isolated from a cDNA library of 1-hour-dehydrated plants of Arabidopsis thaliana L . The clone (3150 bp) contains an open reading frame of 946 amino acid residues with greater than 34% sequence identity to the regulatory subunit of the Clp ATP-dependent protease in Escherichia coli and contains a putative chloroplast-targeting signal at the N-terminus . Southern blot analysis suggested the presence of additional ERD1-related genes in A . thaliana . The expression of ERD1 gene was strongly induced by dehydration-stress but not by heat-, cold-, or heavy-metal-stress . In addition ERD1 gene-expression was not strongly affected by treatment with plant growth regulators, such as auxin, cytokinin, abscisic acid, and gibberellic acid, or by starvation-stress for 10 hours.

Proc Natl Acad Sci U S A, 1993 Nov 15, 90(22), 10653 - 7
Permeability properties of a large gated channel within the ferric enterobactin receptor, FepA; Liu J et al.; FepA is an Escherichia coli outer membrane receptor protein for the siderophore ferric enterobactin . Prior studies conducted in vivo suggested that FepA and other TonB-dependent outer membrane proteins transport ligands by a gated-channel mechanism . To corroborate and extend these findings we have determined the permeability properties of the FepA channel in vitro, by measuring the diffusion rates of hydrophilic nonelectrolytes through the FepA channel in liposome swelling experiments . Like porins, the FepA deletion mutant delta RV showed a size-dependent permeability to oligosaccharides, indicating that it forms a nonspecific, hydrophilic pore . Unlike OmpF and other E . coli porins, however, delta RV proteoliposomes transported stachyose (666 Da) and ferrichrome (740 Da) . These data, and other uptake results with a series of maltodextrins of increasing size, confirm the existence of a channel domain within FepA that is considerably larger than OmpF-type pores . These results represent a reconstitution of the channel function of a TonB-dependent receptor protein and establish that FepA contains the largest channel that has been characterized in the E . coli outer membrane.

J Med Chem, 1993 Nov 12, 36(23), 3542 - 5
Aminoacyl analogs of chloramphenicol: examination of the kinetics of inhibition of peptide bond formation; Drainas D et al.; Two aminoacyl analogs and one peptidyl analog of chloramphenicol (1, Cl2CHCO-CA) were prepared . These are 2 (L-Phe-CA), 3 (Gly-CA), and 4 (L-Phe-Gly-CA) . The kinetics of inhibition of peptide bond formation by these analogs were examined in a cell-free system which was derived from E . coli and used previously for the study of 1 (Drainas; et al . Eur . J . Biochem . 1987, 164, 53-58) . In the absence of inhibitor, the reaction follows first-order kinetics for the entire course of the reaction . In the presence of the analog the reaction gives biphasic log-time plots . The kinetic information pertaining to the initial slope of the plot is analyzed (initial-slope analysis) . This information differentiates the analogs from the parent compound 1 . The parent compound 1 gives complex inhibition kinetics; increasing the concentration of 1 changes the inhibition from competitive to mixed noncompetitive (Drainas; et al . Eur . J . Biochem . 1987, 164, 53-58) . In contrast, the analogs give competitive kinetics even at high concentrations of the inhibitor . The following Ki values have been determined: 18.0 microM for 2, 5.5 microM for 3, 1.5 microM for 4 . If we were to assume that compounds 2, 3 and 4 behave as classical competitive inhibitors, we could say that 4 is 12 times more potent than 3 and 4 times more potent than 2 . On this assumption we could also compare 1 with 4 and see that 4 is 2 times weaker than 1 . It is suggested that as compared with 1, the two aminoacyl analogs and the dipeptidyl analog have increased structural similarity to the 3'-terminus of aminoacyl-tRNA or of peptidyl-tRNA and that this similarity results in a more pronounced competitive inhibition.

Nucleic Acids Res, 1993 Nov 11, 21(22), 5110 - 6
Reconstitution of mammalian excision repair activity with mutant cell-free extracts and XPAC and ERCC1 proteins expressed in Escherichia coli; Park CH et al.; Nucleotide excision repair in humans involves the coordinated actions of 8-10 proteins . To understand the roles of each of these proteins in excision it is necessary to develop an in vitro excision repair system reconstituted entirely from purified proteins . Towards this goal we have expressed in E . coli two of the 8 genes known to be essential for the excision reaction . XPAC and ERCC1 were expressed as fusion proteins with the Escherichia coli maltose binding protein (MBP) and purified to > 80% homogeneity by affinity chromatography . The purified proteins either as fusions or after cleavage from the MBP were able to complement the CFE of cells with mutations in the corresponding genes in an excision assay with thymine dimer containing substrate.

Nucleic Acids Res, 1993 Nov 11, 21(22), 5025 - 33
The structure of the initiation complex at the replication origin, oriC, of Escherichia coli; Woelker B et al.; Two distinct regions in the replication origin, oriC, of Escherichia coli are separately distorted upon initiation complex formation by the initiator protein DnaA . The AT-rich region in the left part of oriC and the start site region in the right part of oriC . Chemical modification of single-stranded DNA was observed at both regions whereas endonuclease recognition of DNA mini-bulges specifically occurred in the start site region . We show that the helical phasing of binding sites for DnaA protein in oriC is important for origin function . An insertion or deletion of one helical turn between the two rightmost binding sites does not alter the efficiency of replication initiation, whereas all modifications of distance by less or more than one helical turn result in inactivation of oriC . DnaA binding and helical distortions in the AT-rich region as well as in the start site region are not affected in the distance mutants irrespective of their functionality in vivo . We propose a specific compact nucleoprotein structure for the initiation complex.

Nature, 1993 Nov 11, 366(6451), 178 - 82
Tandem binding in crystals of a trp repressor/operator half-site complex; Lawson CL et al.; The crystal structure of trp repressor tandemly bound in a 2:1 complex to a 16-base-pair palindromic DNA containing a central trp operator half-site has been determined and refined to 2.4 A resolution . Despite dramatically different DNA sequence contexts and crystallization conditions, the protein/DNA interface is essentially identical to that seen in the original trp repressor/operator complex structure . Water-mediated sequence recognition by trp repressor is likely to be related to the unusual end-on approach of the recognition helix (E), which allows sharing of the major groove by tandem dimers . The tandem complex model accounts for the mutational sensitivity of all trp operator base pairs . The structure also provides the first detailed view of the tandem interaction, revealing a key role for the amino-terminal arms.

Nucleic Acids Res, 1993 Nov 11, 21(22), 5050 - 8
Single exchanges of amino acids in the basic region change the specificity of N-Myc; Feldmann T et al.; We exchanged specific amino acids in the basic region of the murine N-Myc protein and tested the mutant proteins for their DNA binding specificity . The amino acids we exchanged were chosen in analogy to residues of the homologous basic regions of bHLH and bZIP proteins . Mutant N-Myc peptides were expressed in Escherichia coli and specific DNA binding was monitored by gel shift experiments . For this we used palindromic target sequences with systematic base pair exchanges . Several mutants with altered DNA binding specificity were identified . Amino acid exchanges of residues -14 or -10 of the basic region lead to specificity changes (we define leucine 402 of N-Myc as +1; comparable to GCN4 see (1)) . The palindromic N-Myc recognition sequence 5'CACGTG is no longer recognized by the mutant proteins, but DNA fragments with symmetrical exchanges of the target sequence are . Exchanges at position -15 broaden the binding specificity . These data were used to build a computer based model of the putative interactions of the N-Myc basic DNA binding region with its target sequence.

Nucleic Acids Res, 1993 Nov 11, 21(22), 5074 - 8
Competition between frameshifting, termination and suppression at the frameshift site in the Escherichia coli release factor-2 mRNA; Adamski FM et al.; Competition between frameshifting, termination, and suppression at the frameshifting site in the release factor-2 (RF-2) mRNA was determined in vitro using a coupled transcription-translation system by adding a UGA suppressor tRNA . The expression system was programmed with a plasmid containing a trpE-prfB fusion gene so that each of the products of the competing events could be measured . With increasing concentrations of suppressor tRNA the readthrough product increased at the expense of both the termination and the frameshifting product indicating all three processes are in direct competition . The readthrough at the internal UGA termination codon was greater than that at the natural UGA termination codon at the end of the coding sequence . The results suggest that this enhanced suppression may reflect slower decoding of the internal stop codon by the release factor giving suppression a competitive advantage . The internal UGAC stop signal at the frameshift site has been proposed to be a relatively poor signal, but in addition the release factor may be less able to recognise the signal with the mRNA in such a constrained state . Consequently, the frameshifting event itself will be more competitive with termination in vivo because of this longer pause as the release factor is decoding the stop signal.

Biochim Biophys Acta, 1993 Nov 10, 1203(1), 27 - 35
Physico-chemical characterization of a recombinant cytoplasmic form of lysine: N6-hydroxylase; Thariath AM et al.; A recombinant cytoplasmic preparation of lysine: N6-hydroxylase, IucD398, with a deletion of 47 amino acids at the N-terminus, was purified to homogeneity . IucD398 is capable of N-hydroxylation of L-lysine upon supplementation with FAD and NADPH . The enzyme is stringently specific with L-lysine and (S)-2-aminoethyl-L-cysteine serving as substrates . Protonophores, FCCP and CCCP, as well as cinnamylidene, have been found to serve as potent inhibitors of lysine: N6-hydroxylation by virtue of their ability to interfere in the reduction of the flavin cofactor.

Biochim Biophys Acta, 1993 Nov 10, 1203(1), 155 - 61
Expression, purification and binding to the receptor of human insulin-like growth factor II; Miyagishima T et al.; Human insulin-like growth factor II (IGF-II) was expressed as a fused protein with 14 additive amino acids in Escherichia coli with a high yield by an expression system using T7 RNA polymerase . Purification of the expressed protein was simply performed using only differential ultrafiltrations, giving a homogeneous preparation upon polyacrylamide gel electrophoresis and high-performance liquid chromatography . The expressed peptide was reacted with a monoclonal antibody raised against native IGF-II on a blotted membrane . Furthermore, the peptide was bound to IGF-II receptor in solubilized rat fetus membrane, though the affinity was slightly inferior to that of native IGF-II . In addition, fusion IGF-II immobilized on a gel matrix was useful for one-step purification of the IGF-II receptor with a high yield from solubilized rat fetus membranes.

Biochemistry, 1993 Nov 9, 32(44), 11923 - 8
Functional expression in Escherichia coli of the mitotic regulator proteins p24ran and p45rcc1 and fluorescence measurements of their interaction; Klebe C et al.; The gene products for the mitotic regulator genes RCC1 and Ran, p45rcc1 and p24ran, were expressed in Escherichia coli, purified in large amounts, and characterized for their biochemical properties . p24ran binds guanine nucleotide as a 1:1 complex, which is only slowly released from the protein . p45rcc1 catalyzes the exchange of nucleotide bound to the guanine nucleotide binding protein p24ran in the same way as the protein purified from HeLa cells . Likewise, the nucleotide dissociation from HeLa cell-derived p24ran protein is equally efficient with recombinant and nonrecombinant proteins . The recombinant proteins form a strong complex which contains no bound nucleotide . The kinetics of nucleotide exchange on p24ran in the presence or absence of p45rcc1 can be conveniently monitored either by the direct tryptophan fluorescence of p24ran or by fluorescence energy transfer measurements involving fluorescent nucleotides.

Biochemistry, 1993 Nov 9, 32(44), 11794 - 801
Homodinuclear (Pt,Pt) and heterodinuclear (Ru,Pt) metal compounds as DNA-protein cross-linking agents: potential suicide DNA lesions; Van Houten B et al.; Homodinuclear (Pt,Pt) and heterodinuclear (Ru,Pt) metal compounds having the generalized formula M(a)NH2(CH)4NH2M(b) are shown to form specific DNA lesions which can efficiently cross-link proteins to DNA . In this study, the homodinuclear case is represented by M(a) = M(b) = {cis-Pt(Cl2)-(NH3)} and the heterodinuclear case is represented by M(a) = {cis-RuCl2(DMSO)3} and M(b) = {cis-PtCl2(NH3)} . Native and denaturing polyacrylamide gel electrophoresis was used to show the formation of ternary coordination complexes between the metal-treated 49-bp DNA fragment and the Escherichia coli UvrA and UvrB DNA repair proteins . Treatment with proteinase K results in loss of the DNA-protein cross-links . DNA-protein cross-links formed between UvrA and DNA previously modified with the dinuclear metal compounds are reversible with the reducing agent beta-mercaptoethanol . The DNA lesion responsible for efficient DNA-protein cross-linking is most probably a DNA-DNA interstrand cross-link in which each metal atom is coordinated with one strand of the DNA helix . The formation of DNA repair protein associated DNA cross-links, potential "suicide adducts", suggests a novel action mechanism for these anticancer compounds . In addition, these dinuclear metal compounds should be very useful agents for the investigation of a wide range of protein-DNA interactions.

Biochemistry, 1993 Nov 9, 32(44), 11769 - 75
Solution oligomerization of the rev protein of HIV-1: implications for function; Cole JL et al.; rev is an RNA-binding protein of human immunodeficiency virus-1 and is required for the expression of incompletely spliced viral transcripts . Oligomerization of rev is thought to be associated with RNA binding and rev function . Here, we have characterized the oligomerization of rev using equilibrium analytical centrifugation . rev is predominantly monomeric at low concentrations, but reversibly polymerizes to produce large aggregates at higher concentrations . The data fit well to an unlimited isodesmic self-association model in which the association constants for the addition of a monomer to each aggregate are equal {K = 1.08 x 10(6) M-1 at 4 degrees C} . The association constant is essentially independent of monovalent salt concentration from 0.15 to 2 M at pH 6-9 . Thermodynamic parameters derived from the temperature dependence of the association constant over the limited range of 0-30 degrees C reveal that the primary contribution to the free energy of oligomerization is a large negative enthalpy . Binding of rev to the rev-responsive element of RNA was characterized under the same conditions as the centrifugation experiments using a nitrocellulose filter assay . rev binds to the RRE at a protein concentration where rev is predominantly monomeric, suggesting that solution multimerization of rev is not required for rev function.

FEBS Lett, 1993 Nov 8, 334(1), 55 - 9
Expression of fully active ammodytoxin A, a potent presynaptically neurotoxic phospholipase A2, in Escherichia coli; Liang NS et al.; A cDNA encoding the most presynaptically neurotoxic phospholipase A2, ammodytoxin A, from the venom of the long-nosed viper (Vipera ammodytes ammodytes) has been expressed in Escherichia coli . Ammodytoxin A was produced as a fusion protein with the 81 N-terminal residues of adenylate kinase followed by the tetrapeptide recognition site for factor Xa (IEGR) just preceding the first amino acid residue of the toxin . The fusion protein was expressed under the control of tac promoter without IPTG induction in the form of insoluble inclusion bodies . It was dissolved in guanidine hydrochloride, S-sulfonated and refolded in a reoxidation mixture including a reduced/oxidized glutathione redox couple . Ammodytoxin A was fully activated by limited hydrolysis with trypsin that preferentially cleaves the fusion protein at the factor Xa recognition site and purified by cation-exchange chromatography . The correct N-terminus was confirmed by protein sequencing . Recombinant ammodytoxin A has been proved to be indistinguishable from the native toxin in its enzymatic activity and toxicity.

FEBS Lett, 1993 Nov 8, 334(1), 1 - 2
A comment on the absence of calcium regulation of human thioredoxin reductase; Oblong JE et al.; It has been previously suggested that human thioredoxin reductase activity is regulated by calcium . However, the activity of a purified form of human placental thioredoxin reductase was found to not be affected by mM concentrations of calcium, well above intra- and extracellular physiological levels . Furthermore, the suggestion that an E-F hand is present in Escherichia coli thioredoxin reductase is strongly contested . These current results suggest that human thioredoxin reductase is not regulated by calcium.

Biochim Biophys Acta, 1993 Nov 7, 1179(2), 134 - 40
Primed pentose cycle activity supports production and elimination of superoxide anion in Kupffer cells from rats treated with endotoxin in vivo; Spolarics Z et al.; Glucose use and pentose cycle activity were determined in freshly isolated rat Kupffer cells 3 h after an i.v . injection of Escherichia coli endotoxin (0.1 mg/kg body weight), by using {1-14C}, {6-14C} and {2-3H}glucose . Endotoxin treatment in vivo caused a 5-fold increase in the basal glucose uptake in Kupffer cells . Pentose cycle activity was elevated from 8.7 to 13.6 nmol/h per 10(7) cells after endotoxin . In vitro treatment of the cells from saline- and endotoxin-treated animals with phorbol ester (10(-6) M) increased pentose cycle activity 2-fold and 8-fold, respectively . Phorbol ester caused a 50% increase in glucose uptake in both groups . t-Butyl hydroperoxide (0.5 mM) caused a similar increase in pentose cycle activity as phorbol ester . Glucose oxidation in the Krebs cycle was also doubled after endotoxin . KC from endotoxin-treated animals produced O2- spontaneously, and were primed to produce additional large amounts of O2- upon phorbol ester treatment . Addition of t-butyl hydroperoxide inhibited O2- production by Kupffer cells . Depletion of glutathione by N-ethylmaleimide (0.1 mM), or inhibition of NADPH oxidase by diphenyliodonium (0.1 mM) inhibited both the pentose cycle activity and the O2- production . Increasing the concentration of exogenous glucose in the cell medium elevated the glycolytic rate, while pentose cycle flux was not affected either under basal conditions or following subsequent challenges by phorbol ester or t-butyl hydroperoxide . Our data suggest that the endotoxin-induced elevated glucose use in Kupffer cells is accompanied by a primed state of the pentose cycle . This condition supports superoxide and macromolecule synthesis and could also represent a potentiated protective mechanism against oxidative cellular injury during bacterial infections.

J Chromatogr A, 1993 Nov 5, 653(2), 241 - 6
Characterization of further association of the trimeric membrane protein porin by low-angle laser-light scattering photometry coupled with high-performance gel chromatography; Watanabe Y et al.; Porin (OmpF), a trimeric membrane protein, in an extract of the outer membrane of Escherichia coli gave a twin-peaked elution pattern on Sephacryl S-300HR gel chromatography in the presence of sodium dodecyl sulphate . The species eluting earlier and later were found to be the hexamer and trimer, respectively, from molecular mass determination by low-angle laser-light scattering photometry coupled with TSK-G3000SWXL gel chromatography . As the hexamer was dissociated into the trimer under the conditions of sodium dodecyl sulphate polyacrylamide gel electrophoresis, its presence had been overlooked . The addition of lipopolysaccharide, another component of the outer membrane, and subsequent dialysis induced association of the trimer, the product containing an appreciable amount of the hexamer.

J Chromatogr A, 1993 Nov 5, 653(2), 207 - 18
Anion-exchange chromatographic behavior of recombinant rat cytochrome b5 . Thermodynamic driving forces and temperature dependence of the stoichiometric displacement parameter Z; Roush DJ et al.; The HPLC anion-exchange isocratic retention behavior of the recombinant soluble core of wild type rat cytochrome b5 on Mono Q HR 5/5 was investigated as a function of temperature and sodium chloride concentration at fixed eluent flow-rates . Retention was measured over a range of eluent flow-rates at a specified temperature to determine if true adsorption equilibrium could be approximated by the HPLC method . Apparent Van 't Hoff enthalpies of adsorption obtained from the HPLC retention data were positive, indicating an entropically driven spontaneous adsorption process, and were found to decline with increasing ionic strength . The retention results were interpreted in terms of the stoichiometric displacement model to obtain the apparent number of binding sites in the contact region, Z, as a function of temperature and of protein concentration . Z was found to depend significantly on temperature, even under conditions of nearly complete protein recovery, but did not depend on protein concentration at the low loadings studied.

Science, 1993 Nov 5, 262(5135), 867 - 73
Multiple RNA polymerase conformations and GreA: control of the fidelity of transcription; Erie DA et al.; Pre-steady state kinetics of misincorporation were used to investigate the addition of single nucleotides to nascent RNA by Escherichia coli RNA polymerase during transcription elongation . The results were fit with a branched kinetic mechanism that permits conformational switching, at each template position, between an activated and an unactivated enzyme complex, both of which can bind nucleotide triphosphates (NTPs) from solution . The complex exists most often in the long-lived activated state, and only becomes unactivated when transcription is slowed . This model permits multiple levels of nucleotide discrimination in transcription, since the complex can be "kinetically trapped" in the unactivated state in the absence of the correct NTP or if the 3' terminal residue is incorrectly matched . The transcription cleavage factor GreA (or an activity enhanced by GreA) increased the fidelity of transcription by preferential cleavage of transcripts containing misincorporated residues in the unactivated state of the elongation complex . This cleavage mechanism by GreA may prevent the formation of "dead-end" transcription complexes in vivo.

J Mol Biol, 1993 Nov 5, 234(1), 87 - 98
A regulatory cascade in the induction of rhaBAD; Egan SM et al.; The RhaS and RhaR regulatory proteins are encoded in the Escherichia coli L-rhamnose gene cluster . We used complementation analysis and DNA mobility shift assays to show that RhaR is not the direct activator of the L-rhamnose catabolic operon, rhaBAD . An in-frame deletion of rhaS (rhaS-rhaR+) eliminated expression from the rhaBAD promoter, pBAD, while overexpression of rhaS greatly speeded the normally slow induction of transcription from pBAD . Expression from pBAD in a coupled transcription-translation assay was only detected when rhaS+ DNA was added to allow synthesis of RhaS protein . RhaS thus appears to be the direct L-rhamnose-specific activator of rhaBAD expression . Deletion mapping located the binding site for the L-rhamnose-specific regulator to a region overlapping position -70 relative to the rhaBAD transcription start site . Deletion mapping and DNA mobility shift assays located a CRP binding site just upstream from the binding site for the L-rhamnose-specific regulator . Quantitative primer extension analysis showed that induction of both the rhaBAD and rhaSR messages was unusually slow, requiring 40 to 50 minutes to reach a steady-state level . Induction of rhaBAD apparently involves a regulatory cascade in which RhaR first induces rhaSR expression, then RhaS accumulates and induces rhaBAD expression.

J Mol Biol, 1993 Nov 5, 234(1), 8 - 13
Assembly of multimeric proteins . Effect of mutations in the alpha-subunit on membrane assembly and activity of pyridine nucleotide transhydrogenase; Ahmad S et al.; The pyridine nucleotide transhydrogenase of Escherichia coli is an inner membrane protein of two different subunits (alpha and beta) . It functions as a proton pump . The highly hydrophilic carboxy-terminal tail of ten amino acid residues in the alpha-subunit determines the correct folding and proper assembly of the beta-subunit leading to a functional enzyme . Premature termination of the alpha-subunit six amino acid residues from the carboxy-terminal end abolishes the activity completely . Although the two subunits are still assembled into the membrane, the conformation of the beta-subunit is perturbed . Systematic truncation and site-directed substitutions revealed that at least one positive charge in the carboxy-terminal region is required for efficient assembly of the two subunits to give a functional enzyme, while a phenylalanine residue, essential for activity, has no apparent effect on the extent of assembly of the two subunits.

J Mol Biol, 1993 Nov 5, 234(1), 72 - 86
Negative co-dominant inhibition of recA protein function . Biochemical properties of the recA1, recA13 and recA56 proteins and the effect of recA56 protein on the activities of the wild-type recA protein function in vitro; Lauder SD et al.; We have investigated the biochemical properties of several Escherichia coli mutant recA proteins that display a null phenotype . These are the recA1, recA13 and recA56 proteins, each of which carries a single missense mutation . These proteins all share a common defect which is the inability to adopt the high affinity DNA binding state normally elicited by the nucleotide cofactor ATP . Consequently, other than the ability to bind ssDNA, they possess none of the in vitro enzymatic activities of recA protein . However, each protein has characteristics that are unique, leading to the conclusion that the observed mutant phenotypes arise through fundamentally different mechanisms . Despite the magnitude of these defects, the recA56 protein is able to differentially inhibit various activities of wild-type recA protein . Incorporation of recA56 protein into a presynaptic filament with the wild-type recA protein does not affect the ability of the wild-type protein to hydrolyze ATP, as judged by the turnover number (kcat), provided that the ssDNA concentration is not limiting; however, the affinity of wild-type recA protein for ATP is lowered by the presence of recA56 protein . Similarly, the ability to cleave lexA protein is only modestly inhibited . However, both the ability to compete with SSB protein for ssDNA binding sites and the DNA strand exchange activity of wild-type recA protein are severely inhibited by the presence of recA56 protein . These results suggest that individual monomeric components of the recA protein-DNA filament are translated through protein-protein contacts to become macroscopic properties of the filament.

J Mol Biol, 1993 Nov 5, 234(1), 14 - 27
Translation initiation complex formation with 30 S ribosomal particles mutated at conserved positions in the 3'-minor domain of 16 S RNA; Ringquist S et al.; Escherichia coli 30 S ribosomal subunits containing in vitro (phage T7 RNA polymerase-generated) 16 S rRNA, both wild-type and mutant, were examined by toeprinting . These synthetic particles were used to compare the effects of the absence of base modification and of specific nucleotide substitutions in conserved sequence regions of the RNA on the assembly of mRNA, tRNAs and 30 S particles into a translational initiation complex . Initiation factor-3-dependent selection of tRNA(fMet) from a mixture of tRNA(fMet) and tRNA(Phe) occurred with all particles, although 20 times less initiation factor-3 was needed for the synthetic particles, including the mutants . Whereas isolated 30 S particles or those reconstituted with isolated RNA did not distinguish between tRNA(fMet) and tRNA(Phe) for ternary complex formation in the absence of initiation factor-3 (intrinsic selection ability), the synthetic particles preferred tRNA(fMet) . The difference between the natural and synthetic particles appears to be due to the absence of certain base modifications, but not m2(6)A, in the synthetic RNA . Synthetic particles containing the mutation U1512C, which converts the universal U.G pair to C.G enhanced both tRNA(fMet) binding and selectivity, although other mutations at that site, namely U1512G, G1523A and U1512C/C1524U, had no such effect . Mutants U1498G and G1401C/C1501G, both located in a highly conserved single-stranded region of the 3'-minor domain, also enhanced tRNA(fMet) selectivity, in this case by reducing complex formation with elongator tRNA . Complex formation between elongator tRNA and the G1401C/C1501G mutant was reduced to almost undetectable levels . The results also indicated that the association rate for initiation complex formation for G1401C/C1501G was considerably lower than for the wild-type sequence . This result had not been detected by standard tRNA-30 S binding assays . Overall, the data suggest that (some of) the 16 S rRNA base modifications as well as the tertiary structure around the decoding site act to desensitize the intrinsic selection ability of the ribosome for tRNA(fMet).

J Immunol Methods, 1993 Nov 5, 166(1), 1 - 10
Detection and quantification of secreted soluble Fc gamma RIIA in human sera by an enzyme-linked immunosorbent assay; Astier A et al.; Fc gamma RIIA can be produced in a soluble form that contains both the extracellular and intracellular regions of the receptor, due to an alternative splicing of the transmembrane domain-coding exon . We have developed an enzyme-linked immunosorbent assay (ELISA) that permits the specific detection and quantification in human sera of this secreted soluble Fc gamma RIIA . It uses the monoclonal antibody (MAb) IV.3 as capture antibody and rabbit polyclonal IgG directed against the intracellular region of Fc gamma RIIA as detector antibodies . The enzymatic reaction was amplified using an NADH/NAD+ amplification system . As little as 0.8-1.5 ng/ml (20-38 pM) of purified recombinant secreted Fc gamma RIIA could be detected . The serum levels of secreted sFc gamma RIIA ranged from 0 to 30 ng/ml in sera from 51 healthy donors . The mean value was 11.9 ng/ml +/- 6.55 (297 pM +/- 163) and the median value was 10.6 ng/ml (265 pM) (range: 0-764 pM).

J Biol Chem, 1993 Nov 5, 268(31), 23685 - 96
Characterization of the structural requirements for assembly and nucleotide binding of an ATP-binding cassette transporter . The maltose transport system of Escherichia coli; Panagiotidis CH et al.; The periplasmic maltose-binding protein-dependent, maltose transport system of Escherichia coli is a well studied member of the ATP-binding cassette family of transport ATPases . In addition to the water-soluble maltose-binding protein, the system comprises three membrane proteins, MalF, MalG, and MalK, which form a heterotetrameric complex (FGK2) in the cytoplasmic membrane . The purified complex exhibits transport-associated ATPase activity . To characterize the requirements for nucleotide binding and hydrolysis by the FGK2 complex, we used plasmids to express different combinations of the individual subunits as well as mutant forms of the MalK subunit . Prior to measuring nucleotide binding, we examined membrane preparations for the presence of each subunit from strains that contained all possible permutations of the three structural genes, malF, malG, and malK . We found that when all three genes were present or when malF and malK were present together, the corresponding antigens were detected easily on Western immunoblots and were soluble in the non-ionic detergent, Triton X-100 . In contrast, all other permutations resulted in decreased amounts of antigen or antigen that was Triton X-100-insoluble . We relied on photocross-linking with 8-azido-{32P}ATP and ATP hydrolysis as indicators of the ability of the transport complex to interact with purine nucleotides . 8-Azido-{32P}ATP was photocross-linked to the MalK subunit . Photolabeling of MalK was inhibited by ATP, ADP, and GTP and not by other nucleotides . Photolabeling of MalK required the presence of MalF but not MalG . Mutations in malK that affect amino acid residues thought to be directly involved in nucleotide binding did indeed abolish labeling and resulted in loss of transport activity without affecting protein stability . In general, ATP hydrolysis correlated with the photocross-linking . A notable exception is the MalK941 mutant protein which retained the ability to be labeled by 8-azido-{32P}ATP but was unable to catalyze detectable levels of ATP hydrolysis . Some, but not all, of the malK mutations were dominant to wild type . To study the mechanism of dominance we devised a means of measuring the ability of different wild-type and mutant MalK proteins to interact with the MalF and MalG subunits . This assay relies on the fact that, when a bifunctional MalK-LacZ hybrid protein is associated with the MalF and MalG subunits, it is membrane-bound . Excess MalK competed with the MalK-LacZ hybrid protein for sites in the membrane and resulted in the hybrid fractionating as a soluble protein.(ABSTRACT TRUNCATED AT 400 WORDS)

J Biol Chem, 1993 Nov 5, 268(31), 23580 - 4
Calcium-dependent activation of protein kinase C . The role of the C2 domain in divalent cation selectivity; Luo JH et al.; Activation of certain isoforms of protein kinase C (cPKCs) requires Ca2+ and is associated with a conserved C2 domain that is not present in Ca(2+)-independent isoforms (nPKCs) . The site(s) of Ca2+ binding and the role of the C2 domain have not been previously identified . We have analyzed phosphatidylserine-dependent Ca2+ binding to fusion proteins expressed in Escherichia coli that carry various modifications in the regulatory region of cPKC beta 1 or nPKC epsilon . Ca2+ is bound mainly to the C1 domain of PKC beta 1, but the C2 domain confers specificity for Ca2+ binding when compared with Mg2+ and Mn2+ . We propose that in cPKCs there is selective binding of Ca2+ to a pocket formed by the C1 and C2 domains . This induces a change in conformation that activates the enzyme . In nPKCs, the cation binding pocket is less specific for Ca2+ because it lacks the C2 domain . Therefore, divalent cations like Mg2+ can bind to it, thereby abrogating the requirement of Ca2+ for enzyme activation.

J Biol Chem, 1993 Nov 5, 268(31), 23524 - 30
Cloning and expression of cDNA for a human enzyme that hydrolyzes 8-oxo-dGTP, a mutagenic substrate for DNA synthesis; Sakumi K et al.; 8-Oxoguanine (8-oxo-7, 8-dihydroguanine) is produced in DNA, as well as in nucleotide pools of cells, by active oxygen species normally formed during cellular metabolic processes . 8-Oxoguanine nucleotide can pair with cytosine and adenine nucleotides at almost equal efficiencies, and transversion mutation ensues . Human cells contain enzyme activity, which hydrolyzes 8-oxo-dGTP to 8-oxo-dGMP, and this enzyme is responsible for preventing misincorporation of 8-oxoguanine into DNA . We purified this particular human enzyme to physical homogeneity and determined a partial amino acid sequence . We then cloned the cDNA for human 8-oxo-dGTPase and examined its nucleotide sequence . The human protein comprises 156 amino acid residues and has some sequence homology with the Escherichia coli MutT protein, which has a distinct 8-oxo-dGTPase activity . When the human cDNA was expressed in E . coli mutT- mutant cells, there was a significant amount of 8-oxo-dGTPase activity . In such cells, the frequency of spontaneous mutation was greatly reduced . We propose that the human 8-oxo-dGTPase protects genetic information from the untoward effects of endogenous oxygen radicals.

J Biol Chem, 1993 Nov 5, 268(31), 23519 - 23
Reconstitution of recombinant 40-kDa subunit of the clathrin-coated vesicle H(+)-ATPase; Peng SB et al.; We have proposed a model of the ATP hydrolytic sector of the clathrin-coated vesicle H(+)-ATPase wherein significant catalysis requires four subunits of molecular masses of 70, 58, 40, and 33 kDa (Xie, X.-S., and Stone, D . K . (1988) J . Biol . Chem . 263, 9859-9867) . We have cloned and expressed the 40-kDa component in Escherichia coli and have purified the recombinant protein to homogeneity . This subunit lacks ATP hydrolytic capacity, but when reconstituted to a 40 kDa-depleted hydrolytic sector, there is a greater than 20-fold increase in calcium-activated, N-ethylmaleimide-sensitive ATP hydrolysis, indicating that this subunit is required for vacuolar-type proton pump function.

J Biol Chem, 1993 Nov 5, 268(31), 23477 - 82
Two modes of transcription initiation in vitro at the rrnB P1 promoter of Escherichia coli; Borukhov S et al.; The rrnB P1 promoter of Escherichia coli (starting sequence C-4-A-3-C-2-C-1-A+1-C+2-U+3-G+4) forms a binary complex with RNA polymerase that is highly unstable and requires the presence of transcription substrates ATP and CTP for stabilizing the enzyme-DNA association (Gourse, R . L . (1988) Nucleic Acids Res . 16, 9789-9809) . We show that in the absence of UTP and GTP the stabilization is accomplished by short RNA oligomers synthesized in an unusual "-3-->" mode whereby the primer initiated at the +1 site presumably slips back by three nucleotides into the -3 site and is then extended yielding stable ternary complexes . By contrast, short oligomers initiated in the conventional "+1-->" mode without slippage do not exert the stabilization effect and are readily aborted from the promoter complex . The stable -3-->ternary complexes carry sigma factor but otherwise resemble elongation complexes in their high salt stability and in the fact that they are formed with a mutant RNA polymerase deficient in promoter binding . A model is proposed explaining the stability of the -3-->ternary complexes by RNA slipping into a putative "tight RNA binding site" in RNA polymerase which is normally occupied by RNA during elongation.

J Biol Chem, 1993 Nov 5, 268(31), 23427 - 34
The replication fate of R- and S-styrene oxide adducts on adenine N6 is dependent on both the chirality of the lesion and the local sequence context; Latham GJ et al.; In order to deduce the biological fate of adducts formed by the reaction of styrene oxide, a suspected carcinogen, with DNA, four oligodeoxynucleotides were synthesized which contained either R- or S-styrene oxide lesions on the N6 position of neighboring adenines within the human N-ras codon 61 . When these adducted oligodeoxynucleotides were ligated into the single-stranded vector M13mp7L2 and the modified DNA used to transform repair-deficient Escherichia coli, the resultant plaque-forming abilities were found to vary as much as 300-fold, depending on the stereochemical configuration of the styrene oxide lesion and the sequence context in the vicinity of the damage . The frequency of mutations caused by the various styrene oxide adducts were similarly dependent on both their chirality and local sequence context . Oligodeoxynucleotide templates bearing these same four adducts were also constructed in order to evaluate their replication in vitro by the Klenow fragment . Three of the four styryl-modified templates yielded significant levels of fully extended primer upon polymerization . In contrast, the template containing R-styrene oxide at the second position of N-ras 61 was a very poor substrate for replication, a result which correlates well with the observed lethality of this lesion in vivo.

J Biol Chem, 1993 Nov 5, 268(31), 23239 - 49
Dependence of trp repressor-operator affinity, stoichiometry, and apparent cooperativity on DNA sequence and size; Liu YC et al.; A series of chemically synthesized trp and mutant operator DNAs was employed to examine trp repressor binding . Although only a single repressor-operator complex was observed for most DNAs as reported previously, varying DNA sequence revealed two retarded complexes with an additional band of faster mobility . The relative intensity of the two retarded bands with varying repressor concentrations suggests that cooperative interactions between dimers may occur in the formation of the predominant repressor-operator complex . Direct stoichiometry measurements demonstrated that a 2:1 stoichiometry (two dimers per operator) is found in the primary repressor-operator complex band and that a 1:1 stoichiometry is observed, when present, for the minor repressor-operator complex band of faster mobility . Similar retardation patterns with a single complex of 2:1 stoichiometry were observed for 40-base pair (bp) trp operators corresponding to TrpEDCBA, aroH, and Trp-PL (a derivative of TrpEDCBA with increased symmetry) operator sequences as well as to hybrid operators containing a half-binding site from TrpEDCBA in conjunction with lac operator sequences, although the apparent affinity for the half-site DNAs was diminished by 10-fold . In contrast, the prominence of the 1:1 dimer-operator complex for Trp-PR, a different derivative of TrpEDCBA with increased symmetry, suggests that sequence context may diminish cooperativity between dimers . The stoichiometry observed was also dependent on the length of TrpEDCBA operator DNA used, shifting from primarily 2:1 for 40- and 33-bp TrpEDCBA DNA to primarily 1:1 for 29-, 26-, and 20-bp TrpEDCBA DNAs . In addition, the stability of the repressor-operator complex to electrophoresis is reduced for DNA lengths of 33, 29, and 26 bp . Based on the binding data and footprinting patterns for two hybrid 40-bp TrpEDCBA/lac half-binding site DNAs, it appears that repressor associates tightly to the specific trp half-site, whereas the nonspecific half of the DNA is more loosely bound . These results suggest that repressor dimer-dimer interaction may be an important feature in the trp repressor-operator interaction.

J Biol Chem, 1993 Nov 5, 268(31), 23148 - 56
Overexpression of fetal human pigment epithelium-derived factor in Escherichia coli . A functionally active neurotrophic factor; Becerra SP et al.; Pigment epithelium-derived factor (PEDF) is a neurotrophic protein present in low amounts in conditioned medium of cultured fetal human retinal pigment epithelial cells . Recently, the PEDF cDNA has been cloned from a fetal human cDNA library, and its derived amino acid sequence identified it as a member of the serine protease inhibitor (serpin) supergene family (Steele, F . R., Chader, G . J., Johnson, L . V., and Tombran-Tink, J . (1993) Proc . Natl . Acad . Sci . U . S . A . 90, 1526-1530) . We have prepared recombinant expression constructs from the fetal human PEDF cDNA and obtained milligram amounts of biologically active PEDF from Escherichia coli . The full-length open reading frame (Met1-Pro418) and a truncated form (Asp44-Pro418) were used in our constructs . Induction from a vector containing the truncated PEDF version, named pEV-BH, produced a protein (BH) of expected size (M(r) 42,800) associated with inclusion bodies, which contained 25-40% of expressed protein . After solubilization, BH was highly purified by gel filtration and cation exchange chromatography . The NH2-terminal sequence of the purified protein matched that of the pEV-BH construct . We have conducted neurite outgrowth assays in a human retinoblastoma Y-79 cell culture system . Recombinant PEDF (BH) demonstrated neurotrophic activity, as reported for the native PEDF . Thus, unfolded and refolded in vitro BH retained a potent biological activity . In parallel experiments, protease inhibition assays were performed . Recombinant PEDF did not have an effect on trypsin, chymotrypsin, elastase, cathepsin G, endoproteinase Lys-C, endoproteinase Glu-C, or subtilisin activity, suggesting that inhibition of known serine proteases is not the biochemical pathway for the PEDF neutrophic activity.

J Biol Chem, 1993 Nov 5, 268(31), 23139 - 47
Molecular characterization and outer membrane association of a Chlamydia trachomatis protein related to the hsp70 family of proteins; Raulston JE et al.; One route by which Chlamydia trachomatis is internalized into host endometrial epithelial cells is receptor-mediated endocytosis . Although this implies an adhesin-receptor interaction exists, specific chlamydial surface molecules have not been identified . We are investigating potential adhesin molecules using an in vitro functional assay to select for chlamydial recombinant Escherichia coli expressing an adherent phenotype . We have previously shown that E . coli JM109(pPBW58) attaches to epithelial cells by a specific process paralleling C . trachomatis and expresses at least three plasmid-encoded proteins (18, 28, and 82 kDa; Schmiel, D . H., Knight, S . T., Raulston, J . E., Choong, J., Davis, C . H., and Wyrick, P . B . (1991) Infect . Immun . 59, 4001-4012) . In this report, we demonstrate that (i) the 82-kDa protein is associated with the outer membrane of both E . coli JM109-(pPBW58) and C . trachomatis serovar E elementary bodies; (ii) the plasmid-encoded protein is identical to the native chlamydial protein by mass, charge, antigenicity, and partial proteolytic peptide profiles; (iii) a highly homologous protein is present in C . trachomatis biovariant lymphogranuloma venereum; (iv) the 82-kDa protein is not covalently linked by disulfide bonds to other protein species in either E . coli JM109(pPBW58) or C . trachomatis; (v) sequence analysis of the open reading frame indicates this protein is a relative of the heat shock 70 family of proteins; and (vi) the inferred amino acid sequence contains a contiguous 73-amino acid region having 51% identity with the extracellular sperm receptor binding domain in Strongylocentrosus purpuratus (Foltz, K . R., Partin, J . S., and Lennarz, W . J . (1993) Science 259, 1421-1425) . The potential involvement of an hsp70 protein in attachment may provide new insight on adherence mechanisms by obligate intracellular pathogens.

J Biol Chem, 1993 Nov 5, 268(31), 23132 - 8
Function of the active-site lysine in Escherichia coli serine hydroxymethyltransferase; Schirch D et al.; Serine hydroxymethyltransferase has a conserved lysine residue (Lys-229) that forms the internal aldimine with pyridoxal 5'-phosphate . In other pyridoxal 5'-phosphate enzymes investigated so far, this conserved lysine residue also plays a catalytic role as a base that removes the alpha-proton from the amino acid substrate . Three mutant forms of Escherichia coli serine hydroxymethyltransferase (K229Q, K229R, and K229H) were constructed, expressed, and purified . The absorbance spectra, rapid reaction kinetics, and thermal denaturation of the mutant analogs were studied . Only the K229Q mutant serine hydroxymethyltransferase resembled the wild-type enzyme . The results indicate that Lys-229 plays a critical role in expelling the product by converting the external aldimine to an internal aldimine . In the absence of Lys-229, ammonia can also catalyze the same function at a much slower rate . However, Lys-229 apparently is not the base that removes the alpha-proton from the amino acid substrate . The K229Q mutant enzyme could catalyze one turnover of either serine to glycine or glycine to serine at rates approaching those of the wild-type enzyme . After one turnover, the mutant enzyme could not expel the product and bind new substrate . The K229Q mutant enzyme can also transaminate D-alanine, which, like the hydroxymethyltransferase activity, also requires removing the alpha-proton from the substrate . The absorbance spectra of the K229R and K229H serine hydroxymethyltransferases showed that their pyridoxal 5'-phosphate could not readily form an external aldimine with substrates, suggesting that Lys-229 in the wild-type enzyme may never bear a positive charge, further evidence that it is not the base that removes the alpha-proton.

J Biol Chem, 1993 Nov 5, 268(31), 23128 - 31
Effect of the relative position of the UGA codon to the unique secondary structure in the fdhF mRNA on its decoding by selenocysteinyl tRNA in Escherichia coli; Chen GF et al.; The fdhF mRNA for formate dehydrogenase H of Escherichia coli contains a UGA codon at position 140 . This termination codon is decoded by selenocysteinyl tRNA (the selC product) with the aid of its own specific elongation factor, SelB . For this decoding, a unique secondary structure immediately downstream of the UGA codon has been shown to be essential (Zinoni, F., Heider, J., and Bock, A . (1990) Proc . Natl . Acad . Sci . U . S . A . 87, 4660-4664) . We examined the positional effect of the UGA codon relative to the secondary structure on its decoding using a fdhF-lacZ fusion gene . When the UGA codon was separated by one codon (position -1) from the secondary structure, the UGA decoding, as measured by the beta-galactosidase activity, dropped to approximately 76% of the normal level but was still almost as fully dependent upon selC and selenium in the culture medium as in the case of the UGA codon in the normal position (position 0) . However, when the UGA codon was separated by two codons (position -2), the decoding level further dropped to 20% of the normal level, and in addition, became dependent only on selC but independent of selenium . When the UGA codon was further separated by three codons (position -3), the decoding level of UGA (-3) became higher than the decoding of UGA (-2) and was completely independent from selC and selenium, indicating that the UGA codon was nonspecifically suppressed . A similar nonspecific suppression was observed for the UGA codon at position -4, but at a lower level . When two UGA codons were tandemly placed at positions 0 and -1, they were still able to be decoded at 17% of the normal level in a selC- and selenium-dependent manner . In the absence of the SelB function, the decoding level of UGA(0) dropped to 1.6% of the normal level, whereas the UGA(-1) decoding dropped to 7.5% . These results indicate that the UGA codon at position 0 is not only most effectively decoded by selenocysteinyl tRNA but also tightly blocked from its nonspecific suppression in the absence of any components required for the decoding.

Cell, 1993 Nov 5, 75(3), 557 - 66
Protein-protein interactions in gene regulation: the cAMP-CRP complex sets the specificity of a second DNA-binding protein, the CytR repressor; Sogaard-Andersen L et al.; Maximal repression by the CytR protein depends on the formation of nucleoprotein complexes in which CytR interacts with DNA and with cAMP-cAMP receptor protein (CRP) . Here we demonstrate that CytR regulates transcription from deoP2 promoters in which the entire CytR recognition sequence has been eliminated . Furthermore, CytR proteins deleted for the DNA-binding domain repress deoP2 in vivo and interact with deoP2 in vitro in a strictly cAMP-CRP-dependent fashion . These experiments show that the site of action of CytR can be specified by protein-protein interactions to cAMP-CRP, whereas CytR-DNA interactions may primarily serve to stabilize the nucleo-protein complex . This type of specificity mechanism may represent a general concept in the recruitment of DNA-binding proteins in combinatorial regulatory systems.

J Biol Chem, 1993 Nov 5, 268(31), 23585 - 92
Nuclease activities of Moloney murine leukemia virus reverse transcriptase . Mutants with altered substrate specificities; Blain SW et al.; RNases H are traditionally thought to degrade RNA only in RNA-DNA hybrid form . We found that the wild-type Moloney murine leukemia virus (M-MuLV) reverse transcriptase (RT) was capable of degrading RNA in RNA-RNA duplexes as well as in RNA-DNA hybrids, as assayed by in situ gel techniques . Escherichia coli RNase H does not degrade the RNA-RNA duplex in this assay, while E . coli RNase III, a double-strand-specific ribonuclease, does . The apparent specific activity of M-MuLV RT on RNA-RNA duplexes is similar to that on RNA-DNA hybrids . Neither the DNA polymerase domain nor the RNase H domain of RT expressed individually exhibited this RNA-RNA activity . We have generated a series of mutations in the RNase H domain of M-MuLV RT, expressed the mutant enzymes in E . coli, and assayed these mutants for various activities . All RTs were as active as the wild type in the oligo(dT):poly(rA) DNA polymerase assay, and many retained both nuclease activities . Two enzymes with mutations at the carboxyl terminus of the RNase H domain retained RNA-DNA activity, but not RNA-RNA activity . Another mutant enzyme showed the opposite phenotype, retaining RNA-RNA, but not RNA-DNA, nuclease activity . Thus, we were able to genetically separate the two activities . These results may be helpful in defining enzyme-substrate interactions.

Regul Pept, 1993 Nov 3, 48(3), 301 - 7
Vasoactive intestinal peptide and helodermin inhibit phospholipase A2 activity in vitro; Trotz ME et al.; We recently reported that the widely distributed neuropeptide vasoactive intestinal peptide (VIP) reduces inflammatory lung injury due to a variety of agents and inhibits the associated generation of cyclo-oxygenase and lipoxygenase products . We therefore investigated whether VIP may inhibit phospholipase A2 activity, thus reducing the release of arachidonic acid, the common precursor of all eicosanoids . VIP dose-dependently inhibited PLA2 of porcine pancreas and of Naja naja venom, as assessed by the release of free {3H}oleic acid from labeled Escherichia coli phospholipids . The potency of VIP was similar to that of mepacrine, with 50% inhibition at 400-500 microM . The closely related peptide helodermin produced 50% inhibition at 200 microM, but secretin and peptide histidine isoleucineamide produced little or no inhibition . The results suggest that VIP and helodermin selectively inhibit PLA2 in vitro . If this activity is exerted in vivo, it may contribute to the anti-inflammatory activity of these two peptides.

Biochim Biophys Acta, 1993 Nov 2, 1183(1), 161 - 70
The proton pumping activity of H(+)-ATPases: an improved fluorescence assay; Rottenberg H et al.; A new method for the estimation of steady-state delta pH, and the rate of acidification, by H(+)-ATPases (and other proton transporters) in inverted membrane vesicles is described . The method is based on a combination of two widely used fluorescent delta pH probes, 9-aminoacridine and 9-amino-6-chloro-2-methoxyacridine . It is demonstrated that 9-amino-6-chloro-2-methoxyacridine fluorescence quenching, which is very sensitive to small pH gradients, is not sensitive to the magnitude of large pH gradient, while 9-aminoacridine, which does not sense small gradients, is very sensitive to large pH gradients . A proper mixture of the two probes provides a method which is equally sensitive to pH gradients from very small values up to 3.5 pH units . The probe response was evaluated by titrations of the fluorescence signal with nigericin and adjusted by changing the concentration ratio and the emission wavelength . In liposomes, submitochondrial particles and bacterial vesicles an almost linear dependence of quenching on delta pH over the entire range can be obtained with this method . It is demonstrated that the new method can be used to obtain more reliable estimates of the rate of acidification as well as the magnitude of delta pH, whereas each of these and similar probes, by themselves are not as reliable . A determination of the ratio delta Gp/delta muH over a wide range of values reveal that this ratio is not constant but decreases with delta Gp . This finding should be taken into consideration when attempting to estimate the H+/ATP ratio form the measurement of delta Gp/delta muH.

Biochemistry, 1993 Nov 2, 32(43), 11618 - 26
Identification of important residues within the putative nucleoside binding site of HSV-1 thymidine kinase by random sequence selection: analysis of selected mutants in vitro; Black ME et al.; Random sequence mutagenesis in conjunction with genetic complementation was used to map the function of amino acid residues within the putative nucleoside binding site of the herpes simplex virus type 1 (HSV-1) thymidine kinase (TK) . Six codons of the putative nucleoside binding site of the HSV-1 tk were substituted by a duplex of extended oligonucleotides containing 20% random sequences . Approximately 260 mutants were screened for the ability to genetically complement a TK-deficient Escherichia coli . Of those screened, 32% conferred TK activity . Approximately 60% of the TK positive clones contained single amino acid changes, 23% contained double changes, and 13.4% encoded the wild-type TK amino acid sequence . A small percentage of clones, 2.4% and 1.2%, contained triple or quadruple alterations, respectively . Three residues (D162, H163, and R164) appeared to be highly conserved especially with regard to the type of residues able to substitute . Secondary screening results indicated that several of the mutants had higher affinities for acyclovir and/or 3'-azido-3'-deoxythymidine than thymidine in complementation assays . In addition, a number of clones were unable to form colonies on selection medium at elevated temperatures (42 degrees C) . Eight selected mutants were subcloned into an in vitro transcription vector and the derived transcripts used to program a rabbit reticulocyte lysate cell-free translation system . Biologically active translation products were then analyzed in vitro for thymidine kinase activity, for thermal stability, and for the ability to phosphorylate selected nucleoside analogues . Two of the eight mutants had an elevated thymidine kinase activity, two were significantly thermolabile, and three exhibited enhanced efficiency in phosphorylation of nucleoside analogues.

Biochemistry, 1993 Nov 2, 32(43), 11569 - 74
Reduction and loss of the iron center in the reaction of the small subunit of mouse ribonucleotide reductase with hydroxyurea; Nyholm S et al.; Ribonucleotide reductase is a key enzyme for DNA synthesis in living cells, and the mechanisms for its reactions with inhibitors are of interest because the inhibitors are potential antiproliferative agents . Protein R2, the small subunit of mouse ribonucleotide reductase, contains a pair of mu-oxo-bridged ferric ions and a tyrosyl free radical in each of its two polypeptide chains . Light absorption spectroscopy was used to probe the reactions of these redox centers with hydroxyurea (HU), a potent inhibitor of iron containing ribonucleotide reductases . In Escherichia coli protein R2, HU reacts with the tyrosyl radical without affecting the iron center . In contrast to the case for the E . coli protein, HU destroys the specific absorbance bands of both the iron center and the radical on a similar time scale in mouse protein R2, and this is accompanied by release of iron from the protein . Anaerobic experiments with the iron chelator bathophenanthroline present during the HU reaction indicate that the iron is released from the mouse R2 protein in the ferrous form after treatment with HU . The reduced iron center, formed by reaction of Fe2+ with mouse apoprotein R2 under anaerobic conditions, was found to be much less stable than the native Fe3+ site in the presence of suitable iron chelators . The observations are of importance for understanding the mode of action of HU on mammalian cells and for the general question of the stability of the iron center of mouse protein R2 in different redox states.

Biochemistry, 1993 Nov 2, 32(43), 11548 - 58
Interaction with arginine 597 of NADPH-cytochrome P-450 oxidoreductase is a primary source of the uniform binding energy used to discriminate between NADPH and NADH; Sem DS et al.; Site-directed mutagenesis has been used in conjunction with pH and alternate substrate/inhibitor studies to characterize the interactions between NADPH-cytochrome P-450 oxidoreductase (P-450R) and the 2'-phosphate of NADP(H) that provide P-450R with its strong nicotinamide nucleotide specificity . It is known that the 2'-phosphate of NADP(H) is bound to P-450R as the dianion and that interactions between it and residues on P-450R provide 5 kcal/mol of essentially uniform binding energy (preceding paper in this issue) . In order to probe these interactions further, Arg597 of P-450R, which is homologous to Arg235 of ferredoxin-NADP+ reductase that forms a salt bridge with the 2'-phosphate of 2'-phospho-AMP in the crystal structure of that complex {Karplus, P . A., Daniels, M . J., & Herriott, J . R . (1991) Science 251, 60}, was mutated to methionine . The mutant protein, P-450R (R597M), does not appear to have a grossly perturbed tertiary structure on the basis of the observation of similar 31P-NMR chemical shifts for FAD (pyrophosphate) bound to it and wild-type (WT) P-450R, although it is more unstable to urea denaturation . P-450R (R597M) has a Km for NADPH that is 150 times that of P-450R (WT) and a Ki for NADP+ that is 240 times that of P-450R (WT) . In contrast, the R597M mutation has only a modest effect on the Km for NADH (0.8 WT) and the Ki for NAD+ (2.9 WT), indicating that Arg597 must have been interacting specifically with the 2'-phosphate of NADP(H) . The R597M mutation has relatively little effect on kcat for NADPH (1.2 WT) or NADH (0.6 WT), indicating that the mutation is affecting ground and transition states to essentially the same degree, by removing 3 kcal/mol of uniform binding energy . The NADP+ pKi profile for P-450R (R597M) shows a pKa of 5.78 for the 2'-phosphate of NADP+, which is bound to P-450R (R597M) as the dianion, but the pKa of 9.5 for the preferentially protonated enzymic group observed in the P-450R (WT) profile is no longer present . It is argued then that the 2'-phosphate binding pocket of P-450R (WT) has a high positive charge density (> + 2) and that Arg597, which is in this binding pocket, has a highly perturbed pKa of 9.5 . Finally, a general theoretical treatment of the thermodynamic consequences of individual and combined perturbations to complementary interacting groups on enzyme and substrate is presented (see Appendix).(ABSTRACT TRUNCATED AT 400 WORDS)

Biochemistry, 1993 Nov 2, 32(43), 11539 - 47
Enzyme-substrate binding interactions of NADPH-cytochrome P-450 oxidoreductase characterized with pH and alternate substrate/inhibitor studies; Sem DS et al.; The pH dependence of the kinetic parameters for the reaction catalyzed by NADPH-cytochrome P-450 oxidoreductase (P-450R) has been determined, using various substrates and inhibitors . All Vmax and (V/K) profiles show pKas of 6.2-7.3, for an acidic group that is preferentially unprotonated for catalysis, and of 8.1-9.6, for a basic group that is preferentially protonated for catalysis . The presence of the wrong ionization state for both of these groups is tolerated more at lower ionic strength (300 mM) than at higher ionic strength (850 mM) . Ionization of the basic group has a more pronounced effect on binding of substrate (cytochrome c or dichloroindophenol) than on catalysis, since ionization has only a 2-fold effect on Vmax with cytochrome c, and only a 5-fold effect on Vmax with dichloroindophenol, while (V/K) for both substrates continues to drop at high pH with no sign of reaching a plateau . Therefore, this basic group affects predominantly substrate binding and, to a lesser extent, catalysis . It is most likely located on the surface of the protein at the cytochrome c/dichloroindophenol binding site, near the FMN prosthetic group . The NADP+ pKi profile shows a pKa of 5.95 for the 2'-phosphate of NADP+, which is bound to P-450R as the dianion, and a pKa of 9.53 for an enzyme group that must be protonated in order to bind NADP+.(ABSTRACT TRUNCATED AT 250 WORDS)

Biochemistry, 1993 Nov 2, 32(43), 11524 - 9
Site-directed mutants of the cytochrome bo ubiquinol oxidase of Escherichia coli: amino acid substitutions for two histidines that are putative CuB ligands; Calhoun MW et al.; The bo-type ubiquinol oxidase of Escherichia coli is a member of the superfamily of structurally related heme-copper respiratory oxidases . The members of this family, which also includes the aa3-type cytochrome c oxidases, contain at least two heme prosthetic groups, a six-coordinate low-spin heme, and a high-spin heme . The high-spin heme is magnetically coupled to a copper, CuB, forming a binuclear center which is the site of oxygen reduction to water . Vectorial proton translocation across the membrane bilayer appears to be another common feature of this superfamily of oxidases . It has been proposed previously that the two adjacent histidines in putative transmembrane helix VII (H333 and H334 in the E . coli sequence) of the largest subunit of the heme-copper oxidases are ligands to CuB . Previously reported mutagenesis studies of the E . coli bo-type oxidase and the aa3-type oxidase of Rhodobacter sphaeroides supported this model, as substitutions at these two positions produced nonfunctional enzymes but did not perturb the visible spectra of the two heme groups . In this work, six different amino acids, including potential copper-liganding residues, were substituted for H333 and H334 of the E . coli oxidase . All of the mutations resulted in inactive, but assembled, oxidase with both of the heme components present . However, cryogenic Fourier transform infrared (FTIR) spectroscopy of the CO adducts revealed that dramatic changes occur at the binuclear center as a result of each mutation and that CuB appears to be absent.(ABSTRACT TRUNCATED AT 250 WORDS)

Mol Cell Biol, 1993 Nov, 13(11), 7045 - 55
The ZEBRA activation domain: modular organization and mechanism of action; Chi T et al.; An RNA polymerase II activator often contains several regions that contribute to its potency, an organization ostensibly analogous to the modular architecture of promoters and enhancers . The regulatory significance of this parallel organization has not been systematically explored . We considered this problem by examining the activation domain of the Epstein-Barr virus transactivator ZEBRA . We performed our experiments in vitro so that the activator concentrations, stabilities, and affinities for DNA could be monitored . ZEBRA and various amino-terminal deletion derivatives, expressed in and purified from Escherichia coli, were assayed in a HeLa cell nuclear extract for the ability to activate model reporter templates bearing one, three, five, and seven upstream ZEBRA binding sites . Our data show that ZEBRA contains four modules that contribute to its potency in vitro . The modules operate interchangeably with promoter sites to determine the transcriptional response such that the loss of modules can be compensated for by increasing promoter sites . Potassium permanganate footprinting was used to show that transcriptional stimulation is a consequence of the activator's ability to promote preinitiation complex assembly . Kinetic measurements of transcription complex assembly in a reconstituted system indicate that ZEBRA promotes formation of a subcomplex requiring the TFIIA and TFIID fractions, where TFIIA acts as an antirepressor . We propose a model in which the concentration of DNA-bound activation modules in the vicinity of the gene initiates synergistic transcription complex assembly.

Mol Cell Biol, 1993 Nov, 13(11), 6841 - 8
Short artificial hairpins sequester splicing signals and inhibit yeast pre-mRNA splicing; Goguel V et al.; To examine the stability of yeast (Saccharomyces cerevisiae) pre-mRNA structures, we inserted a series of small sequence elements that generated potential RNA hairpins at the 5' splice site and branch point regions . We analyzed spliceosome assembly and splicing in vitro as well as splicing and nuclear pre-mRNA retention in vivo . Surprisingly, the inhibition of in vivo splicing approximately paralleled that of in vitro splicing . Even a 6-nucleotide hairpin could be shown to inhibit splicing, and a 15-nucleotide hairpin gave rise to almost complete inhibition . The in vitro results indicate that hairpins that sequester the 5' splice site have a major effect on the early steps of spliceosome assembly, including U1 small nuclear ribonucleoprotein binding . The in vivo experiments lead to comparable conclusions as the sequestering hairpins apparently result in the transport of pre-mRNA to the cytoplasm . The observations are compared with previous data from both yeast and mammalian systems and suggest an important effect of pre-mRNA structure on in vivo splicing.

J Immunol, 1993 Nov 1, 151(9), 4964 - 72
Study of natural lipocortin I . A potent mediator for macrophage-mediated immunosuppression in tumor-bearing mice; Sakata T et al.; We previously reported a possible role of lipocortin I secreted from Mac-1+, -2+ macrophages in immunosuppression in tumor-bearing mice . In this study, we purified natural lipocortin I from the spleens of tumor-bearing mice and compared its immunosuppressive activity with recombinant lipocortin I produced by Escherichia coli . The culture supernatants of splenic macrophages from tumor-bearing mice suppressed the mitogenic responses in splenic lymphocytes . The culture supernatants contained higher levels of lipocortin I, but not PGE2, which was considered as a major immunosuppressive factor . Anti-lipocortin I antiserum neutralized these inhibitory activities . Natural lipocortin I purified from the spleens of tumor-bearing mice showed a potent suppressive activity, which was > or = 200 times higher than that of recombinant mouse lipocortin I at protein level . Alkaline phosphatase treatment of natural lipocortin I decreased immunosuppressive activity to the level of recombinant lipocortin I . N-glycosidase F treatment failed to decrease the immunosuppressive activities . These results suggest that the protein modification of lipocortin I, possibly phosphorylation but not glycosylation, is critical for the potent immunosuppressive activity in tumor-bearing mice.

Infect Immun, 1993 Nov, 61(11), 4540 - 5
Organization of two invariant surface glycoproteins in the surface coat of Trypanosoma brucei; Ziegelbauer K et al.; The surface coat of Trypanosoma brucei, formed by about 10(7) molecules of the membrane-form variant surface glycoprotein (mfVSG) per cell, is generally considered to constitute a barrier against the access of antibodies directed to invariant surface proteins . The recent characterization of two invariant surface glycoproteins (ISGs) with apparent molecular masses of 65 and 75 kDa (ISG65 and ISG75; 70,000 and 50,000 molecules per cell, respectively), which are both predicted to be composed of large extracellular domains, single transmembrane alpha-helices, and small intracellular domains, enabled a critical test of this hypothesis . Although ISG65 is distributed over the entire surface of the parasites, it is not accessible to antibodies or to the proteinase trypsin in live cells provided the mfVSG is also proteinase resistant . ISG75 is similarly distributed; its accessibility to antibodies depends on the expressed mfVSG, and it is sensitive to trypsin in a variant clone in which the mfVSG is proteinase resistant . Vaccination experiments using recombinant proteins to a mixture of the native ISGs were unsuccessful . ISG65 but not ISG75 elicited an antibody response in chronically infected mice . The results strengthen the view of the protective properties of the variant surface glycoprotein coat by steric hindrance and suggest that additional factors such as low abundance or low immunogenicity of invariant surface proteins may prevent a control of the disease by the humoral immune response.

Plant Cell, 1993 Nov, 5(11), 1651 - 9
Molecular characterization of a vacuolar processing enzyme related to a putative cysteine proteinase of Schistosoma mansoni; Hara-Nishimura I et al.; Proproteins of various vacuolar proteins are post-translationally processed into mature forms by the action of a unique vacuolar processing enzyme . If such a processing enzyme is transported to vacuoles together with proprotein substrates, the enzyme must be a latent form . Immunocytochemical localization of a vacuolar processing enzyme, a 37-kD cysteine proteinase, in the endosperm of maturing castor bean seeds places the enzyme in the vacuolar matrix, where a variety of proproteins is also present . To characterize a molecular structure of vacuolar processing enzyme, we isolated a cDNA for the enzyme . Deduced primary structure of a 55-kD precursor is 33% identical to a putative cysteine proteinase of the human parasite Schistosoma mansoni . The precursor is composed of a signal peptide, a 37-kD active processing enzyme domain, and a propeptide fragment . Although the precursor expressed in Escherichia coli has no vacuolar processing activity, a 36-kD immunopositive protein expressed in E . coli is active . These results suggest that the activation of the vacuolar processing enzyme requires proteolytic cleavage of a 14-kD C-terminal propeptide fragment of the precursor.

Plant Cell, 1993 Nov, 5(11), 1529 - 39
An Arabidopsis myb homolog is induced by dehydration stress and its gene product binds to the conserved MYB recognition sequence; Urao T et al.; An Arabidopsis cDNA (Atmyb2) that contains a sequence that encodes a transcription factor, which is a homolog of MYB, was cloned from a cDNA library prepared from dehydrated Arabidopsis rosette plants . A gene (Atmyb2) corresponding to the Atmyb2 cDNA was also cloned and its nucleotide sequence was determined . RNA gel blot analysis showed that the Atmyb2 mRNA was induced by dehydration and disappeared upon rehydration . The Atmyb2 mRNA also accumulated upon salt stress and with the onset of treatment with abscisic acid . A beta-glucuronidase reporter gene driven by the Atmyb2 promoter was induced by dehydration and salt stress in transgenic Arabidopsis plants . These observations indicate that Atmyb2 is responsive to dehydration at the transcriptional level . The putative protein (ATMYB2) encoded by Atmyb2 has 274 amino acids, a molecular mass of 32 kD, and a putative DNA binding domain that shows considerable homology to plant MYB-related proteins, such as maize C1 . A fusion protein that included ATMYB2 was expressed in Escherichia coli, and it bound specifically to oligonucleotides that contained a consensus MYB recognition sequence (TAACTG), such as is found in the simian virus 40 enhancer and the maize bronze-1 promoter . Binding was sequence specific, as indicated by a gel mobility shift experiment . These results suggest that a MYB-related transcription factor is involved in the regulation of genes that are responsive to water stress in Arabidopsis.

Protein Eng, 1993 Nov, 6(8), 981 - 8
Cloning, sequencing and expression of the Fab fragment of a monoclonal antibody to the herbicide atrazine; Ward VK et al.; The Fab region of an IgG2b antibody (AM7B2.1) reactive to the herbicide atrazine was cloned into a plasmid vector using the polymerase chain reaction and two sets of degenerate oligonucleotide primers designed to mimic the amino acid variation at the N-termini of kappa L-chains and gamma H-chains . These primers also provide a secretion signal fused precisely to the antibody gene sequence for secretion of the mature antibody . A further set of universal oligonucleotide primers was developed for the direct sequencing of the VH and CH1 regions of gamma H-chains and the VL and CL regions of kappa L-chains without subcloning and were used to determine the sequence of this antibody . The kappa L-chain was found to not possess a conserved Cys residue at position 23 and the implications of this observation are discussed . The cloned genes were expressed in Escherichia coli using a commercially available T7 RNA polymerase-based plasmid . The clones were also expressed in a T7 RNA polymerase-based system containing an attenuated version of the T7 RNA polymerase promoter, plus a lac promoter placed in an antisense orientation, to enhance plasmid stability . The expressed products were confirmed as atrazine reactive by binding to an atrazine derivative conjugated with alkaline phosphatase.

Protein Eng, 1993 Nov, 6(8), 901 - 6
Identification by site-directed mutagenesis of amino acids in the B2 subsite of bovine pancreatic ribonuclease A; Tarragona-Fiol A et al.; In addition to hydrolysing RNA, bovine pancreatic ribonuclease splits esters of pyrimidine nucleoside 3'-phosphates, including dinucleotides . For a series of 3':5'-linked dinucleotides of general structure CpN, where N is a 5' linked nucleoside, kcat for the release of N varies enormously with the precise structure of N . Structural studies have been interpreted to indicate that the group N interacts with a subsite, B2, on the enzyme that comprises Gln69, Asn71 and Glu111 . We report studies by site-directed mutagenesis that indicate that Gln69 is not involved in productive interactions with any of the dinucleotide substrates and that Asn71 is an important component of subsite B2 for all dinucleotide substrates tested . Glu111 appears to be functionally involved in catalysis for dinucleotide substrates solely when N is guanosine.

J Cell Sci, 1993 Nov, 106 ( Pt 3), 919 - 28
In vitro assembly properties of vimentin mutagenized at the beta-site tail motif; Kouklis PD et al.; The intermediate filament (IF) proteins vimentin, desmin and peripherin share a 9-residue sequence motif (beta-site) located near the end of their COOH-terminal tail domain . Peptide inhibition experiments have previously suggested that the beta-site is involved in interactions that limit the lateral growth of IFs and prevent inappropriate filament-filament associations . To investigate this question further, we have constructed and expressed, in Escherichia coli, hamster vimentin bearing different mutations in the beta-site . We show here that a single exchange of glycine 450 with a valine residue, or an internal deletion of amino acids 444-452, strongly interferes with the normal assembly of IFs under in vitro conditions . These mutants polymerize into irregular fibrils that have a strong tendency to anastomose and laterally aggregate under isotonic conditions . In contrast, a non-conservative substitution of arginine 448 for glutamic acid does not significantly interfere with filament structure and yields subunits that polymerize into long, smooth filaments that show a slight aberration in thickness . All mutant proteins are soluble in low salt and form oligomers similar to the ones formed by wild-type vimentin . On the basis of these findings and on related observations, we propose that the tail domain of type III IF proteins contains important structural elements involved in lateral protofilament-protofilament interactions.

Can J Microbiol, 1993 Nov, 39(11), 1066 - 70
Detection of Escherichia coli by the nutrient agar plus 4-methylumbelliferyl beta-D-glucuronide (MUG) membrane filter method; Shadix LC et al.; A two-step membrane filter procedure was evaluated to determine the ability to differentiate Escherichia coli from other coliform bacteria recovered from water . M-Endo LES agar incubated at 35 degrees C for 24 +/- 2 h was used as the initial isolation medium . Membranes containing coliform colonies were transferred to nutrient agar plus 4-methylumbelliferyl beta-D-glucuronide (MUG) and incubated for an additional 4 h at 35 degrees C . Escherichia coli colonies were distinguished by fluorescence when viewed under a long-wavelength ultraviolet light . A total of 119 MUG-positive colonies were isolated from 15 water sources, of which 115 (96.6%) were identified as E . coli . An examination of 182 pure culture environmental E . coli isolates revealed that 167 isolates (91.8%) exhibited fluorescence on the nutrient agar plus MUG medium . Survivors of E . coli cultures exposed to chlorination were also capable of producing a positive MUG reaction.

Mol Biol Cell, 1993 Nov, 4(11), 1145 - 59
Genetic interactions between KAR2 and SEC63, encoding eukaryotic homologues of DnaK and DnaJ in the endoplasmic reticulum; Scidmore MA et al.; KAR2 encodes the yeast homologue of mammalian BiP, the endoplasmic reticulum (ER) resident member of the HSP70 family . Kar2p has been shown to be required for the translocation of proteins across the ER membrane as well as nuclear fusion . Sec63, an ER integral membrane protein that shares homology with the Escherichia coli DnaJ protein, is also required for translocation . In this paper we describe several specific genetic interactions between these two proteins, Kar2p and Sec63p . First, temperature-sensitive mutations in KAR2 and SEC63 form synthetic lethal combinations . Second, dominant mutations in KAR2 are allele-specific suppressors for the temperature-sensitive growth and translocation defect of sec63-1 . Third, the sec63-1, unlike other translocation defective mutations, results in the induction of KAR2 mRNA levels . Taken together, these genetic interactions suggest that Kar2p and Sec63p interact in vivo in a manner similar to that of the E . coli HSP70, DnaK, and DnaJ . We propose that the interaction between these two proteins is critical to their function in protein translocation.

Mol Biol Cell, 1993 Nov, 4(11), 1121 - 32
Autonomous replication of human chromosomal DNA fragments in human cells; Masukata H et al.; We have examined whether a human chromosome has distinct segments that can replicate autonomously as extrachromosomal elements . Human 293S cells were transfected with a set of human chromosomal DNA fragments of 8-15 kilobase pairs that were cloned on an Escherichia coli plasmid vector . The transfected cells were subsequently cultured in the presence of 5-bromodeoxyuridine during two cell generations, and several plasmid clones labeled in both of the daughter DNA strands were isolated . Efficiency of replication of these clones, as determined from the ratios of heavy-heavy and one-half of heavy-light molecules to total molecules recovered from density-labeled cells, was 9.4% per cell generation on the average . Replication efficiency of control clones excluded during the selection was about 2.2% and that of the vector plasmid alone was 0.3% . A representative clone p1W1 replicated in a semiconservative manner only one round during the S phase of the cell cycle . It replicated extrachromosomally without integration into chromosome . The human segment of the clone was composed of several subsegments that promoted autonomous replication at different efficiencies . Our results suggest that certain specific nucleotide sequences are involved in autonomous replication of human segments.

Bioconjug Chem, 1993 Nov-Dec, 4(6), 549 - 53
Synthesis and ribosome binding properties of model mRNAs modified with undecagold cluster; Skripkin E et al.; The synthesis and purification of short model messenger RNAs modified with undecagold cluster are described . A monoamino undecagold cluster was introduced on the oxidized 3' cis-glycol group of the mRNA followed by reduction of the formed Schiff's base . The stability of the modified mRNA under the conditions used for in vitro messenger RNA translation is studied . The possibility of the formation of a specific translational initiation complex with bacterial ribosomes and modified mRNAs is shown . The results of these experiments indicate that the attachment of an undecagold cluster to a mRNA is a useful tool for electron microscopic and crystallographic studies.

Mol Gen Mikrobiol Virusol, 1993 Nov-Dec, (6), 23 - 7
{High plasmid pBR322 multimerization in Escherichia coli B}; Viacheslavov LG et al.; In bacteria E . coli B (wild) plasmid pBR322 is present as a set of circular supercoiled multimers formed of up to 20 head-to-tail linked monomers . The highest degree of plasmid multimerization was achieved when the bacteria were grown in a mineral-glucose medium based on 0.15 M phosphate buffer (pH 7.5) . In E . coli K12 grown under similar conditions, plasmid pBR322 was mainly present as monomers . A kinetic experiment (initially only monomeric plasmids were present in the cells) has shown that multimerization of plasmids occurs step by step, that is, by means of interplasmid recombinations . Therefore, in E . coli B the efficiency of interplasmid recombinations is higher in comparison with E . coli K12 . This result reflects some differences between basic systems of genetic recombination operating in these two lines of E . coli.

Anticancer Res, 1993 Nov-Dec, 13(6A), 2037 - 43
The genotoxicity of unsubstituted and nitrated polycyclic aromatic hydrocarbons; Mersch-Sundermann V et al.; To determine the DNA damaging properties of unsubstituted and substituted polycyclic hydrocarbons, 61 aromatic and heterocyclic compounds were examined for the induction of the SOS system in E . coli PQ37 . PAH such as benzo{ghi}fluoranthene, benzo{j}fluoranthene, benzo{c}phenanthrene, benzo{a}pyrene, chrysene, dibenzo{a,1}pyrene, fluoranthene and triphenylene showed relatively high genotoxicity . With respect to the nitroarenes, the highest genotoxic potencies were exhibited by the dinitropyrenes . The SOS-inducing potency of nitroarenes increased from the bicyclic to the tetracyclic ring system . Additionally, it was seen that any increase in the extent of nitration is paralleled by an increase of genotoxicity . Whereas PAH required metabolic activation by hepatic cytochrome P-450 enzymes, nPAH were direct-acting genotoxicants.

Genetics, 1993 Nov, 135(3), 643 - 54
Use of high and low level overexpression plasmids to test mutant alleles of the recF gene of Escherichia coli K-12 for partial activity; Sandler SJ et al.; We showed that sufficient overexpression of the wild-type recF gene interfered with three normal cell functions: (1) UV induction of transcription from the LexA-protein-repressed sulA promoter, (2) UV resistance and (3) cell viability at 42 degrees . To show this, we altered a low-level overexpressing recF+ plasmid with a set of structurally neutral mutations that increased the rate of expression of recF . The resulting high-level overexpressing plasmid interfered with UV induction of the sulA promoter, as did the low-level overexpressing plasmid . It also reduced UV resistance more than its low level progenitor and decreased viability at 42 degrees, an effect not seen with the low-level plasmid . We used the high-level plasmid to test four recF structural mutations for residual activity . The structural alleles consisted of an insertion mutation, two single amino acid substitution mutations and a double amino acid substitution mutation . On the Escherichia coli chromosome the three substitution mutations acted similarly to a recF deletion in reducing UV resistance in a recB21 recC22 sbcB15 sbcC201 genetic background . By this test, therefore, all three appeared to be null alleles . Measurements of conjugational recombination revealed, however, that the three substitution mutations may have residual activity . On the high-level overexpressing plasmid all three substitution mutations definitely showed partial activity . By contrast, the insertion mutation on the high-level overexpressing plasmid showed no partial activity and can be considered a true null mutation . One of the substitutions, recF143, showed a property attributable to a leaky mutation . Another substitution, recF4101, may block selectively two of the three interference phenotypes, thus allowing us to infer a mechanism for them.

Genetics, 1993 Nov, 135(3), 631 - 42
A sister-strand exchange mechanism for recA-independent deletion of repeated DNA sequences in Escherichia coli; Lovett ST et al.; In the genomes of many organisms, deletions arise between tandemly repeated DNA sequences of lengths ranging from several kilobases to only a few nucleotides . Using a plasmid-based assay for deletion of a 787-bp tandem repeat, we have found that a recA-independent mechanism contributes substantially to the deletion process of even this large region of homology . No Escherichia coli recombination gene tested, including recA, had greater than a fivefold effect on deletion rates . The recA-independence of deletion formation is also observed with constructions present on the chromosome . RecA promotes synapsis and transfer of homologous DNA strands in vitro and is indispensable for intermolecular recombination events in vivo measured after conjugation . Because deletion formation in E . coli shows little or no dependence on recA, it has been assumed that homologous recombination contributes little to the deletion process . However, we have found recA-independent deletion products suggestive of reciprocal crossovers when branch migration in the cell is inhibited by a ruvA mutation . We propose a model for recA-independent crossovers between replicating sister strands, which can also explain deletion or amplification of repeated sequences . We suggest that this process may be initiated as post-replicational DNA repair; subsequent strand misalignment at repeated sequences leads to genetic rearrangements.

Appl Environ Microbiol, 1993 Nov, 59(11), 3771 - 7
Characterization of enzymatic reduction of hexavalent chromium by Escherichia coli ATCC 33456; Shen H et al.; Chromium reduction by Escherichia coli ATCC 33456 quantitatively transferred hexavalent chromium, Cr(VI), to trivalent chromium, Cr(III) . The reduced chromium was predominantly present in the external medium . Supernatant fluids of cell extract, obtained by centrifugation at 12,000 and 150,000 x g, showed almost the same Cr(VI) reduction activity, indicating that Cr(VI) reduction by E . coli ATCC 33456 was a largely soluble reductase activity . In studies with respiratory inhibitors, no inhibitory effects on aerobic and anaerobic Cr(VI) reduction were demonstrated by addition of cyanide, azide, and rotenone into both intact cell cultures and supernatant fluids of E . coli ATCC 33456 . Although cytochromes b and d were identified in the membrane fraction of cell extracts, Cr(VI) was not reduced by the membrane fraction alone . The cytochrome difference spectra analysis also indicated that these cytochromes of the respiratory chain require the presence of the soluble Cr(VI) reductase to mediate electron transport to Cr(VI) . Stimulation of Cr(VI) reduction by an uncoupler, 2,4-dinitrophenol, indicated that the respiratory-chain-linked electron transport to Cr(VI) was limited by the rate of dissipation of the proton motive force.

Appl Environ Microbiol, 1993 Nov, 59(11), 3564 - 71
Cloning and characterization of a cDNA from Aspergillus parasiticus encoding an O-methyltransferase involved in aflatoxin biosynthesis; Yu J et al.; Aflatoxins are polyketide-derived secondary metabolites produced by the fungi Aspergillus flavus and Aspergillus parasiticus . Among the catalytic steps in the aflatoxin biosynthetic pathway, the conversion of sterigmatocystin to O-methylsterigmatocystin and the conversion of dihydrosterigmatocystin to dihydro-O-methylsterigmatocystin are catalyzed by an S-adenosylmethionine-dependent O-methyltransferase . A cDNA library was constructed by using RNA isolated from a 24-h-old culture of wild-type A . parasiticus SRRC 143 and was screened by using polyclonal antiserum raised against a purified 40-kDa O-methyltransferase protein . A clone that harbored a full-length cDNA insert (1,460 bp) containing the 1,254-bp coding region of the gene omt-1 was identified by the antiserum and isolated . The complete cDNA sequence was determined, and the corresponding 418-amino-acid sequence of the native enzyme with a molecular weight of 46,000 was deduced . This 46-kDa native enzyme has a leader sequence of 41 amino acids, and the mature form of the enzyme apparently consists of 377 amino acids and has a molecular weight of 42,000 . Direct sequencing of the purified mature enzyme from A . parasiticus SRRC 163 showed that 19 of 22 amino acid residues were identical to the amino acid residues in an internal region of the deduced amino acid sequence of the mature protein . The 1,460-bp omt-1 cDNA was cloned into an Escherichia coli expression system; a Western blot (immunoblot) analysis of crude extracts from this expression system revealed a 51-kDa fusion protein (fused with a 5-kDa beta-galactosidase N-terminal fragment).(ABSTRACT TRUNCATED AT 250 WORDS)

Appl Environ Microbiol, 1993 Nov, 59(11), 3534 - 44
New medium for the simultaneous detection of total coliforms and Escherichia coli in water; Brenner KP et al.; A new membrane filter agar medium (MI agar) containing a chromogen, indoxyl-beta-D-glucuronide, and a fluorogen, 4-methylumbelliferyl-beta-D-galactopyranoside, was developed to simultaneously detect and enumerate Escherichia coli and total coliforms (TC) in water samples on the basis of their enzyme activities . TC produced beta-galactosidase, which cleaved 4-methylumbelliferyl-beta-D-galactopyranoside to form 4-methylumbelliferone, a compound that fluoresced under longwave UV light (366 nm), while E . coli produced beta-glucuronidase, which cleaved indoxyl-beta-D-glucuronide to form a blue color . The new medium TC and E . coli recoveries were compared with those of mEndo agar and two E . coli media, mTEC agar and nutrient agar supplemented with 4-methylumbelliferyl-beta-D-glucuronide, using natural water samples and spiked drinking water samples . On average, the new medium recovered 1.8 times as many TC as mEndo agar, with greatly reduced background counts (< or = 7%) . These differences were statistically significant (significance level, 0.05) . Although the overall analysis revealed no statistically significant difference between the E . coli recoveries on MI agar and mTEC agar, the new medium recovered more E . coli in 16 of 23 samples (69.6%) . Both MI agar and mTEC agar recovered significantly more E . coli than nutrient agar supplemented with 4-methylumbelliferyl-beta-D-glucuronide . Specificities for E . coli, TC, and noncoliforms on MI agar were 95.7% (66 of 69 samples), 93.1% (161 of 173 samples), and 93.8% (61 of 65 samples), respectively . The E . coli false-positive and false-negative rates were both 4.3% . This selective and specific medium, which employs familiar membrane filter technology {corrected} to analyze several types of water samples, is less expensive than the liquid chromogen and fluorogen media and may be useful for compliance monitoring of drinking water.

Mol Biol (Mosk), 1993 Nov-Dec, 27(6), 1386 - 93
{Recognition of T-T cyclobutane pyrimidine dimers in the form of primers by DNA polymerases}; Kolocheva TI et al.; A study was made of the efficiency of primer conversion catalyzed by human placenta DNA polymerase on the length of primer . The dependence of -log Kd and Vmax on the number of mononucleotide units (n) of primer were shown to be linear up to n <--> 10 . Each mononucleotide unit of the primer enhanced its affinity by a factor of 2.3 and the maximal polymerization rate by a factor of 1.3 . For the first time estimation was made for the contribution of replication enzymes: human placenta DNA polymerase and E . coli DNA polymerase I (as well as AMV reverse transcriptase) to a decrease in the amount of T-T dimers in DNA emerging after UV- or gamma-irradiation . The efficiency of elongation of the d(pT)10 primer was shown to decrease with an increase in T-T dimers in the primer . When the d(pT)10 primer contains about 2.6 T-T dimers per molecule, the efficiency of its elongation decreases by a factor of 8-18.

Mol Biol (Mosk), 1993 Nov-Dec, 27(6), 1380 - 5
{Recognition of primers, containing noncomplementary nucleotides and units, lacking bases, by the Klenow fragment of Escherichia coli DNA polymerase I}; Kolocheva TI et al.; A comparison was carried out for the Km and maximal rates of conversion on (Vmax) with primers containing noncomplementary bases, as well as primers without several bases, and fully complementary primers of the same length . The number of complementary bases from the 3' end to the noncomplementary nucleotide was shown to determine the efficiency of interaction (and conversion) of the primers containing noncomplementary bases with enzyme . The DNA polymerase practically does not discriminate between the primers without one or two bases and the fully complementary primers . Elimination of one base in any position from the 3' end of the primer is equivalent to shortening of the primer by one nucleotide unit, and leads to a decrease in the affinity by a factor of 1.8 . We suppose that DNA polymerase does not participate in primer mistake correction during the repair process if the DNA contains apurinic or apyrimidinic nucleotide units.

Mol Biol Evol, 1993 Nov, 10(6), 1380 - 95
Molecular mechanisms of colicin evolution; Riley MA; This review explores features of the origin and evolution of colicins in Escherichia coli . First, the evolutionary relationships of 16 colicin and colicin-related proteins are inferred from amino acid and DNA sequence comparisons . These comparisons are employed to detail the evolutionary mechanisms involved in the origin and diversification of colicin clusters . Such mechanisms include movement of colicin plasmids between strains of E . coli and subsequent plasmid cointegration, transposition- and recombination-mediated transfer of colicin and related sequences, and rapid diversification of colicin and immunity proteins through the action of positive selection . The wealth of information contained in colicin sequence comparisons makes this an ideal system with which to explore molecular mechanisms of evolutionary change.

J Gen Microbiol, 1993 Nov, 139 ( Pt 11), 2711 - 4
Dimensional rearrangement of Escherichia coli B/r cells during a nutritional shift-down; Zaritsky A et al.; In a search for the mechanism underlying dimensional changes in bacteria, the glucose analogue methyl alpha-D-glucoside was used to effect a rapid reduction in the mass growth rate of Escherichia coli by competitively inhibiting glucose uptake, a so-called nutritional shift-down . The new steady-state cell mass and volume were reached after 1 h, during which the rate of cell division was maintained; rearrangement of the linear dimensions (cell length, diameter), however, required an additional 2 h and caused an undershoot in cell length, consistent with the view that E . coli is slow to modify its diameter . The results are compared with the overshoot in cell length that occurs following nutritional shift-up.

J Gen Microbiol, 1993 Nov, 139 ( Pt 11), 2677 - 84
Characterization of regulatory mutations causing anaerobic derepression of the sodA gene in Escherichia coli K12: cooperation between cis- and trans-acting regulatory loci; Beaumont MD et al.; The genetic loci leading to anaerobic derepression of a sodA::lacZ protein fusion in a UV-generated mutant strain (UV14) of Escherichia coli were identified . The mutant (UV14) was found to harbour two altered loci: one is in the trans-regulatory gene fnr (fumarate nitrate reduction) where leucine-129 was changed to glutamine (fnr14), and the second (sodA14) is in the promoter region (cis) of the sodA gene apparently affecting the binding of the Fur (ferric uptake regulation) protein . Introduction of an fnr+ gene into UV14 restored anaerobic repression of sodA::lacZ and restored the ability of the cells to reduce nitrate . However, when either the fnr14 or the sodA14 mutation was introduced into an otherwise wild-type background, only slight anaerobic derepression of sodA was observed . When both the cis- and trans-acting mutations (i.e . sodA14 and fnr14) were combined simultaneously in an otherwise wild-type background, the specific activity of sodA::lacZ expression was comparable to that of the original mutant strain (UV14) . Furthermore, a genetically confirmed fur fnr double mutant was also similarly derepressed in anaerobic sodA::lacZ expression . The data presented suggest that the cis-mutation in UV14 (sodA14) affects the Fur-binding site in the sodA promoter, while having no effect on Fnr or Arc mediated repression . Also, a second putative Fnr-binding site that straddles the ribosomal binding-site was identified in the sodA gene.

Kansenshogaku Zasshi, 1993 Nov, 67(11), 1108 - 14
{Inhibitory effect of mouse antiendotoxin monoclonal antibody (E5) on hypotension induced by endotoxin and TNF-alpha}; Inada K et al.; Mouse anti-endotoxin monoclonal antibody (E5) is now under clinical trial (Phase II) in Japan by assessing clinical findings and plasma endotoxin levels . We attempted to evaluate an inhibitory effect of E5 on hypotension induced by endotoxin and TNF-alpha . Systolic blood pressure was measured with a programmable sphygmomanometer using the tail-cuff method . The mixture of endotoxin (E . coli O111:B4, Sigma, 2 micrograms/mouse) and recombinant mouse TNF-alpha (Genzyme, 8000 units) were injected into mice (ddY, 6-8-weeks-olds), and the change of blood pressure was observed for 3 hrs . The mixture significantly decreased the blood pressure to about 70% of the control . E5 (20 micrograms/mouse), which was injected 10 min before, significantly abrogated the effect of endotoxin and TNF-alpha . The hypotension induced by the mixture was definitely inhibited by the injection of E5 (50, 100 micrograms/mouse), injected 10 min later, or the injection of E5 (100 micrograms/mouse) 30 min later . We previously found that E5 prevents the lethality induced by a concomitant injection of endotoxin, TNF-alpha and IL-2 . These results suggest that E5 effective for inhibition of endotoxin activity in vivo.

J Dairy Sci, 1993 Nov, 76(11), 3428 - 36
Severity of experimental Escherichia coli mastitis in ketonemic and nonketonemic dairy cows; Kremer WD et al.; The severity of experimental Escherichia coli mastitis in relation to in vitro chemotaxis of polymorphonuclear leukocytes was investigated in cows during negative energy balance . The negative energy balance was induced by feed restriction . Cows were classified into two groups, ketonemic and nonketonemic, based on the beta-hydroxybutyrate concentration in the peripheral blood at the moment of inoculation . Bacterial growth in the inoculated quarter was used as a parameter to indicate the severity of experimental mastitis . In the nonketonemic cows, experimental mastitis ranged from moderate to severe . Severity of experimental mastitis was negatively related to preinfection chemotactic response of polymorphonuclear leukocytes . In contrast, the course of experimental mastitis in the ketonemic group was relatively severe in all cows, regardless of preinfection chemotactic response.

J Virol Methods, 1993 Nov, 45(1), 49 - 66
Use of bacterially-expressed antigen for detection of antibodies to the EBV-specific deoxyribonuclease in sera from patients with nasopharyngeal carcinoma; Chen JY et al.; A cDNA clone, BG9, corresponding to the open reading frame BGLF5 of Epstein-Barr virus (EBV) DNase was inserted into an E . coli expression vector, pET3a, to generate a recombinant plasmid, pDNase 5 . High level of expression of a DNase activity was detected in the E . coli transformed with pDNase 5 following induction with IPTG . The enzyme activity was purified using DEAE-cellulose, phosphocellulose and DNA-cellulose column chromatography . The purified protein appeared to be nearly homogeneous in SDS-PAGE using Coomassie blue staining . The requirement for divalent cations and optimum pH as well as inhibitory concentrations of ionic strength and polyamines for the purified enzyme activity were determined and seemed to be very similar to those of the enzyme activity purified from an EBV producing lymphoblastoid cell line . Using the purified enzyme as an antigen and anti-IgA as the secondary antibody, 82% (64/78) and 91% (71/78) of sera from patients with nasopharyngeal carcinoma (NPC) were shown to be positive by dot immunobinding assay and ELISA, respectively . The results suggest that purified E . coli expressed EBV DNase may be useful for preparing specific test for large scale screening of patients with NPC.

FEMS Microbiol Lett, 1993 Nov 1, 113(3), 279 - 84
Synthesis of the K5 (group II) capsular polysaccharide in transport-deficient recombinant Escherichia coli; Bronner D et al.; The genes directing the expression of group II capsules in Escherichia coli are organized into three regions . The central region 2 is type specific and thought to determine the synthesis of the respective polysaccharide, whilst the flanking regions 1 and 3 are common to all group II gene clusters and direct the surface expression of the capsular polysaccharide . In this communication we analyze the involvement of region 1 and 3 genes in the synthesis of the capsular KS polysaccharide . Recombinant E . coli strains harboring all KS specific region 2 genes and having various combinations of region 1 and 3 genes were studied using immunoelectron microscopy . Membranes from these bacteria were incubated with UDP{14C}GlcA and UDPGlcNAc in the absence or presence of KS polysaccharide as an exogenous acceptor . It was found that recombinant strains with only gene region 2 did not produce the K5 polysaccharide . Membranes of such strains did not synthesize the polymer and did not elongate K5 polysaccharide added as an exogenous acceptor . An involvement of genes from region 1 (notably kpsC and kpsS) and from region 3 (notably kpsT) in the K5 polysaccharide synthesis was apparent and is discussed.

FEMS Microbiol Lett, 1993 Nov 1, 113(3), 273 - 8
Stationary phase-specific expression of the fic gene in Escherichia coli K-12 is controlled by the rpoS gene product (sigma 38); Utsumi R et al.; The fic gene, near pabA located at 75 min of the Escherichia coli chromosome, was previously identified as the regulatory factor of cell division . In this paper we have examined how fic gene expression is controlled during the growth cycle using a fic-lacZ protein fusion plasmid (pFL1) . Its expression was induced at stationary phase while it was nearly abolished in rpoSmutants . Using a RNase protection assay, fic transcript at stationary phase was detected in rpoS+ strains, but not in the rpoS mutants . Furthermore, primer extension analysis indicated that the fic transcript controlled by RpoS initiates at a G located 185 bp upstream from ATG of the fic coding region . Compared with the sigma 70 recognition sequence, the -10 region of fic promoter resembled the Pribnow box, but no homologous sequence was observed at the -35 region . These results were consistent with the characteristic sequence profile of fic promoter recognized specifically by RpoS in vitro, which is the only example of the type III promoter so far detected in vitro and in vivo.

Circ Shock, 1993 Nov, 41(3), 185 - 96
Transmigration routes and a delayed systemic hypotension in rats after intraperitoneal injection of endotoxin from Escherichia coli; Sugimoto K et al.; An intraperitoneal injection of endotoxin (ETX; 3 mg/kg) to rats caused gradual decrease in the systemic arterial blood pressure for up to 3 hr, together with decrease in heart rate, increase in hematocrit, and changes in the core temperature (an initial increase and a subsequent decrease) . Pretreatment of rats with indomethacin (10 mg/kg, p.o.) prevented the decrease in the systemic blood pressure and the changes in other three parameters . The intraperitoneal injection of ETX also induced a gradual increase in exudation of plasma for up to 3 hr, with increased levels of prostaglandin (PG) E2 and 6-keto-PGF1 alpha in the peritoneal exudate . Indomethacin inhibited the exudation of plasma . The levels of ETX in the arterial and portal venous plasmas began to increase 5 min after the intraperitoneal injection of ETX, and reached levels on the order of micrograms per milliliter plasma 10-20 min after the injection . The levels of ETX in the right and left thoracic lymph nodes, but not in the mesenteric lymph nodes, increased in parallel with those in the systemic arterial plasma . In conclusion, the delayed hypotension may be attributable to the mesenteric vasodilatation induced by PGs generated in the peritoneal cavity, and the ETX injected entered the systemic circulation mainly through lymphatic vessels, but in the initial stage, a part of ETX may be transmigrated into portal vein through damaged intestine.

Transgenic Res, 1993 Nov, 2(6), 325 - 9
Cis effect of lacZ sequences in transgenic mice; Paldi A et al.; Transgenic mice carrying the 3-hydroxy-3-methylglutarylCoA reductase (HMG) promoter driving the Escherichia coli beta-galactosidase (lacZ) gene did not display the expected ubiquitous and constitutive expression in HMG-lacZ transgenic mice . The same promoter is however able to drive ubiquitous expression of the chloramphenicol acetyltransferase (cat) gene . Two lines of double HMG-lacZ and HMG-cat transgenic mice were obtained in which the two constructs were integrated at the same genomic sites . These mice expressed both reporter genes, but exclusively in the testes . These results suggest that the lacZ sequence might interfere negatively with the expression of the adjacent HMG-cat transgene.

Protein Sci, 1993 Nov, 2(11), 1959 - 65
Heme biosynthesis in mammalian systems: evidence of a Schiff base linkage between the pyridoxal 5'-phosphate cofactor and a lysine residue in 5-aminolevulinate synthase; Ferreira GC et al.; 5-Aminolevulinate synthase is the first enzyme of the heme biosynthetic pathway in nonplant higher eukaryotes . Murine erythroid 5-aminolevulinate synthase has been purified to homogeneity from an Escherichia coli overproducing strain, and the catalytic and spectroscopic properties of this recombinant enzyme were compared with those from nonrecombinant sources (Ferreira, G.C . & Dailey, H.A., 1993, J . Biol . Chem . 268, 584-590) . 5-Aminolevulinate synthase is a pyridoxal 5'-phosphate-dependent enzyme and is functional as a homodimer . The recombinant 5-aminolevulinate synthase holoenzyme was reduced with tritiated sodium borohydride and digested with trypsin . A single peptide contained the majority of the label . The tritiated peptide was isolated, and its amino acid sequence was determined; it corresponded to 15 amino acids around lysine 313, to which pyridoxal 5'-phosphate is bound . Significantly, the pyridoxyllysine peptide is conserved in all known cDNA-derived 5-aminolevulinate synthase sequences and is present in the C-terminal (catalytic) domain . Mutagenesis of the 5-aminolevulinate synthase residue, which is involved in the Schiff base linkage with pyridoxal 5'-phosphate, from lysine to alanine or histidine abolished enzyme activity in the expressed protein.

Protein Sci, 1993 Nov, 2(11), 1938 - 47
A 19F-NMR study of the membrane-binding region of D-lactate dehydrogenase of Escherichia coli; Sun ZY et al.; D-Lactate dehydrogenase (D-LDH) is a membrane-associated respiratory enzyme of Escherichia coli . The protein is composed of 571 amino acid residues with a flavin adenine dinucleotide (FAD) cofactor, has a molecular weight of approximately 65,000, and requires lipids or detergents for full activity . We used NMR spectroscopy to investigate the structure of D-LDH and its interaction with phospholipids . We incorporated 5-fluorotryptophan (5F-Trp) into the native enzyme, which contains five tryptophan residues, and into mutant enzymes, where a sixth tryptophan is substituted into a specific site by oligonucleotide-directed mutagenesis, and studied the 5F-Trp-labeled enzymes using 19F-NMR spectroscopy . In this way, information was obtained about the local environment at each native and substituted tryptophan site . Using a nitroxide spin-labeled fatty acid, which broadens the resonance from any residue within 15 A, we have established that the membrane-binding area of the protein includes the region between Tyr 228 and Phe 369, but is not continuous within this region . This conclusion is strengthened by the results of 19F-NMR spectroscopy of wild-type enzyme labeled with fluorotyrosine or fluorophenylalanine in the presence and absence of a nitroxide spin-labeled fatty acid . These experiments indicate that 9-10 Phe and 3-4 Tyr residues are located near the lipid phase.

Protein Sci, 1993 Nov, 2(11), 1853 - 61
Tryptophan replacements in the trp aporepressor from Escherichia coli: probing the equilibrium and kinetic folding models; Mann CJ et al.; Mutants of the dimeric Escherichia coli trp aporepressor are constructed by replacement of the two tryptophan residues in each subunit in order to assess the effects on equilibrium and kinetic fluorescence properties of the folding reaction . The three kinetic phases detected by intrinsic tryptophan fluorescence in refolding of the wild-type aporepressor are also observed in folding of both Trp 19 to Phe and Trp 99 to Phe single mutants, demonstrating that these phases correspond to global rather than local conformational changes . Comparison of equilibrium fluorescence (Royer, C.A., Mann, C.J., & Matthews, C.R., 1993, Protein Sci . 2, 1844-1852) and circular dichroism transition curves induced by urea shows that replacement of either Trp 19 or Trp 99 results in noncoincident behavior . Unlike the wild-type protein (Gittelman, M.S . & Matthews, C.R., 1990, Biochemistry 29, 7011-7020), tertiary and/or quaternary structures are disrupted at lower denaturant concentration than is secondary structure . The equilibrium results can be interpreted in terms of enhancement in the population of a monomeric folding intermediate in which the lone tryptophan residue is highly exposed to solvent, but in which substantial secondary structure is retained . The location of both mutations at the interface between the two subunits (Zhang, R.G., et al., 1987, Nature 327, 591-597) provides a simple explanation for this phenomenon.

Protein Sci, 1993 Nov, 2(11), 1844 - 52
Resolution of the fluorescence equilibrium unfolding profile of trp aporepressor using single tryptophan mutants; Royer CA et al.; Single tryptophan mutants of the trp aporepressor, tryptophan 19-->phenylalanine (W19F) and tryptophan 99-->phenylalanine (W99F), were used in this study to resolve the individual steady-state and time-resolved fluorescence urea unfolding profiles of the two tryptophan residues in this highly intertwined, dimeric protein . The wild-type protein exhibits a large increase in fluorescence intensity and lifetime, as well as a large red shift in the steady-state fluorescence emission spectrum, upon unfolding by urea (Lane, A.N . & Jardetsky, O., 1987, Eur . J . Biochem . 164, 389-396; Gittelman, M.S . & Matthews, C.R., 1990, Biochemistry 29, 7011-7020; Fernando, T . & Royer, C.A., 1992, Biochemistry 31, 6683-6691) . Unfolding of the W19F mutant demonstrated that Trp 99 undergoes a large increase in intensity and a red shift upon exposure to solvent . Lifetime studies revealed that the contribution of the dominant 0.5-ns component of this tryptophan tends toward zero with increasing urea, whereas the longer lifetime components increase in importance . This lifting of the quenching of Trp 99 may be due to disruption of the interaction between the two subunits upon denaturation, which abolishes the interaction of Trp 99 on one subunit with the amide quenching group of Asn 32 on the other subunit (Royer, C.A., 1992, Biophys . J . 63, 741-750) . On the other hand, Trp 19 is quenched in response to unfolding in the W99F mutant . Exposure to solvent of Trp 19, which is buried at the hydrophobic dimer interface in the native protein, results in a large red shift of the average steady-state emission.(ABSTRACT TRUNCATED AT 250 WORDS)

Cell Mol Biol (Noisy-le-grand), 1993 Nov, 39(7), 791 - 800
Endocytosis of lipopolysaccharide in mouse macrophages; Kriegsmann J et al.; Lipopolysaccharide binding sites of mouse peritoneal macrophages were demonstrated by means of immunogold technique . Resident peritoneal macrophages identified by peroxidatic activity in the nuclear envelope and in the rough endoplasmic reticulum show moderate and constant specific binding of bacterial lipopolysaccharide from E . coli to cell surface structures . Labeling of peritoneal macrophages with LPS-gold particles (LPS-Au) at 4 degrees C followed by incubation of the cells at 37 degrees C permits the investigation of LPS endocytosis . After various incubation times LPS-Au was detected in different endocytic compartments . LPS was internalized via coated pits and coated and uncoated vesicles (5 min.) . After 60 min, incubation time LPS-Au occurred in electron lucent endosomes, multivesicular bodies, tubulo-reticular structures and in lysosomes . Gold particles appeared mainly in lysosomes after a longer incubation time (240 min.) . The results of LPS binding and internalization are in accordance with a postulated LPS-receptor binding.

Dtsch Tierarztl Wochenschr . 1993 Nov;100(11):454.
Isolation of Chlamydia spp . from ostriches (Struthio camelus) (short report); Kolb J et al.; Post mortem examination of ostrich chicks (Struthio camelus) originating from a flock with a high mortality rate is described . The main pathological lesions found, indicated colibacillosis which was further corroborated by the isolation of pure culture E . coli . Furthermore Chlamydia spp . was isolated for the first time from ostriches . A detailed isolation procedure is presented . The association of colibacillosis with Chlamydia and the latency of Chlamydia is discussed.

Dtsch Tierarztl Wochenschr, 1993 Nov, 100(11), 434 - 9
{Physiological function of the gastrointestinal tract and pathophysiological changes in neonatal diarrhea of calves}; Kaske M; Firstly, the basic principles of absorption and secretion in the intestine of healthy animals are described . The etiology and the pathophysiologic mechanisms of neonatal diarrhea in the calf due to E . coli and rota-/coronavirus are discussed . Enterotoxins of E . coli stimulate primarily the secretion of chloride (and thereby water) in the small intestine without damaging the intestinal mucosa . The sodium-dependent transport systems for the absorption of glucose and amino acids remain intact . In contrast, infections with rota- or coronavirus lead primarily to a disturbance of absorption processes in the intestine due to villous atrophy . Crypt cells are indirectly affected by inflammation mediators . Intestinal motility is inhibited during most diarrheic episodes; the application of para-sympatholytics in therefore not favourable.

Int Immunol, 1993 Nov, 5(11), 1383 - 92
Induction of IFN-gamma in macrophages by lipopolysaccharide; Fultz MJ et al.; In this paper we report that macrophages can be stimulated to express detectable levels of IFN-gamma-specific mRNA . Macrophages from lipopolysaccharide (LPS)-responsive, C3H/OuJ mice are induced by LPS to increase steady-state levels of IFN-gamma-specific mRNA, while those from LPS-hyporesponsive C3H/HeJ mice are not . This interstrain variation is apparently the result of LPS-specific signal differences since macrophages derived from both Lpsn and Lpsd mouse strains are able to produce comparable levels of IFN-gamma-specific mRNA following stimulation with polyinosinic-polycytidylic acid . The identity of the cell type responsible for this IFN-gamma message appears to be the macrophage as IFN-gamma-specific mRNA was also detectable following T and natural killer cell depletion, in the LPS-stimulated RAW 264.7 cell line, and in a homogeneous population of mature macrophages propagated in vitro by stimulation of bone marrow progenitors with recombinant colony stimulating factor-1 . Immunofluorescent staining of fixed and permeabilized LPS-stimulated macrophages confirmed the presence of immunoreactive IFN-gamma protein . The possible significance of IFN-gamma production by macrophages is discussed in the context of normal macrophage differentiation as well as the inflammatory immune response.

Transfusion, 1993 Nov-Dec, 33(11), 919 - 24
Production of modified crosslinked cell-free hemoglobin for human use: the role of quantitative determination of endotoxin contamination; Roth RI et al.; In vivo toxicity remains a major barrier to the successful use of cell-free hemoglobin (Hb) as an oxygen carrier in humans . Bacterial endotoxin (lipopolysaccharide, LPS) is known to contribute to the in vivo toxicity of Hb preparations, and the prevention of LPS contamination is a critical aspect of the effort to create an efficacious Hb blood substitute . Limulus amebocyte lysate assays for endotoxin were performed on multiple Hb samples from 26 independent production runs for the preparation of human crosslinked cell-free hemoglobin (alpha alpha Hb) . High levels of LPS contamination (1- > 100 ng/mL) of alpha alpha Hb solutions were detected in multiple samples during many of the initial production runs . It was observed that LPS contamination of alpha alpha Hb solutions could occur at any step during the production sequence . Substantial enhancement by alpha alpha Hb of the biologic effects of LPS was demonstrated by two independent assays for endotoxin (the Limulus amebocyte lysate test and a mononuclear cell procoagulant assay), whereas LPS biologic activity was only slightly increased by human serum albumin and substantially diminished by IgG . These results suggest that the prevention of LPS-related toxicities in vivo may be more important to the clinical use of Hb solutions than to the use of other intravenous protein products . Therefore, it was encouraging to note that, with the careful monitoring for LPS in the production facility and in multiple samples during cell-free Hb production, sources of LPS contamination were recognized and the appropriate sites were made endotoxin-free.(ABSTRACT TRUNCATED AT 250 WORDS)

Anal Chem, 1993 Nov 1, 65(21), 3053 - 60
Characterization of immobilized Escherichia coli alkaline phosphatase reactors in flow injection analysis; Shan Y et al.; The characterization of immobilized Escherichia coli alkaline phosphatase reactors used in flow injection analysis is reported for factors such as optimum pH, activity, ionic strength, product inhibition, and substrate specificity . The kinetics of the immobilized enzyme was studied, and mathematical descriptions were developed for the use of an immobilized enzyme packed-bed reactor to evaluate the kinetic parameters and the number of active sites on the immobilized enzyme . Suppression of phosphatase activity by orthophosphate was found to be significantly reduced, and the Michaelis-Menten constant increased when the enzyme was immobilized and packed in a reactor . Immobilized E . coli alkaline phosphatase exhibited similar activity at pH 8 in Tris-HCl, NaHCO3 and borate-HCl buffers but slightly lower activity in NH3H2O-NH4Ac buffer . The performance of the immobilized enzyme reactor was not affected by the presence of up to 10 M Mg(II), Ni(II), Cd(II), Co(II), Mn(II), Cu(II), or urea, 1 M Fe(II), or 0.1 M Fe(III) in the substrate stream . The chelating agent EDTA, however, gradually deactivated the immobilized enzyme . The periodic restoration of enzyme activity was achieved following the removal and addition of zinc ions . The immobilized E . coli alkaline phosphatase packed-bed reactor was used to measure the alkaline phosphatase available phosphorus content of a number of model organophosphorus compounds . p-Nitrophenyl phosphate showed a linear response in the range of 1.6 x 10(-7)-1.6 x 10(-4) M . This study forms part of a larger program to develop enzymatic systems for water quality measurement.

Anal Chem, 1993 Nov 1, 65(21), 3015 - 23
Identification of the facile gas-phase cleavage of the Asp-Pro and Asp-Xxx peptide bonds in matrix-assisted laser desorption time-of-flight mass spectrometry; Yu W et al.; Abundant ions corresponding to the gas-phase cleavage of the Asp-Pro and Asp-Xxx bonds of peptides in the process of matrix-assisted laser desorption were observed using a time-of-flight mass spectrometer equipped with both linear and reflector mass analyzers . Peptides containing the N-terminal sequence, Asp-Pro .. . from an endoproteinase Asp-N digest yielded one peak in the molecular ion region in the linear mode and two equally abundant peaks in the reflector mode TOF mass spectra . The lower molecular masses in the reflector mode mass spectra could be eliminated by removing the Asp residue or derivatizing its side-chain carboxyl group . The observed mass differences did not correspond to any amino acid; however, by lowering the potential of the reflector to correct for the energy loss the mass difference was determined to be 115 Da, i.e., Asp . The extent and rate of this decomposition was compared with that obtained using a four-sector tandem mass spectrometer in the MS/MS mode of operation without and with a collision gas at collision cell potentials of 3.0 and 9.86 kV . These data suggest the Asp-Pro peptide bond is more labile than other peptide bonds in the gas phase . Abundant metastable decomposition of internal Asp-Pro bonds was also observed in larger peptides and proteins . Based on these latter data, a mechanism for this gas-phase cleavage is proposed.(ABSTRACT TRUNCATED AT 250 WORDS)

Plant Mol Biol, 1993 Nov, 23(4), 905 - 9
A conjugative plasmid vector for promoter analysis in several cyanobacteria of the genera Synechococcus and Synechocystis; Marraccini P et al.; A promoter-probe vector, pSB2A, based on the plasmid RSF1010 and the promoterless chloramphenicol acetyl transferase (cat) reporter gene, has been constructed . pSB2A appeared to be most efficiently transferred by conjugation to the widely used cyanobacteria Synechocystis strains PCC6803 (S.6803) and PCC6714 (S.6714) and Synechococcus strains PCC7942 (S.7942) and PCC6301 (S.6301), where it replicates stably even though it contains no cyanobacterial DNA . Using pSB2A we found that (1) a light-regulated promoter from S.6803 remains controlled by light intensity in S.7942 while it is silent in Escherichia coli, and (2) the E . coli tac promoter behaves as a strong and light-independent promoter in the four cyanobacterial hosts tested.

Plant Mol Biol, 1993 Nov, 23(4), 881 - 8
Cloning, sequence analysis and expression of a cDNA encoding active phosphoenolpyruvate carboxylase of the C3 plant Solanum tuberosum; Merkelbach S et al.; A cDNA coding for phosphoenolpyruvate carboxylase (PEPC) was isolated from a cDNA library from Solanum tuberosum and the sequence of the cDNA was determined . It was inserted into a bacterial expression vector and a PEPC- Escherichia coli mutant could be complemented by the cDNA construct . A functional fusion protein could be synthesized in E . coli . The properties of this PEPC protein clearly resembled those of typical C3 plant enzymes.

Br J Anaesth, 1993 Nov, 71(5), 702 - 8
Dose-response relationship for inhaled nitric oxide in experimental pulmonary hypertension in sheep; Dyar O et al.; We have examined the effect of inhaled nitric oxide 4-512 p.p.m . in six sheep with pulmonary hypertension induced first with hypoxia and then with 6 micrograms kg-1 of E . coli endotoxin . A similar dose-dependent reduction in pulmonary artery pressure occurred in pulmonary hypertension induced by hypoxia or endotoxin, with a maximum effect of 25-30% decrease with nitric oxide 64 p.p.m . Increasing the dose to 512 p.p.m . had no further effect . The ED50 for inhaled nitric oxide was 39 p.p.m . for pulmonary hypertension induced by hypoxia and 48 p.p.m . for endotoxin-induced pulmonary hypertension . A dose-dependent increase in arterial oxygenation, which reached a maximum with nitric oxide 64 p.p.m., was seen with inhaled nitric oxide after endotoxin infusion . If the toxicity of inhaled nitric oxide can be determined, it may prove useful in the treatment of pulmonary hypertension secondary to the adult respiratory distress syndrome.

Mol Gen Genet, 1993 Nov, 241(3-4), 399 - 408
Identification, molecular cloning and characterization of the gene encoding the chi subunit of DNA polymerase III holoenzyme of Escherichia coli; Carter JR et al.; We have identified a previously reported open reading frame (ORF13) that maps between pepA and valS at 96.6 centisomes of the Escherichia coli genome as the structural gene for the chi subunit of DNA polymerase III holoenzyme . This conclusion is supported by a perfect match of the amino-terminal 24 residues of chi with the DNA sequence of ORF13 and a demonstration that ORF13 directs expression of a protein that co-migrates with authentic chi on SDS-polyacrylamide gels . ORF13, designated holC, was isolated from the E . coli chromosome and inserted into a tac promoter-based expression plasmid to direct production of the chi subunit to 5-7% of the total soluble protein . The 3' end of holC was sequenced to resolve discrepancies between two published versions.

J Gen Virol, 1993 Nov, 74 ( Pt 11), 2453 - 7
Localization of a single-stranded RNA-binding domain in the movement protein of red clover necrotic mosaic dianthovirus; Osman TA et al.; Mutant movement proteins of red clover necrotic mosaic dianthovirus (RCNMV), consisting of in-frame deletions or fusions with a maltose-binding protein, were produced in Escherichia coli using expression vectors . The ability of the mutant proteins to bind to ssRNA was tested by photochemical cross-linking and gel retardation . The results showed that the region between amino acids 181 and 225 of the RCNMV movement protein contains an ssRNA-binding domain.

J Gen Virol, 1993 Nov, 74 ( Pt 11), 2317 - 24
Mapping and sequence of the gene encoding the African swine fever virion protein of M(r) 11500; Alcami A et al.; The gene encoding the African swine fever virus protein of M(r) 11,500, present in the virus particle, has been mapped and sequenced in the genome of the Vero cell-adapted virus strain BA71V . A serum raised against virion proteins of M(r) 12,000 to 13,000 isolated from polyacrylamide gels was used to screen a plasmid expression library, containing viral DNA random fragments, that expresses viral polypeptides fused to beta-galactosidase . Using this method, we have identified and sequenced the open reading frame (ORF) A137R, which initiates at the right end of the EcoRI A restriction fragment and extends into the EcoRI F fragment . Expression of the protein in Escherichia coli has confirmed that ORF A137R encodes a protein with an M(r) of about 12,000 . A specific serum was raised against the E . coli-expressed protein, and has been used to identify the protein encoded by the ORF, which is translated at late times of infection and incorporated into the virus particle . Immunofluorescence experiments have shown that the protein localizes in virus factories.

Biopolymers, 1993 Nov, 33(11), 1747 - 55
Structural changes in 16S RNA from Escherichia coli upon unfolding by urea; Timchenko AA et al.; The urea-induced unfolding of 16S RNA at low ionic strength has been studied by dynamic light scattering, uv spectroscopy, and some hydrodynamic methods . Three components could be resolved in the photon correlation spectra of scattered light, using the inverse Laplace transform SIPP program {G.R . Danovich and I.N . Serdyuk (1983) in Photon Correlation Techniques in Fluid Mechanics, vol . B38, E.O . Schulz-Dubois, Ed., Springer, Berlin/Heidelberg, New York, p . 315} . One component is assigned to the center-of-mass translation of the RNA, another one to a combination of translational and internal motion, and the last to diffusion of urea clusters . The hydrodynamic dimensions of RNA increase strongly upon transition from 4 to 6 M urea . We conclude that up to 2 M urea, 16S RNA is highly elongated, and coiled above 4 M urea, with a great increase of the hydrodynamic dimensions of RNA being observed upon transition from 4 to 6 M urea . A scheme for RNA unfolding is proposed.

Biochem J, 1993 Nov 1, 295 ( Pt 3), 719 - 24
Purification and biochemical characterization of recombinant human placental growth hormone produced in Escherichia coli; Igout A et al.; The hGH-V (or hGH-2) gene codes for human placental growth hormone (hPGH) . Secretion of hPGH is continuous, in contrast with the pulsed secretion of pituitary growth hormone (hGH) which it progressively replaces in the maternal bloodstream . hGH-V cDNA has previously been cloned and isolated . Analysis of its nucleotide sequence has revealed a 191-residue protein, hPGH, differing from hGH at 13 positions . The calculated pI is more basic than that of the pituitary hormone . Here we have inserted hGH-V cDNA into the pIN-III-ompA3 plasmid in order to produce hPGH in its native form in Escherichia coli D1210 . Expression of hGH-V cDNA in E . coli is significantly lower than that of hGH cDNA with the same expression system . The hPGH produced in E . coli was purified in quantities sufficient to allow its biochemical and immunochemical characterization . The molecular mass of the protein was determined by electrospray m.s . The determined mass, 22,320 Da, agrees well with the molecular mass calculated from the translated cDNA sequence, assuming the presence of two disulphide bridges . Having established the technique for producing hPGH with a primary structure identical to the natural, non-glycosylated, 22 kDa isoform, we can now plan the full physicochemical and pharmaceutical characterization of this new hormonal entity.

Biochem J, 1993 Nov 1, 295 ( Pt 3), 645 - 8
Glucosamine-6-phosphate deaminase from Escherichia coli has a trimer of dimers structure with three intersubunit disulphides; Altamirano MM et al.; Glucosamine-6-phosphate deaminase is an oligomeric protein composed of six identical 29.7 kDa subunits . Each subunit has four cysteine residues located at positions 118, 219, 228 and 239 . We have previously shown that Cys-118 and Cys-239 form a pair of vicinal thiols, the reactivity of which changes with the allosteric transition . The site-directed mutations Cys-->Ser corresponding to the other two cysteine residues have been constructed, as well as some selected multiple mutations involving the four cysteines . Thiol and disulphide measurements on the wild-type and mutant enzymes indicate that thiols from Cys-219 are oxidized and form interchain disulphide bonds . The disulphide-linked dimer was demonstrated by SDS/PAGE . This result is consistent with preliminary crystallographic data and thermal denaturation studies, and strongly suggests that glucosamine-6-phosphate deaminase is a trimer of disulphide-linked dimers . The mutant forms of the deaminase lacking the interchain disulphide bond or the thiol at Cys-228 are both stable hexamers showing the same sensitivity to urea denaturation as the wild-type protein . Furthermore, these Cys-->Ser mutants display the same kinetics and allosteric properties as those already described for the wild-type enzyme.

Am J Physiol, 1993 Nov, 265(5 Pt 2), R1121 - 5
Role of prostaglandins in exercise-induced core temperature elevation in female Sprague-Dawley rats; Rowsey PJ et al.; Female Sprague-Dawley rats (12:12-h photoperiod; body temperature, BT, measured with biotelemetry) with access to running wheels for 6 wk have an elevated BT (compared with rats with no access to exercise wheels, i.e, sedentary) both during the period of voluntary exercise (nighttime) (0.5 degree C, P = 0.0001) and the nonexercise period (daytime) (0.3 degree C, P = 0.002) . To determine whether prostaglandin (PG) E was responsible for any portion of this daytime rise in BT, we injected a dose of sodium salicylate (300 mg/kg), which was shown to produce complete antipyresis in rats injected with lipopolysaccharide (LPS), into exercised and sedentary rats 4 h after the onset of the lights-on period . The injections of sodium salicylate led to a fall in body temperature in both the exercised and sedentary rats of similar amounts (-0.88 degree C vs . -0.61 degree C at 2 h postinjection, P = 0.59) . We conclude that the increase in daytime BT of exercised female rats is not mediated by prostaglandins.

Proc Natl Acad Sci U S A, 1993 Nov 1, 90(21), 9993 - 7
Activation by nitric oxide of an oxidative-stress response that defends Escherichia coli against activated macrophages; Nunoshiba T et al.; Nitric oxide is a free radical (NO) formed biologically through the oxidation of L-arginine by nitric oxide synthases . NO is produced transiently in mammalian cells for intercellular signaling and in copious quantities to cause cytostasis and cytotoxicity . In the latter situation, NO is a deliberate cytotoxic product of activated macrophages, along with other reactive oxygen species such as hydrogen peroxide (H2O2) and superoxide (O2-) . Escherichia coli has a complex set of responses to H2O2 and O2- that involves approximately 80 inducible proteins; we wondered whether these bacteria might induce analogous defenses against nitric oxide . We show here that a multigene system controlled by the redox-sensitive transcriptional regulator SoxR is activated by NO in vivo . This induction confers bacterial resistance to activated murine macrophages with kinetics that parallel the production of NO by these cells . Elimination of specific SoxR-regulated genes diminishes the resistance of these bacteria to the cytotoxic macrophages . The required functions include manganese-containing superoxide dismutase, endonuclease IV (a DNA-repair enzyme for oxidative damage), and micF, an antisense regulator of the outer membrane porin OmpF . These results demonstrate that SoxR is a sensor for cellular exposure to NO, and that the soxRS response system may contribute to bacterial virulence.

Proc Natl Acad Sci U S A, 1993 Nov 1, 90(21), 9837 - 41
Local breathing and global unfolding in hydrogen exchange of barnase and its relationship to protein folding pathways; Clarke J et al.; We have measured the rate constants for exchange of amide protons in 15N-labeled wild-type barnase and a disulfide mutant that is more stable by 2 kcal.mol-1 . The relative rate constants for exchange for wild type and mutant should reflect the changes in the equilibrium constants for local or global unfolding . The values for regions whose structure has been shown to be unaffected by the mutation fall into three subsets: those that are essentially unaffected by the mutation and so presumably exchange by local breathing; those where the energies change by close to 2 kcal.mol-1 and so presumably require global unfolding for exchange; and intermediate values that probably reflect a mixture of local and global unfolding in wild-type barnase . Amide protons that require the full change in unfolding energy are predominantly in the beta-sheet, which forms early in folding, but also include two that are involved in tertiary interactions that are known not to be formed until late in the folding pathway . Exchange in the major helix, which is known to form early, is largely unaffected by mutation and so exchanges by local breathing . There is thus no direct relationship between hydrogen-exchange behavior and the protein folding pathway . However, experiments on mutants of varying stability may provide further evidence on the sequence of events in folding.

Proc Natl Acad Sci U S A, 1993 Nov 1, 90(21), 9828 - 31
Escherichia coli ribosomal protein L7/L12 dimers remain fully active after interchain crosslinking of the C-terminal domains in two orientations; Oleinikov AV et al.; Cysteine site-directed mutagenesis was used to create variants of Escherichia coli ribosomal protein L7/L12 that have single cysteine substitutions, at residues 63 or 89, located in different exposed loops in the structure of the globular C-terminal domain indicated by the crystallographic structure . That structure shows a possible dimer interaction in which the two sites of cysteine substitution appear to be too distant for disulfide bond formation . After mild oxidation in solution both of the overexpressed purified cysteine-substituted proteins formed interchain disulfide crosslinked dimers in high yield . Both crosslinked dimers were fully active in restoring activity in poly(U)-directed polyphenylalanine synthesis to ribosomal core particles depleted of wild-type L7/L12 . These results show that the two C-terminal domains have independent mobility . The activity of dimeric L7/L12 does not require the independent movement of the two globular C-terminal domains in an L7/L12 dimer; moreover, it appears independent of their mutual orientation when joined by crosslinking at the two loops . A third variant with a cysteine substitution at residue 33 near the junction between the alpha-helical N-terminal domain and the flexible hinge was prepared and tested . This protein was active in the protein synthesis assay in the reduced state . Oxidation produced the interchain crosslinked dimer in high yield, but this crosslinked dimer was inactive in polyphenylalanine synthesis . The inactivation was due to the inability of the Cys33-Cys33 oxidized dimer to bind to the core particle.

Proc Natl Acad Sci U S A, 1993 Nov 1, 90(21), 9823 - 7
Repair by human cell extracts of single (6-4) and cyclobutane thymine-thymine photoproducts in DNA; Szymkowski DE et al.; One cis-syn cyclobutane thymine dimer or one (6-4) thymine-thymine photoproduct was built into an identical sequence of a closed-circular M13 duplex DNA, and nucleotide excision repair synthesis carried out by human cell extracts in the area containing each lesion was determined . Extracts from normal cells repaired the (6-4) photoproduct with a patch size of approximately 20-30 nucleotides, but repair was at least 10-fold lower at the cyclobutane dimer . The (6-4) lesion was repaired with comparable efficiency to a single acetylamino-fluorene-guanine adduct in a similar location . Extract from nucleotide excision repair-deficient xeroderma pigmentosum group A cells could not remove any of these adducts but could complete repair of the lesions after incision with Escherichia coli UvrABC proteins . This direct comparison of repair of two UV photoproducts, in an in vitro system where chromatin assembly and transcription are absent, suggests that the more rapid repair of the (6-4) lesion observed in the mammalian cell genome overall is due in part to a significant difference in the ability of the repair complex to locate and incise these lesions in DNA.

Proc Natl Acad Sci U S A, 1993 Nov 1, 90(21), 10350 - 4
An ethoxyquin-inducible aldehyde reductase from rat liver that metabolizes aflatoxin B1 defines a subfamily of aldo-keto reductases; Ellis EM et al.; Protection of liver against the toxic and carcinogenic effects of aflatoxin B1 (AFB1) can be achieved through the induction of detoxification enzymes by chemoprotectors such as the phenolic antioxidant ethoxyquin . We have cloned and sequenced a cDNA encoding an aldehyde reductase (AFB1-AR), which is expressed in rat liver in response to dietary ethoxyquin . Expression of the cDNA in Escherichia coli and purification of the recombinant enzyme reveals that the protein exhibits aldehyde reductase activity and is capable of converting the protein-binding dialdehyde form of AFB1-dihydrodiol to the nonbinding dialcohol metabolite . We show that the mRNA encoding this enzyme is markedly elevated in the liver of rats fed an ethoxyquin-containing diet, correlating with acquisition of resistance to AFB1 . AFB1-AR represents the only carcinogen-metabolizing aldehyde reductase identified to date that is induced by a chemoprotector . Alignment of the amino acid sequence of AFB1-AR with other known and putative aldehyde reductases shows that it defines a subfamily within the aldo-keto reductase superfamily.

Proc Natl Acad Sci U S A, 1993 Nov 1, 90(21), 10330 - 4
Formation of functional peptide complexes of class II major histocompatibility complex proteins from subunits produced in Escherichia coli; Altman JD et al.; Class II major histocompatibility complex molecules play a major role in the immune response by binding peptide fragments of exogenous antigens and displaying them on the surfaces of antigen-presenting cells, where they can be recognized by T cells . To facilitate structural and functional studies of these molecules, we have produced truncated alpha and beta chains of the murine class II molecule I-Ek in Escherichia coli (Ec-I-Ek) and have developed conditions to fold them in the presence of specific peptides with yields of complex approaching 2% . Reconstitution is specific since only unlabeled peptide known to bind I-Ek compete with biotinylated peptide, as assessed by ELISA . Complexes of the refolded heterodimer (Ec-I-Ek) with either of two different peptide antigens remain associated during nonreducing SDS/PAGE . Immobilized Ec-I-Ek-peptide complexes stimulate lymphokine production by three T-cell clones in an antigen-specific manner with a dose-response relation comparable to previously described soluble I-Ek molecules produced in CHO cells . These results demonstrate that folding of Ek alpha and Ek beta polypeptides does not require any other protein to produce the biologically relevant conformation and that carbohydrate modification of this class II molecule is not necessary for alpha beta T-cell recognition.

Proc Natl Acad Sci U S A, 1993 Nov 1, 90(21), 10325 - 9
Conformation-sensitive gel electrophoresis for rapid detection of single-base differences in double-stranded PCR products and DNA fragments: evidence for solvent-induced bends in DNA heteroduplexes; Ganguly A et al.; Several techniques have recently been developed to detect single-base mismatches in DNA heteroduplexes that contain one strand of wild-type and one strand of mutated DNA . Here we tested the hypothesis that an appropriate system of mildly denaturing solvents can amplify the tendency of single-base mismatches to produce conformational changes, such as bends in the double helix, and thereby increase the differential migration of DNA heteroduplexes and homoduplexes during gel electrophoresis . The best separations of heteroduplexes and homoduplexes were obtained with a standard 6% polyacrylamide gel polymerized in 10% ethylene glycol/15% formamide/Tris-taurine buffer . As predicted by the hypothesis of solvent-induced bends, when the concentration of either ethylene glycol or formamide was increased, the differential migration decreased . Also, single-base mismatches within 50 bp of one end of a heteroduplex did not produce differential migration . Sixty of 68 single-base mismatches in a series of PCR products were detected in some 59 different sequence contexts . The eight mismatches not detected were either within 50 bp of the nearest end of the PCR product or in isolated high-melting-temperature domains . Therefore, it was possible to predict in advance the end regions and sequence contexts in which mismatches may be difficult to detect . The procedure can be applied to any PCR products of 200-800 bp and requires no special equipment or preparation of samples.

Proc Natl Acad Sci U S A, 1993 Nov 1, 90(21), 10230 - 4
mu opiate receptor: cDNA cloning and expression; Wang JB et al.; mu opiate receptors recognize morphine with high affinity . A 2.1-kb rat brain cDNA whose predicted translation product displays 63% identity with recently described delta and kappa opiate receptor sequences was identified through polymerase chain reaction and cDNA homology approaches . This cDNA recognizes a 10.5-kb mRNA that is expressed in thalamic neurons . COS-cell expression confers naloxonazine-, Na(+)-, and GTP-sensitive binding of mu but not delta or kappa opioid ligands . Expressing cells bind morphine, {D-Ala2,N-methyl-Phe4,glyol5}enkephalin (DAMGO), and {D-Ala2,D-Leu5}enkephalin (DADLE) with nanomolar or subnanomolar affinities, defining a mu opiate receptor that avidly recognizes analgesic and euphoric opiate drugs and opioid peptides.

Proc Natl Acad Sci U S A, 1993 Nov 1, 90(21), 10216 - 20
Control of folding and membrane translocation by binding of the chaperone DnaJ to nascent polypeptides; Hendrick JP et al.; Recent evidence supports the view that cellular protein folding may be mediated by molecular chaperones . A fundamental question concerns the stage in its biogenesis at which the folding protein makes first contact with these components . We show here by crosslinking that the chaperone DnaJ binds nascent ribosome-bound polypeptide chains as short as 55 residues . Cotranslational binding of DnaJ to firefly luciferase and chloramphenicol acetyltransferase resulted in an arrest of folding as long as the functional partners of DnaJ in Escherichia coli, DnaK and GrpE, were missing . Protein uptake into microsomes and mitochondria was also interrupted by DnaJ . Both folding and post-translational translocation recommenced upon addition of DnaK and GrpE . We propose that DnaJ protects nascent polypeptide chains against aggregation and, in cooperation with Hsp70, controls their productive folding once a complete polypeptide or a polypeptide domain has been synthesized.

Proc Natl Acad Sci U S A, 1993 Nov 1, 90(21), 10115 - 9
Serine-173 of the Epstein-Barr virus ZEBRA protein is required for DNA binding and is a target for casein kinase II phosphorylation; Kolman JL et al.; An Epstein-Barr virus-encoded protein, ZEBRA, mediates the switch from latency to the viral lytic life cycle . ZEBRA's domain structure and DNA binding specificity resemble that of cellular transcriptional activators such as c-Fos/c-Jun . We show that ZEBRA, like c-Jun, is phosphorylated by casein kinase II (CKII) . The principal site of phosphorylation is serine-173 (S173), five amino acids upstream of the basic DNA recognition domain . CKII phosphorylation abrogated ZEBRA's capacity to bind its target DNA sequences . S173 is a functional component of ZEBRA's DNA binding domain, since mutation of S173 to alanine (S173A) reduced DNA binding in vitro to 10% of wild-type levels . Transcriptional activation of a native viral promoter in vivo by mutant S173A was also reduced markedly . Reversible phosphorylation of S173 is likely to be an important means of regulating ZEBRA's activity in vivo.

Proc Natl Acad Sci U S A, 1993 Nov 1, 90(21), 10056 - 60
Molecular cloning and functional expression of a cDNA encoding glycosylation-inhibiting factor; Mikayama T et al.; By using probes based on partial amino acid sequence of glycosylation-inhibiting factor (GIF) from a mouse T-cell hybridoma, a full-length cDNA encoding mouse GIF was isolated . A cDNA clone encoding human GIF was isolated from cDNA libraries of a GIF-producing human T-cell hybridoma by using mouse GIF cDNA as a probe . The cDNAs encode a putative 12.5-kDa peptide of 115 amino acids . Northern blot analysis demonstrated a single, 0.6-kb transcript . Polyclonal rabbit antibodies against the Escherichia coli-derived recombinant 13-kDa peptide bound hybridoma-derived GIF . Although the peptide did not contain a signal peptide sequence, transfection of the cDNA into COS-1 cells resulted in secretion of 13-kDa peptide, but the peptide had substantially less bioactivity than the hybridoma-derived GIF . However, expression of a chimeric cDNA encoding a fusion protein consisting of the N-terminal pro region of calcitonin precursor and human GIF and cotransfection with furin cDNA to allow intracellular cleavage of the fusion protein resulted in secretion of 13-kDa peptide that was comparable to hybridoma-derived GIF in its bioactivity . Both the 13-kDa peptide and GIF bioactivity in the transfected COS-1 supernatant bound to a monoclonal antibody against hybridoma-derived human GIF . These results indicate that the 13-kDa peptide represents recombinant GIF, but posttranslational modification of the peptide is important for generation of the bioactivity . The GIF cDNA had high homology with the cDNA encoding macrophage migration inhibitory factor . However, the recombinant GIF failed to inhibit migration of human monocytes, and recombinant human macrophage migration inhibitory factor did not have GIF bioactivity.

Mol Immunol, 1993 Nov, 30(16), 1529 - 41
Native and recombinant Fel dI as probes into the relationship of allergen structure to human IgE immunoreactivity; Bond JF et al.; To delineate the relationship between the structural conformation and the stability of an allergen and its antigenicity, we have chosen the major allergen from cat dander, Fel dI . From protein sequence analysis data we have examined the structure of the naturally occurring Fel dI and we have found it to exist as an anti-parallel heterodimer . We have used ELISA, RAST, Western blot and histamine release techniques to compare the IgE reactivity of a set of cat allergic patient samples to purified, native Fel dI and the E . coli expressed chains 1 and 2 . Results from these studies demonstrate a significant level of IgE reactivity to all forms when examined for direct binding . However, both blot and ELISA competition assays show a much higher reactivity to Fel dI in solution compared to the separate recombinant chains and this is supported by the histamine release data . Although native Fel dI chain 2 contains an N-linked carbohydrate moiety, this does not seem to play a role in the reactivity of IgE to chain 2 . Denaturation of Fel dI with alkali conditions leads to a dramatic decrease in IgE reactivity, even though measurable changes to the backbone structure of the protein are minimal . One proposed explanation is that both chains possess a core region determined by their primary structures and that the major IgE epitopes are dependent upon them . The relative reactivity amongst these allergen forms varied with the method of analysis, implying that the conformational requirements for IgE antibody binding are best studied by the application of more than one experimental protocol . Results from these qualitative analyses afford insight into the allergenicity of this exceptionally stable cat pelt protein.

Mol Immunol, 1993 Nov, 30(16), 1491 - 8
Antibody recognition of the recombinant human nuclear antigens RNP 70 kD, SS-A, SS-B, Sm-B, and Sm-D by autoimmune sera; Wagatsuma M et al.; Five human nuclear antigens, RNP 70 kD, SS-A, SS-B, Sm-B and Sm-D, were produced in E . coli using the expression vector pSEM . cDNAs encoding these antigens were ligated to a truncated lacZ' gene of the vector and the beta-galactosidase fusion proteins were efficiently expressed as intracellular inclusion bodies after isopropyl-beta-thiogalactopyranoside induction . The antibody reactivities of these fusion proteins were evaluated by Western blot and by ELISA employing panel sera from patients with autoimmune diseases such as systemic lupus erythematosus, Sjogren's syndrome or mixed connective tissue disease . The three fusion proteins, RNP 70 kD, SS-B, and Sm-B, showed good reactivities in both systems, whereas the other two fusion proteins, SS-A and Sm-D, showed poor and no reactivity in both systems, respectively . It can be concluded that RNP 70 kD, SS-B and Sm-B recombinant antigens are useful reagents for the differential diagnosis of the autoimmune diseases.

J Surg Res, 1993 Nov, 55(5), 465 - 72
Altered hepatic production of apolipoproteins B and E in the fasted septic rat: factors in the development of hypertriglyceridemia; Tripp RJ et al.; The etiology of hypertriglyceridemia associated with sepsis remains unclear, but we will attempt to elucidate its character by studying the hepatic production of apolipoproteins B and E . Male Lewis rats (260-330 g) were assigned to two groups, control (n = 5) and septic (n = 5) . The septic group was injected with 2 x 10(8) live Escherichia coli colonies/100 g body wt . Food was removed from all rats after injections . Twenty-four hours later a recirculating in situ liver perfusion was performed for 120 min with KRB buffer, containing L-{35S}methionine . The production of apolipoprotein B (apo B), apolipoprotein E (apo E), albumin, and transferrin was determined by immunoprecipitation . The septic rats showed a protein-specific response to sepsis . The total protein secreted increased throughout each perfusion, septic greater than control . Apo B production was increased 2.6-fold in the septic versus control groups (P = 0.037), while apo E production was decreased by 2.9 times control (P = 0.036) . Albumin production was decreased 2-fold in the septic group (P = 0.002) . The increased hepatic production of apo B represents an increased number of very low density lipoprotein (VLDL) particles and contributes to the elevated VLDL triglyceride levels seen in sepsis . In contrast, decreased apo E production may result in a diminished ability for peripheral and/or hepatic receptor recognition of VLDL and VLDL remnants, respectively . Each of these changes are factors in the development of hypertriglyceridemia in sepsis.

J Neurosci, 1993 Nov, 13(11), 4764 - 75
Several extracellular domains of the neural cell adhesion molecule L1 are involved in neurite outgrowth and cell body adhesion; Appel F et al.; The neural cell adhesion molecule L1 is a multidomain protein that plays important roles in cell adhesion, migration, and neurite outgrowth . To analyze structure-function relationships of L1 in neurite outgrowth and cell body adhesion, we have expressed and purified a set of different fragments of the extracellular part of this glycoprotein in CHO cells and in Escherichia coli . When neurite outgrowth from small cerebellar neurons was measured on substrate-coated L1 or L1 fragments, neurite outgrowth was promoted by the immunoglobulin-like domains I-II, III-IV, and V-VI, and by the fibronectin type III homologous repeats 1-2, while the fibronectin type III homologous repeats 3-5 were ineffective . In contrast, cell bodies of small cerebellar neurons adhered mostly to the immunoglobulin-like domains I-II and V-VI, and to the fibronectin type III homologous repeats 3-5, but less to the immunoglobulin-like domains III-IV and fibronectin type III homologous repeats 1-2 . In both assays, the neuronal cell surface receptor for all active protein fragments was identified as L1 . No significant differences in functional activities were found between fragments with and without carbohydrate structures . These findings indicate that L1 uses several domains for homophilic interactions overlapping for the two functions analyzed here, but also showing some regional specialization . Furthermore, we show that a homophilic molecule uses several domains in one function, with neurite outgrowth requiring more domains than adhesion for maximal activity.

J Clin Invest, 1993 Nov, 92(5), 2546 - 52
Endotoxin increases parathyroid hormone-related protein mRNA levels in mouse spleen . Mediation by tumor necrosis factor; Funk JL et al.; Parathyroid hormone-related protein (PTHrP) causes hypercalcemia in malignancy . However, the role and regulation of PTHrP in normal physiology is just beginning to be explored . PTHrP is found in the spleen and has several other features common to cytokines . Since endotoxin (LPS) causes many of its effects indirectly by inducing cytokines, studies were undertaken to determine whether LPS might also induce splenic PTHrP expression . LPS (100 ng/mouse) increased splenic PTHrP mRNA levels 3.6-fold in C3H/OuJ mice . This effect was maximal at 2 h and returned to baseline by 4 h . PTHrP peptide levels also increased 3.3-fold in splenic extracts in response to LPS (1 microgram/mouse) . Murine TNF-alpha and human IL-1 beta, cytokines that mediate many of the effects of LPS, also increased splenic PTHrP mRNA levels . LPS-resistant C3H/HeJ mice, which produce minimal amounts of TNF and IL-1 in response to LPS, were resistant to LPS induction of splenic PTHrP mRNA, while TNF-alpha and IL-1 beta readily increased PTHrP mRNA levels in C3H/HeJ mice . Anti-TNF antibody blocked LPS induction of splenic PTHrP mRNA in C3H/OuJ mice by 68%, indicating that TNF is a mediator of the LPS induction of PTHrP levels . In contrast, an IL-1 receptor antagonist (IL-1ra) was ineffective . The increase in PTHrP in the spleen during the immune response suggests that PTHrP may play an important role in immune modulation, perhaps by mediating changes in lymphocyte proliferation and/or function.

J Clin Invest, 1993 Nov, 92(5), 2394 - 400
Gene transfer into respiratory epithelial cells by targeting the polymeric immunoglobulin receptor; Ferkol T et al.; A system for targeting foreign DNA to epithelial cells in vitro has been developed by exploiting receptor-mediated endocytosis . The polymeric immunoglobulin receptor transports dimeric immunoglobulin A and immunoglobulin M through epithelial cells, including those of the respiratory tract, by binding the immunoglobulins at the basolateral surface and transporting them across the cell . Fab fragments of antibodies directed against the extracellular portion of the receptor, secretory component, are similarly transported . Anti-human secretory component Fab fragments were covalently linked to a polycation, and complexed to various expression plasmids . When bound to an expression plasmid containing the Escherichia coli lacZ gene ligated to the Rous sarcoma virus promoter, the complexes transfected HT29.74 human colon carcinoma cells induced to express polymeric immunoglobulin receptor, but not those lacking the receptor . Primary cultures of human tracheal epithelial cells grown on collagen gels, which induce the expression of polymeric immunoglobulin receptor, were also transfected with the complexes . From 5 to 66% of the respiratory epithelial cells had beta-galactosidase activity after treatment, comparable to the percentage of cultured human tracheal epithelial cells that express polymeric immunoglobulin receptor (8-35%) . The addition of excess human secretory component (Fab ligand) to the culture medium at the time of transfection blocked the delivery of DNA . The expression plasmid, either alone, complexed to the polycation, or complexed to a carrier based on an irrelevant Fab fragment, was not effective in transfecting either cell type . This DNA carrier system introduces DNA specifically into epithelial cells that contain pIgR in vitro.

J Cell Biol, 1993 Nov, 123(4), 963 - 76
R2D5 antigen: a calcium-binding phosphoprotein predominantly expressed in olfactory receptor neurons; Nemoto Y et al.; R2D5 is a mouse monoclonal antibody that labels rabbit olfactory receptor neurons . Immunoblot analysis showed that mAb R2D5 recognizes a 22-kD protein with apparent pI of 4.8, which is abundantly contained in the olfactory epithelium and the olfactory bulb . We isolated cDNA for R2D5 antigen and confirmed by Northern analysis and neuronal depletion technique that R2D5 antigen is expressed predominantly, but not exclusively, in olfactory receptor neurons . Analysis of the deduced primary structure revealed that R2D5 antigen consists of 189 amino acids with calculated M(r) of 20,864 and pI of 4.74, has three calcium-binding EF hands, and has possible phosphorylation sites for Ca2+/calmodulin-dependent protein kinase II (CaM kinase II) and cAMP-dependent protein kinase (A kinase) . Using the bacterially expressed protein, we directly examined the biochemical properties of R2D5 antigen . R2D5 antigen binds Ca2+ and undergoes a conformational change in a manner similar to calmodulin . R2D5 antigen is phosphorylated in vitro by CaM kinase II and A kinase at different sites, and 1.81 and 0.80 mol of Pi were maximally incorporated per mol of R2D5 antigen by CaM kinase II and A kinase, respectively . Detailed immunohistochemical study showed that R2D5 antigen is also expressed in a variety of ependymal cells in the rabbit central nervous system . Aside from ubiquitous calmodulin, R2D5 antigen is the first identified calcium-binding protein in olfactory receptor neurons that may modulate olfactory signal transduction . Furthermore our results indicate that olfactory receptor neurons and ependymal cells have certain signal transduction components in common, suggesting a novel physiological process in ependymal cells.

J Cell Biol, 1993 Nov, 123(3), 619 - 26
The major myosin-binding domain of skeletal muscle MyBP-C (C protein) resides in the COOH-terminal, immunoglobulin C2 motif; Okagaki T et al.; A common feature shared by myosin-binding proteins from a wide variety of species is the presence of a variable number of related internal motifs homologous to either the Ig C2 or the fibronectin (Fn) type III repeats . Despite interest in the potential function of these motifs, no group has clearly demonstrated a function for these sequences in muscle, either intra- or extracellularly . We have completed the nucleotide sequence of the fast type isoform of MyBP-C (C protein) from chicken skeletal muscle . The deduced amino acid sequence reveals seven Ig C2 sets and three Fn type III motifs in MyBP-C . alpha-chymotryptic digestion of purified MyBP-C gives rise to four peptides . NH2-terminal sequencing of these peptides allowed us to map the position of each along the primary structure of the protein . The 28-kD peptide contains the NH2-terminal sequence of MyBP-C, including the first C2 repeat . It is followed by two internal peptides, one of 5 kD containing exclusively spacer sequences between the first and second C2 motifs, and a 95-kD fragment containing five C2 domains and three fibronectin type III motifs . The C-terminal sequence of MyBP-C is present in a 14-kD peptide which contains only the last C2 repeat . We examined the binding properties of these fragments to reconstituted (synthetic) myosin filaments . Only the COOH-terminal 14-kD peptide is capable of binding myosin with high affinity . The NH2-terminal 28-kD fragment has no myosin-binding, while the long internal 100-kD peptide shows very weak binding to myosin . We have expressed and purified the 14-kD peptide in Escherichia coli . The recombinant protein exhibits saturable binding to myosin with an affinity comparable to that of the 14-kD fragment obtained by proteolytic digestion (1/2 max binding at approximately 0.5 microM) . These results indicate that the binding to myosin filaments is mainly restricted to the last 102 amino acids of MyBP-C . The remainder of the molecule (1,032 amino acids) could interact with titin, MyBP-H (H protein) or thin filament components . A comparison of the highly conserved Ig C2 domains present at the COOH-terminus of five MyBPs thus far sequenced (human slow and fast MyBP-C, human and chicken MyBP-H, and chicken MyBP-C) was used to identify residues unique to these myosin-binding Ig C2 repeats.

J Bacteriol, 1993 Nov, 175(22), 7505 - 8
Analysis of the genetic requirements for viability of Escherichia coli K-12 DNA adenine methylase (dam) mutants; Peterson KR et al.; RecBCD protein, necessary for Escherichia coli dam mutant viability, is directly required for DNA repair . Recombination genes recF+, recN+, recO+, and recQ+ are not essential for dam mutant viability; they are required for recBC sbcBC dam mutant survival . mutH, mutL, or mutS mutations do not suppress subinduction of SOS genes in dam mutants.

J Bacteriol, 1993 Nov, 175(22), 7500 - 4
Site-directed mutagenesis reveals the importance of conserved charged residues for the transport activity of the PheP permease of Escherichia coli; Pi J et al.; Site-directed mutagenesis has been used to identify a number of charged residues essential for the transport activity of the PheP protein . These residues are highly conserved in the cluster of amino acid transporters . However, some other conserved residues and a number of aromatic residues have been shown not to be essential for transport activity.

J Bacteriol, 1993 Nov, 175(22), 7492 - 4
Negative autoregulation by the Escherichia coli SoxS protein: a dampening mechanism for the soxRS redox stress response; Nunoshiba T et al.; The soxRS redox stress regulon of Escherichia coli is triggered in two stages, with the activated SoxR protein stimulating the soxS gene, whose product then triggers transcription of approximately 10 promoters . Genetic and biochemical experiments presented here show that SoxS protein also limits soxS transcription in vivo and binds the soxS promoter in vitro.

J Bacteriol, 1993 Nov, 175(22), 7441 - 9
The glnB region of the Escherichia coli chromosome; Liu J et al.; We present sequences of the glnB gene of Escherichia coli and of two open reading frames (ORFs) located directly upstream of glnB and transcribed in the same direction . The major transcriptional start sites for glnB are located between ORF-2 and glnB, but some transcription of glnB is initiated at the promoter for ORF-1 . The putative amino acid sequence of the ORF-2 product has high homology to that of response regulators which by phosphorylation acquire the ability to activate transcription of sigma 54-dependent promoters . The product of ORF-1 showed no similarity to other known proteins . The product of neither ORF-1 nor ORF-2 is necessary for the ability of PII, the product of glnB, to bring about the repression of glutamine synthetase in response to nitrogen excess . On the other hand, the product of hmpA, a gene located on the other side of glnB and transcribed in the opposite direction, appears to play an auxiliary role in this process.

J Bacteriol, 1993 Nov, 175(22), 7373 - 82
Novel mechanism for UV sensitivity and apparent UV nonmutability of recA432 mutants: persistent LexA cleavage following SOS induction; Ennis DG et al.; The recA432 mutant allele was isolated (T . Kato and Y . Shinoura, Mol . Gen . Genet . 156:121-131, 1977) by virtue of its defect in cellular mutagenesis (Mut-) and its hypersensitivity to damage by UV irradiation (UVs), which were phenotypes expected for a recA mutant . However, we found that in a different genetic background (lexA51 sulA211 uvrB+), recA432 mutants expressed certain mutant phenotypes but not the Mut- and UVs phenotypes (D.G . Ennis, N . Ossanna, and D.W . Mount, J . Bacteriol . 171:2533-2541, 1989) . We present several lines of evidence that these differences resulted from the sulA genotype of the cell and that the apparent UVs and Mut- phenotypes of the sulA+ derivatives resulted from lethal filamentation of induced cells because of persistent derepression of sulA . First, transduction of sulA(Def) mutations into the recA432 strains restored cellular mutagenesis and resistance to UV . Second, recA432 sulA+ strains underwent filamentous death following SOS-inducing treatments . Third, cleavage of LexA repressor in a recA432 strain continued at a rapid rate long after UV induction, at a time when cleavage of the repressor in the recA+ parental strain had substantially declined . Fourth, we confirmed that a single mutation (recA432) conferring both the UVs and Mut- phenotypes mapped to the recA gene . These findings indicate that the RecA432 mutant protein is defective in making the transition back to the deactivated state following SOS induction; thus, the SOS-induced state of recA432 mutants is prolonged and can account for an excess of SulA protein, leading to filamentation . These results are discussed in the context of molecular models for RecA activation for LexA and UmuD cleavage and their roles in the control of mutagenesis and cell division in the SOS response.

J Bacteriol, 1993 Nov, 175(22), 7254 - 9
Requirement for the polymerization and 5'-->3' exonuclease activities of DNA polymerase I in initiation of DNA replication at oriK sites in the absence of RecA in Escherichia coli rnhA mutants; Cao Y et al.; In previous studies, we found that the requirement for RecA protein in constitutive stable DNA replication (cSDR) can be bypassed by derepression of the LexA regulon and that DNA polymerase I (DNA PolI) is essential for this Rip (RecA-independent process) pathway of cSDR (Y . Cao, R . R . Rowland, and T . Kogoma, J . Bacteriol . 175:7247-7253, 1993) . In this study, the role of DNA PolI in the Rip pathway was further examined . By using F' plasmids carrying different parts of the polA gene, a series of complementation tests was carried out to investigate the requirement for the three enzymatic activities, polymerization, 3'-->5' exonuclease, and 5'-->3' exonuclease activities, of DNA PolI . The result indicated that both the 5'-->3' exonuclease and polymerization activities of DNA PolI are essential for bypassing the requirement for RecA in cSDR but that the 3'-->5' exonuclease activity can be dispensed with . Complementation experiments with rat DNA Pol beta also supported the hypothesis that a nick translation activity is probably involved in cSDR in the absence of RecA . An analysis of DNA synthesis suggested that DNA PolI is involved in the initiation but not the elongation stage of cSDR . Moreover, the dnaE293(Ts) mutation was shown to render the bypass replication temperature sensitive despite the presence of active DNA PolI, suggesting that DNA PolIII is responsible for the elongation stage of the Rip pathway . A model which describes the possible roles of RecA in cSDR and the possible function of DNA PolI in the Rip pathway is proposed.

J Bacteriol, 1993 Nov, 175(22), 7247 - 53
DNA polymerase I and the bypassing of RecA dependence of constitutive stable DNA replication in Escherichia coli rnhA mutants; Cao Y et al.; In Escherichia coli rnhA mutants, several normally repressed origins (oriK sites) of DNA replication are activated . The type of DNA replication initiated from these origins, termed constitutive stable DNA replication, does not require DnaA protein or the oriC site, which are essential for normal DNA replication . It requires active RecA protein . We previously found that the lexA71(Def)::Tn5 mutation can suppress this RecA requirement and postulated that the derepression of a LexA regulon gene(s) leads to the activation of a bypass pathway, Rip (for RecA-independent process) . In this study, we isolated a miniTn10spc insertion mutant that abolishes the ability of the lexA(Def) mutation to suppress the RecA requirement of constitutive stable DNA replication . Cloning and DNA sequencing analysis of the mutant revealed that the insertion occurs at the 3' end of the coding region of the polA gene, which encodes DNA polymerase I . The mutant allele, designated polA25::miniTn10spc, is expected to abolish the polymerization activity but not the 5'-->3' or 3'-->5' exonuclease activity . Thus, the Rip bypass pathway requires active DNA polymerase I . Since the lethal combination of recA(Def) and polA25::miniTn10spc could be suppressed by derepression of the LexA regulon only when DNA replication is driven by the oriC system, it was suggested that the bypass pathway has a specific requirement for DNA polymerase I at the initiation step in the absence of RecA . An accompanying paper (Y . Cao and T . Kogoma, J . Bacteriol . 175:7254-7259, 1993) describes experiments to determine which activities of DNA polymerase I are required at the initiation step and discusses possible roles for DNA polymerase in the Rip bypass pathway.

J Bacteriol, 1993 Nov, 175(22), 7209 - 15
tRNA modification activity is necessary for Tet(M)-mediated tetracycline resistance; Burdett V; Tet(M) protein interacts with the protein biosynthetic machinery to render this process resistant to the tetracycline in vivo and in vitro (V . Burdett, J . Biol . Chem . 266:2872-2877, 1991) . To understand this process more completely, a mutant of Escherichia coli which is altered in the ability of Tet(M) to confer resistance has been identified . This mutation maps to miaA and displays phenotypes characteristic of previously isolated miaA mutations . The miaA gene product modifies A37 adjacent to the anticodon of several tRNA species . Both the mutant isolated in this work and previously isolated miaA mutants confer tetracycline sensitivity in the presence of functional Tet(M), both share a slow growth phenotype, and in neither case is a wild-type phenotype restored in trans by F'112 carrying the 89- to 98-min region of the chromosome . These similar phenotypes further substantiate the assignment of the mutation described here to the miaA locus.

J Bacteriol, 1993 Nov, 175(22), 7178 - 88
Cotranscription of two genes necessary for ribosomal protein L11 methylation (prmA) and pantothenate transport (panF) in Escherichia coli K-12; Vanet A et al.; Genetic complementation and enzyme assays have shown that the DNA region between panF, which encodes pantothenate permease, and orf1, the first gene of the fis operon, encodes prmA, the genetic determinant for the ribosomal protein L11 methyltransferase . Sequencing of this region identified one long open reading frame that encodes a protein of 31,830 Da and corresponds to the prmA gene . We found, both in vivo and in vitro, that prmA is expressed from promoters located upstream of panF and thus that the panF and prmA genes constitute a bifunctional operon . We located the major 3' end of prmA transcripts 90 nucleotides downstream of the stop codon of prmA in the DNA region upstream of the fis operon, a region implicated in the control of the expression of the fis operon . Although no promoter activity was detected immediately upstream of prmA, S1 mapping detected 5' ends of mRNA in this region, implying that some mRNA processing occurs within the bicistronic panF-prmA mRNA.

J Bacteriol, 1993 Nov, 175(22), 7170 - 7
Stress response of Escherichia coli to elevated hydrostatic pressure; Welch TJ et al.; The response of exponentially growing cultures of Escherichia coli to abrupt shifts in hydrostatic pressure was studied . A pressure upshift to 546 atm (55,304 kPa) of hydrostatic pressure profoundly perturbed cell division, nucleoid structure, and the total rate of protein synthesis . The number of polypeptides synthesized at increased pressure was greatly reduced, and many proteins exhibited elevated rates of synthesis relative to total protein synthesis . We designated the latter proteins pressure-induced proteins (PIPs) . The PIP response was transient, with the largest induction occurring approximately 60 to 90 min postshift . Fifty-five PIPs were identified . Many of these proteins are also induced by heat shock or cold shock . The PIP demonstrating the greatest pressure induction was a basic protein of 15.6 kDa . High pressure inhibits growth but does not inhibit the synthesis of stringently controlled proteins . Cold shock is the only additional signal which has been found to elicit this type of response . These data indicate that elevated pressure induces a unique stress response in E . coli, the further characterization of which could be useful in delineating its inhibitory nature.

J Bacteriol, 1993 Nov, 175(22), 7138 - 41
Conditional synthesis and utilization of 1,5-anhydroglucitol in Escherichia coli; Shiga Y et al.; A cyclic polyol, 1,5-anhydro-D-glucitol (AG), is widely detected in most organisms, although little is known about its metabolism and physiological roles . The present study demonstrates the synthesis of AG in Escherichia coli C600 . The major portion of the synthesized AG was indicated to be derived from glucose retaining all the six carbon atoms, and only 5% was attributed to AG synthesized from C3 compounds . AG synthesis is apparent in an early stage of the stationary phase, and accumulation is transient both in cells and in medium . Evidence is also presented for AG uptake and metabolism and for effects of cyclic AMP.

J Bacteriol, 1993 Nov, 175(22), 7125 - 9
Polarized cells, polar actions; Maddock JR et al.; The recognition of polar bacterial organization is just emerging . The examples of polar localization given here are from a variety of bacterial species and concern a disparate array of cellular functions . A number of well-characterized instances of polar localization of bacterial proteins, including the chemoreceptor complex in both C . crescentus and E . coli, the maltose-binding protein in E . coli, the B . japonicum surface attachment proteins, and the actin tail of L . monocytogenes within a mammalian cell, involve proteins or protein complexes that facilitate bacterial interaction with the environment, either the extracellular milieux or that within a plant or mammalian host . The significance of this observation remains unclear . Polarity in bacteria poses many problems, including the necessity for a mechanism for asymmetrically distributing proteins as well as a mechanism by which polar localization is maintained . Large structures, such as a flagellum, are anchored at the pole by means of the basal body that traverses the peptidoglycan wall . But for proteins and small complexes, whether in the periplasm or the membrane, one must invoke a mechanism that prevents the diffusion of these proteins away from the cell pole . Perhaps the periplasmic proteins are retained at the pole by the presence of the periseptal annulus (35) . The constraining features for membrane components are not known . For large aggregates, such as the clusters of MCP, CheA, and CheW complexes, perhaps the size of the aggregate alone prevents displacement . In most cases of cellular asymmetry, bacteria are able to discriminate between the new pole and the old pole and to utilize this information for localization specificity . The maturation of new pole to old pole appears to be a common theme as well . Given numerous examples reported thus far, we propose that bacterial polarity displays specific rules and is a more general phenomenon than has been previously recognized.

J Bacteriol, 1993 Nov, 175(21), 7097 - 101
Antisense transcription of the ftsZ-ftsA gene junction inhibits cell division in Escherichia coli; Dewar SJ et al.; A 490-bp DNA segment spanning the junction between the ftsA and ftsZ genes inhibits cell division when present in high copy number . We show that this segment contains an antisense promoter and an antisense transcription terminator which define a new gene, stfZ.

J Bacteriol, 1993 Nov, 175(21), 7092 - 6
Isolation and analysis of novel mutants of Escherichia coli prlA (secY); Olsen MK et al.; Plasmid libraries of prlA mutants containing single-base-pair changes throughout the gene were generated by in vitro random mutagenesis . The prlA mutations capable of suppressing the secretion defect of LamB caused by mutations in the LamB signal peptide were selected and analyzed . Together with additional mutations generated by site-directed mutagenesis, a number of novel prlA mutations and/or suppressors were identified . These mutations provide the starting points for studying the relationship of structure and function of PrlA in its interaction with LamB and/or other component(s) in the Escherichia coli protein secretion-translocation complex.

J Bacteriol, 1993 Nov, 175(21), 7066 - 73
purU, a source of formate for purT-dependent phosphoribosyl-N-formylglycinamide synthesis; Nagy PL et al.; A gene designated purU has been identified and characterized . purU is adjacent to tyrT at min 27.7 on the Escherichia coli chromosome . The gene codes for a 280-amino-acid protein . The C-terminal segment of PurU from residues 84 to 280 exhibits 27% identity with 5'-phosphoribosylglycinamide (GAR) transformylase, the product of purN . Primer extension mapping and assays of lacZ in a promoter probe vector identified two promoters giving mono- and bi-cistronic purU mRNA . Neither mRNA was regulated by purines . Mutations in either of two pairs of genes are required to block synthesis of 5'-phosphoribosyl-N-formylglycinamide (FGAR) from GAR: purN purT (purT encodes an alternative formate-dependent GAR transformylase) or purN purU . On the basis of the growth of purU, purN, and purU purN mutants, it appears that PurU provides the major source of formate for the purT-dependent synthesis of FGAR.

J Bacteriol, 1993 Nov, 175(21), 7024 - 32
Mechanism of autophosphorylation of Escherichia coli nitrogen regulator II (NRII or NtrB): trans-phosphorylation between subunits; Ninfa EG et al.; Nitrogen regulator II (NRII or NtrB) is a homodimeric signal-transducing protein kinase/phosphatase responsible for the transcriptional regulation of the Ntr regulon in Escherichia coli . NRII is a member of a large family of proteins that are part of the related two-component signal transduction systems . We studied the mechanism of NRII autophosphorylation by using purified components . Alteration of the site of NRII autophosphorylation to asparagine (H-139-->N {H139N}) or deletion of the C-terminal 59 amino acids of NRII (ter291) resulted in proteins that were not autophosphorylated upon incubation with ATP . Alteration of glycine 313 to alanine resulted in a protein (G313A) that was phosphorylated to a lesser extent than the wild-type protein . Unlike wild-type NRII and H139N, G313A could not be efficiently cross-linked to {alpha-32P}ATP, suggesting that the G313A mutation affects nucleotide binding . Fusion of maltose-binding protein (MBP) to the N-terminal end of NRII resulted in a protein (MBP-NRII) that autophosphorylated normally . We developed a procedure for forming mixed dimers in vitro from these proteins . In mixed dimers consisting of MBP-NRII and H139N, only the MBP-NRII subunit is phosphorylated . In contrast, in mixed dimers consisting of MBP-NRII and G313A, phosphorylation is predominantly on the G313A subunit . We also demonstrated that the G313A and H139N proteins could complement for the autophosphorylation reaction when they were treated so as to permit the formation of mixed dimers and that the wild-type and H139N proteins could phosphorylate the ter291 protein . These results indicate that the autophosphorylation reaction occurs within the dimer by a trans, intersubunit mechanism in which one subunit binds ATP and phosphorylates the other subunit.

J Bacteriol, 1993 Nov, 175(21), 6988 - 95
Regions of maltose-binding protein that influence SecB-dependent and SecA-dependent export in Escherichia coli; Strobel SM et al.; In Escherichia coli, the efficient export of maltose-binding protein (MBP) is dependent on the chaperone SecB, whereas export of ribose-binding protein (RBP) is SecB independent . To localize the regions of MBP involved in interaction with SecB, hybrids between MBP and RBP in SecB mutant cells were constructed and analyzed . One hybrid consisted of the signal peptide and first third of the mature moiety of MBP, followed by the C-terminal two-thirds of RBP (MBP-RBP112) . This hybrid was dependent upon SecB for its efficient export and exhibited a strong export defect in secA mutant cells . A hybrid between RBP and MBP with the same fusion point was also constructed (RBP-MBP116) . The RBP-MBP116 hybrid remained SecB independent and only exhibited a partial export defect in secA mutant cells . In addition, MBP species with specific alterations in the early mature region were less dependent on SecB for their efficient export . The export of these altered MBP species was also less affected in secA mutant cells and in cells treated with sodium azide . These results present additional evidence for the targeting role of SecB.

J Bacteriol, 1993 Nov, 175(21), 6982 - 7
Transcription of ColE1Ap mbeC induced by conjugative plasmids from twelve different incompatibility groups; Selvaratnam S et al.; Although nonconjugative mobilizable plasmids require helping functions of conjugative plasmids in order to be mobilized into recipients, at least some genes from the nonconjugative plasmids may be induced to assist in the DNA transfer process . Conjugative plasmids from 12 different incompatibility groups mobilized the nonconjugative plasmid ColE1Ap between Escherichia coli strains . Introduction of any of the conjugative plasmids into the ColE1Ap-containing strain resulted in an induction of mbeC, the product of which is a component of the mobilization relaxation complex . Each of the conjugative plasmids caused protein to bind specifically to mbe promoter DNA, suggesting a direct regulatory interaction.

J Bacteriol, 1993 Nov, 175(21), 6939 - 44
Control of gluconeogenic growth by pps and pck in Escherichia coli; Chao YP et al.; It is well-known that Escherichia coli grows more slowly on gluconeogenic carbon sources than on glucose . This phenomenon has been attributed to either energy or monomer limitation . To investigate this problem further, we varied the expression levels of pck, encoding phosphoenolpyruvate carboxykinase (Pck), and pps, encoding phosphoenolpyruvate synthase (Pps) . We found that the growth rates of E . coli in minimal medium supplemented with succinate and with pyruvate are limited by the levels of Pck and Pps, respectively . Optimal overexpression of pck or pps increases the unrestricted growth rates on succinate and on pyruvate, respectively, to the same level attained by the wild-type growth rate on glycerol . Since Pps is needed to supply precursors for biosyntheses, we conclude that E . coli growing on pyruvate is limited by monomer supply . However, because pck is required both for biosyntheses and catabolism for cells growing on succinate, it is possible that growth on succinate is limited by both monomer and energy supplies . The growth yield with respect to oxygen remains approximately constant, even though the overproduction of these enzymes enhances gluconeogenic growth . It appears that the constant yield for oxygen is characteristic of efficient growth on a particular substrate and that the yield is already optimal for wild-type strains . Further increases in either Pck or Pps above the optimal levels become growth inhibitory, and the growth yield for oxygen is reduced, indicating less efficient growth.

J Bacteriol, 1993 Nov, 175(21), 6925 - 31
Nucleotide sequence and 3'-end deletion studies indicate that the K(+)-uptake protein kup from Escherichia coli is composed of a hydrophobic core linked to a large and partially essential hydrophilic C terminus; Schleyer M et al.; The kup (formerly trkD) gene from Escherichia coli encodes a minor K(+)-uptake system . The gene is located just upstream of the rbsDACBK operon at 84.5 min on the chromosome and is transcribed clockwise . kup codes for a 69-kDa protein, which may be composed of two domains . The first 440 amino acid residues appear to form an integral membrane protein that might traverse the cell membrane 12 times . The C-terminal 182 amino acid residues are predicted to form a hydrophilic domain located at the cytoplasmic side of the membrane . Deletion studies from the 3' end of kup showed that removal of almost the complete hydrophilic domain of the protein reduced, but did not abolish, K(+)-uptake activity.

J Bacteriol, 1993 Nov, 175(21), 6857 - 66
Induction by ethanol of alcohol dehydrogenase activity in Acetobacter pasteurianus; Takemura H et al.; The membrane-bound alcohol dehydrogenase (ADH) activity of Acetobacter pasteurianus NCI1380 was enhanced more than 10-fold by the addition of ethanol to the medium . In order to elucidate the mechanism of the ethanol induction, a gene cluster encoding the dehydrogenase and cytochrome c subunits of ADH was cloned from this strain, and its nucleotide sequence was determined . Comparison of the deduced amino acid sequences and the NH2-terminal sequences determined with purified proteins showed that the dehydrogenase and cytochrome c subunits contained typical signal peptides of 35 and 26 amino acids, respectively . Transcriptional analysis of the cloned genes by primer extension revealed that the gene cluster was transcribed from two different promoters upstream from the dehydrogenase gene . One (59 bp upstream of the ATG start codon) of the two promoters was used in the presence of ethanol, whereas the other (232 bp upstream of the ATG start codon) was used in the absence of ethanol . Immunoblot analyses showed that almost the same amounts of the cytochrome c and the 15-kDa subunits were produced in both the presence and absence of ethanol and that the amount of the dehydrogenase subunit localized in the membrane was decreased in the absence of ethanol . This incorrect localization of the dehydrogenase subunit might be one of the factors responsible for the low ADH activity in the absence of ethanol.

J Bacteriol, 1993 Nov, 175(21), 6850 - 6
chpA and chpB, Escherichia coli chromosomal homologs of the pem locus responsible for stable maintenance of plasmid R100; Masuda Y et al.; The pem locus is responsible for stable maintenance of plasmid R100 and consists of two genes, pemI and pemK . The pemK gene product is a growth inhibitor, while the pemI gene product is a suppressor of this inhibitory function . We found that the PemI amino acid sequence is homologous to two open reading frames from Escherichia coli called mazE and orf-83, which are located at 60 and 100 min on the chromosome, respectively . We cloned and sequenced these loci and found additional open reading frames, one downstream of each pemI homolog, both of which encode proteins homologous to PemK . The pem locus homolog at 60 min was named chpA and consists of two genes, chpAI and chpAK; the other, at 100 min, was named chpB and consists of two genes, chpBI and chpBK . The distal portion of chpBK was found to be adjacent to the ppa gene that encodes pyrophosphatase, whose map position had not been previously determined . We then demonstrated that the chpAK and chpBK genes encode growth inhibitors, while the chpAI and chpBI genes encode suppressors for the inhibitory function of the ChpAK and ChpBK proteins, respectively . These E . coli pem locus homologs may be involved in regulation of cell growth.

J Bacteriol, 1993 Nov, 175(21), 6836 - 41
Mycobacteriophage L5 integrase-mediated site-specific integration in vitro; Lee MH et al.; Mycobacteriophage L5, a temperate phage of the mycobacteria, forms stable lysogens in Mycobacterium smegmatis via site-specific integration of the phage genome . Recombination occurs within specific phage and bacterial attachment sites and is catalyzed by the phage-encoded integrase protein in vivo . We describe here the overexpression and purification of L5 integrase and its ability to mediate integrative recombination in vitro . We find that L5 integrase-mediated recombination is greatly stimulated by extracts of M . smegmatis but not by Escherichia coli extracts, purified E . coli integration host factor, or purified HU, indicating the presence of a novel mycobacterial integration host factor.

J Bacteriol, 1993 Nov, 175(21), 6797 - 809
Use of the rep technique for allele replacement to construct mutants with deletions of the pstSCAB-phoU operon: evidence of a new role for the PhoU protein in the phosphate regulon; Steed PM et al.; The phosphate regulon is negatively regulated by the PstSCAB transporter and PhoU protein by a mechanism that may involve protein-protein interaction(s) between them and the Pi sensor protein, PhoR . In order to study such presumed interaction(s), mutants with defined deletions of the pstSCAB-phoU operon were made . This was done by construction of M13 recombinant phage carrying these mutations and by recombination of them onto the chromosome by using a rep host (which cannot replicate M13) for allele replacement . These mutants were used to show that delta (pstSCAB-phoU) and delta (pstB-phoU) mutations abolished Pi uptake by the PstSCAB transporter, as expected, and that delta phoU mutations had no effect on uptake . Unexpectedly, delta phoU mutations had a severe growth defect, and this growth defect was (largely) alleviated by a compensatory mutation in the pstSCAB genes or in the phoBR operon, whose gene products positively regulate expression of the pstSCAB-phoU operon . Because delta phoU mutants that synthesize a functional PstSCAB transporter constitutively grew extremely poorly, the PhoU protein must have a new role, in addition to its role as a negative regulator . A role for the PhoU protein in intracellular Pi metabolism is proposed . Further, our results contradict those of M . Muda, N . N . Rao, and A . Torriani (J . Bacteriol . 174:8057-8064, 1992), who reported that the PhoU protein was required for Pi uptake.

Genes Dev, 1993 Nov, 7(11), 2149 - 60
Cell cycle-regulated nuclear localization of MCM2 and MCM3, which are required for the initiation of DNA synthesis at chromosomal replication origins in yeast; Yan H et al.; MCM2 and MCM3 are two genetically interacting and structurally related proteins essential for growth in Saccharomyces cerevisiae . Mutants defective in these proteins affect the stability of minichromosomes in general, but the severity of the defect is dependent on the autonomously replicating sequence (ARS) that drives the replication of that plasmid . In this paper we show by two-dimensional gel electrophoresis that the initiation of DNA synthesis at chromosomal replication origins is also reduced in frequency in these mutants . We show further that the nuclear and subnuclear localizations of the MCM2 and MCM3 proteins are temporally regulated with respect to the cell cycle . These proteins enter the nucleus at the end of mitosis, persist there throughout G1 phase, and disappear from it at the beginning of S phase . Once inside the nucleus, a fraction of the MCM2 and MCM3 proteins becomes tightly associated with DNA . The association of MCM2 and MCM3 with chromatin presumably leads to the initiation of DNA synthesis, and their subsequent disappearance from the nucleus presumably prevents reinitiation of DNA synthesis at replication origins . This temporally and spatially restricted localization of MCM2 and MCM3 in the nucleus may serve to ensure that DNA replication occurs once and only once per cell cycle.

Dev Biol, 1993 Nov, 160(1), 99 - 107
Behavior of the components of maturation-promoting factor, cdc2 kinase and cyclin B, during oocyte maturation of goldfish; Katsu Y et al.; We examined the changes that occurred in the two components of maturation-promoting factor (MPF), cdc2 kinase and cyclin B, during oocyte maturation in goldfish, using monoclonal antibodies against the C-terminal sequence of goldfish cdc2 kinase and Escherichia coli-produced full-length goldfish cyclin B . Immature oocytes contained a 35-kDa inactive cdc2 kinase . In addition to the 35-kDa form, a 34-kDa active cdc2 kinase was detected in oocytes undergoing germinal vesicle breakdown (GVBD) . Cyclin B was absent in immature oocytes and appeared just before GVBD, coinciding exactly with the appearance of the 34-kDa active cdc2 kinase . Precipitation with p13suc1 beads and anticyclin B antibody revealed that cyclin B formed a complex with cdc2 kinase as soon as it appeared . MPF activation was induced by 1 ng cyclin B after introduction into immature oocytes or oocyte extracts . This corresponds to the amount of cyclin B found in mature oocytes (the concentration in the oocyte is 2 micrograms/ml) . These results suggest that MPF activation in fish oocytes is induced by complex formation with preexisting cdc2 kinase and newly synthesized cyclin B during oocyte maturation, a situation differing from that in Xenopus and starfish, in which the cdc2 kinase-cyclin B complex is already present in immature oocytes . Unlike that in Xenopus, an inhibition of protein synthesis in unfertilized mature goldfish oocytes caused a decrease in the cdc2 kinase activity/cyclin B protein level and led to a progression from meiotic metaphase to meiotic anaphase . This result indicates that the mechanisms of maintaining MPF activity in mature goldfish oocytes differ from those in Xenopus.

Fertil Steril, 1993 Nov, 60(5), 826 - 33
Elevated levels of interleukin-6 in ascites and serum from women with ovarian hyperstimulation syndrome; Friedlander MA et al.; OBJECTIVE: To examine the role of pro-inflammatory cytokines in ovarian hyperstimulation syndrome (OHSS) . DESIGN: In vitro laboratory study of serum, peritoneal cells isolated and fluid obtained from ascites removed in the therapeutic management of four patients with OHSS . SETTING: Tertiary care referral teaching hospital . PATIENTS: Four patients with OHSS comprised the study population . Five healthy women at the time of elective laparoscopic tubal ligation served as controls . Control serum was also obtained from healthy adult volunteers, and control peritoneal fluid (PF) was obtained from patients on peritoneal dialysis . INTERVENTIONS: Therapeutic paracentesis was performed on four patients with OHSS . RESULTS: Peritoneal cells were isolated and fluid obtained from ascites removed in the therapeutic management of the women with OHSS . Peritoneal cells were obtained by intraoperative lavage from the control women . The cells were incubated with various concentrations of Escherichia coli lipopolysaccharide (LPS) for 20 and 48 hours . Interleukin-1 beta (IL-1 beta), tumor necrosis factor (TNF), interleukin-6 (IL-6), and prostaglandin E2 (PGE2) were assayed in the incubation supernatants . The release of the three cytokines and PGE2 in response to LPS by peritoneal cells from women with OHSS was not different from the controls . However, both serum and ascitic fluid from women with OHSS contained significantly greater levels of IL-6 than control serum and PF . No significant differences in TNF levels in serum, ascitic fluid, or PF could be found by ELISA or bioassay . CONCLUSIONS: Increased production of IL-6, released into the peritoneal cavity and the circulation, may mediate OHSS.

FEBS Lett, 1993 Nov 1, 333(3), 261 - 7
Escherichia coli single-stranded DNA-binding protein alters the structure of intramolecular triplexes in plasmids; Klysik J et al.; The ability of the Escherichia coli single-stranded DNA-binding protein (SSB) to recognize structural features associated with intramolecular triplex formation in oligopurine.oligopyrimidine (pur.pyr) inserts in recombinant plasmids was evaluated . The SSB protein binds to supercoiled plasmids and causes a site-preferential increase in OsO4 reactivity of the pyrimidine strand involved in the formation of the Hy-3 isomer of the triplex structure . The E . coli RecA protein showed no reaction with triplexes in similar studies . This behavior is consistent with SSB-mediated unpairing of the H-DNA-forming region.

FEBS Lett, 1993 Nov 1, 333(3), 257 - 60
The three-dimensional structure of the colicin E3 immunity protein by distance geometry calculation; Yajima S et al.; The three-dimensional solution structure of the colicin E3 immunity protein (84 residues) was determined by distance geometry calculations . The hydrophilic side of a four-stranded antiparallel beta-sheet constitutes a part of the surface of the protein, and two loops lie on the hydrophobic side of the sheet . All the three specificity-determining residues, which are included in the center of the beta-sheet, display their side groups on the protein surface.

Eur J Biochem, 1993 Nov 1, 217(3), 991 - 9
Catalytic mechanism of 3-deoxy-D-manno-2-octulosonate-8-phosphate synthase . The use of synthetic analogues to probe the structure of the putative reaction intermediate; Baasov T et al.; The proposed mechanistic pathway for the reaction catalyzed by 3-deoxy-D-manno-2-octulosonate-8-phosphate (Kdo8P) synthase was examined in terms of the structure of the putative bisphosphate intermediate . Two 2-deoxy analogues of the product Kdo8P, having been structurally prohibited from undergoing the ring-opening and possessing the stereochemistry of either the alpha-pyranase (compound 1) or the beta-pyranose form (compound 2) of the product, were synthesized and probed as inhibitors for the synthase . It was found that both analogues bind to the enzyme and are competitive inhibitors with respect to phosphoenolpyruvate binding, having Ki values of 470 microM and 303 microM, respectively . Comparison of this data to the Ki value of the tautomeric mixture of the product Kdo8P (Ki = 590 microM) suggests that both the alpha- and the beta-pyranose anomers (65.8% and 3.1%, respectively at neutral pH) bind to the enzyme with a slight (1.13 kJ/mol) preference for the beta-anomer, and that the C2 hydroxyl does not contribute to the binding . This uncertain stereochemical preference exhibited by the enzyme for the stereoisomers at the anomeric carbon suggests that the carboxylate binding site of the product is indistinct, while the hydroxyl and carboxylate binding sites may be interchangeable . More importantly, however, the isosteric phosphonate analogue 2,6-anhydro-3-deoxy-2 beta-phosphonylmethyl-8-phosphate-D-glycero-D-talo-octonate (3), which mimics the topological and electrostatic properties of the proposed cyclic intermediate, was found to be the most potent inhibitor of the enzyme with a Ki value of 5 microM . Two hitherto unrecognized aspects of the mechanism of the synthase were identified . First, the results showing that the cyclic analogues 1, 2 and 3 are inhibitors of the enzyme whereas the previously reported acyclic analogue, which contains no carbonyl group at C2 and may thus resemble the open-chain form of Kdo8P, is not an inhibitor, suggest that the pyranose form and not the open-chain acyclic form of the putative bisphosphate intermediate is handled by the enzyme . Second, since the overall stereochemical course of the transformation mediated by the synthase has been shown to involve si face addition of phosphoenolpyruvate to the re face of the carbonyl of arabinose 5-phosphate, the present observation involving analogue 3 suggest that the bisphosphate intermediate formed during the initial steps of synthesis may have the pyranose structure with the anomeric phosphate located in the beta-configuration.

Eur J Biochem, 1993 Nov 1, 217(3), 839 - 47
Reconstruction of translation . Evidence for the involvement of the rescue protein in the association/dissociation of ribosomal subunits; Ganoza MC et al.; The in vitro reversal of conditionally lethal mutations has greatly aided the study of translation . N4316 is a mutant of Escherichia coli that has a temperature-sensitive defect in a protein called the rescue protein . Without the rescue protein, translation in vivo and in vitro is drastically reduced and frameshift errors, as well as increased read-through of nonsense codons, occurs . Using reversal of temperature-sensitivity as an assay, the rescue protein was purified from a ribosomal eluate of the parental (D10) strain . Composite polyacrylamide/agarose gel electrophoresis and sedimentation on sucrose density gradients were employed to examine the distribution of 70S ribosomes and ribosomal subunits in the mutant (N4316) and the parental (D10) extracts at restrictive (43 degrees C) and non-restrictive (35 degrees C) temperatures . Fewer polysomes and a larger proportion of 70S ribosomes relative to subunits were observed at 43 degrees C with N4316, but not with D10 extracts . Addition of the rescue protein had no effect at 35 degrees C with either strain, but restored the polysome pattern of N4316 at 43 degrees C . The purified rescue protein labelled by methylation retained activity and bound preferentially to 30S subunits . Rescue bound to 30S particles prevented the action of IF-3 fostering formation of 70S ribosomes . Thus the rescue protein enables formation of 70S ribosomes from 30S and 50S subunits . 70S ribosomes which contain the rescue protein are active in translation and resist dissociation induced by high centrifugal fields . We propose that the rescue protein alters the conformation of 70S ribosomes resulting in a tighter association of subunits which, in turn, fosters both higher rates and increased accuracy of translation.

Eur J Biochem, 1993 Nov 1, 217(3), 1049 - 56
The putative zinc-finger protein WZF1 interacts with a cis-acting element of wheat histone genes; Sakamoto A et al.; A nonamer motif (CATCCAACG) that is one of the cis-acting elements identified in the proximal promoter region of some wheat histone genes is included in the region that interacts with the wheat DNA-binding protein, HBP (histone gene-binding protein)-2 . To obtain structural and functional information about this DNA-binding protein, we attempted to isolate a cDNA clone encoding HBP-2 on the basis of its ability to bind to a nonamer-containing 38-bp DNA fragment . Southwestern screening of a wheat cDNA library with concatenated 38-residue oligonucleotides as the probe produced one candidate clone . Nucleotide sequence analyses of this cDNA clone and the corresponding genomic clone showed that the protein deduced from the nucleotide sequence consisted of 261 amino acids and contained a set of zinc-finger motifs similar to those found in many eukaryotic transcription factors . The protein, named WZF1 (wheat zinc-finger protein 1), which was expressed from the cDNA in Escherichia coli cells, bound specifically and metal-ion-dependently to the nonamer-containing oligonucleotide . The WZF1 mRNA was highly expressed in the root apexes of wheat seedlings, but less so in the proximal portion of young leaves; whereas, histone H3 mRNA was highly expressed in both tissues . The expression patterns of the WZF1 and histone H3 genes in the early stages of germination differed, expression of the WZF1 gene being almost constant but not that of the H3 gene . The relationship of WZF1 and HBP-2 and the possible role of WZF1 in the histone gene expression were discussed.

EMBO J, 1993 Nov, 12(11), 4413 - 24
The alpha-helical rod domain of human lamins A and C contains a chromatin binding site; Glass CA et al.; We examined regions of human lamins A and C involved in binding to surfaces of mitotic chromosomes . An Escherichia coli expression system was used to produce full-length lamin A and lamin C, and truncated lamins retaining the central alpha-helical rod domain (residues 34-388) but lacking various amounts of the amino-terminal 'head' and carboxy-terminal 'tail' domains . We found that lamin A, lamin C and lamin fragments lacking the head domain and tail sequences distal to residue 431 efficiently assembled into paracrystals and strongly associated with mitotic chromosomes . Furthermore, the lamin rod domain also associated with chromosomes, although efficient chromosome coating required the pH 5-6 conditions needed to assemble the rod into higher order structures . Biochemical assays showed that chromosomes substantially reduced the critical concentration for assembly of lamin polypeptides into pelletable structures . Association of the lamin rod with chromosomes was abolished by pretrypsinization of chromosomes, and was not seen for vimentin (which possesses a similar rod domain) . These data demonstrate that the alpha-helical rod of lamins A and C contains a specific chromosome binding site . Hence, the central rod domain of intermediate filament proteins can be involved in interactions with other cellular structures as well as in filament assembly.

EMBO J, 1993 Nov, 12(11), 4305 - 15
Depletion of functional ribosomal RNA operons in Escherichia coli causes increased expression of the remaining intact copies; Condon C et al.; The synthesis of ribosomal RNA is a complex and highly regulated process . To study this process, we have used deletion-insertions to disrupt sequentially from one to four of the seven rRNA (rrn) operons on the Escherichia coli genome . Inactivation of four rrn operons caused a 2.3-fold increase in the expression of a chloramphenicol acetyl transferase reporter gene fused to the tandem promoters of rrnA and a similar increase in the expression of the trp tRNA gene at the end of rrnC . This reflected enhanced expression of the remaining operons to compensate for having only three intact copies . The elevated expression was caused by an increase in both transcription initiation and RNA polymerase elongation rates specifically on rrn operons and occurred in the absence of changes in the intracellular concentration of ppGpp, suggesting that ppGpp is not involved in the regulation of this phenomenon . We discuss these results in relation to the ribosome feedback inhibition model described by Nomura and coworkers.

EMBO J, 1993 Nov, 12(11), 4105 - 14
Multiple pathways for protein transport into or across the thylakoid membrane; Cline K et al.; Many thylakoid proteins are cytosolically synthesized and have to cross the two chloroplast envelope membranes as well as the thylakoid membrane en route to their functional locations . In order to investigate the localization pathways of these proteins, we over-expressed precursor proteins in Escherichia coli and used them in competition studies . Competition was conducted for import into the chloroplast and for transport into or across isolated thylakoids . We also developed a novel in organello method whereby competition for thylakoid transport occurred within intact chloroplasts . Import of all precursors into chloroplasts was similarly inhibited by saturating concentrations of the precursor to the OE23 protein . In contrast, competition for thylakoid transport revealed three distinct precursor specificity groups . Lumen-resident proteins OE23 and OE17 constitute one group, lumenal proteins plastocyanin and OE33 a second, and the membrane protein LHCP a third . The specificity determined by competition correlates with previously determined protein-specific energy requirements for thylakoid transport . Taken together, these results suggest that thylakoid precursor proteins are imported into chloroplasts on a common import apparatus, whereupon they enter one of several precursor-specific thylakoid transport pathways.

Crit Care Med, 1993 Nov, 21(11), 1750 - 7
Endotoxin infusion primes elicited neutrophils and Kupffer cells for platelet-activating factor-induced and tripeptide formyl-methionine-leucine-phenylalanine-induced basal free intracellular calcium concentration responses; Hagar AF et al.; OBJECTIVE: To determine whether in vivo infusions of bacterial endotoxin prime rat Kupffer cells and elicited neutrophils by altering the basal free intracellular calcium concentration or the response of free intracellular calcium to platelet-activating factor or the tripeptide formyl-methionine-leucine-phenylalanine (f-met-leu-phe) . DESIGN: Prospective, randomized, controlled study . SETTING: Laboratory of a university medical school . SUBJECTS: Adult male Sprague-Dawley rats . INTERVENTIONS: Rats were infused with sterile saline or with Escherichia coli endotoxin for 3 or 30 hrs via a venous catheter or an osmotic minipump . Rats were anaesthetized, and the livers were perfused in situ with a collagenase-containing buffer . Kupffer cells and elicited neutrophils were isolated by centrifugal elutriation, and were partially purified over Ficoll gradients . MEASUREMENTS AND MAIN RESULTS: Basal free intracellular calcium concentrations were determined with the calcium indicator, fluo-3 . Various concentrations of platelet-activating factor or f-met-leu-phe were then added, and any alterations in free intracellular calcium values were quantified . Neither acute (3-hr) nor chronic (30-hr) infusions of endotoxin altered basal free intracellular calcium values . F-met-leu-phe-induced increases in free intracellular calcium concentrations were positively correlated with the percentage of neutrophils in Kupffer cell fractions . Three-hour infusions of endotoxin, which caused a significant inclusion of neutrophils in the Kupffer cell fraction, resulted in an enhanced response to f-met-leu-phe . This response was considered to be primarily due to the presence of neutrophils, because preparations that were > 95% Kupffer cells did not respond to f-met-leu-phe . Platelet-activating factor-induced increases in free intracellular calcium concentrations were also positively correlated with the percentage of neutrophils . However, Kupffer cells isolated from rats infused for 30 hrs with endotoxin contained < 12% neutrophils, and exhibited a ten-fold greater response to platelet-activating factor than did Kupffer cells isolated from saline-infused rats . CONCLUSIONS: In vivo infusions of endotoxin result in an enhanced response to platelet-activating factor by rat Kupffer cells . F-met-leu-phe does not appear to induce an increase in free intracellular calcium values in rat Kupffer cells, but it appears to do so in endotoxin-elicited neutrophils . These observations indicate that changes in calcium homeostasis may be one mechanism by which endotoxin primes Kupffer cells and elicited neutrophils for enhanced production of superoxide anion and/or nitric oxide.

Blood, 1993 Nov 1, 82(9), 2853 - 64
Stimulation of interleukin-6 and prostaglandin E2 secretion from peritoneal macrophages by polymers of albumin; Shacter E et al.; Interleukin-6 (IL-6) is a multifunctional cytokine that is elevated in vivo during acute infection, chronic inflammation, and some hematopoietic malignancies . To understand how IL-6 becomes elevated in vivo, it is important to identify factors that can stimulate its secretion from effector cells . We found that commercial preparations of bovine serum albumin (BSA) stimulated murine macrophages to secrete high levels of IL-6 . In fact, BSA was at least as potent as bacterial lipopolysaccharide (LPS) in stimulating IL-6 production . Stimulation was clearly visible at concentrations as low as 20 micrograms/mL and reached saturation at 0.5 to 1 mg/mL albumin, at which concentration 1.1 x 10(6) oil-elicited macrophages produced 6,000 +/- 700 B9 units of IL-6 in an overnight incubation . Prostaglandin E2 production was induced by the same concentrations of BSA . Both resident and oil-elicited peritoneal cells were responsive to the albumin . The stimulatory activity did not derive from contamination of the protein with Escherichia coli LPS; when compared directly with LPS, the response to BSA was more rapid, had a higher amplitude, and was not inhibitable by polymyxin B . In addition, macrophages isolated from C3H/HeJ mice, which have an inherited defect in their ability to respond to LPS, secreted IL-6 in response to BSA but not to LPS . The stimulatory activity was stable to heat, mild acid, and reduction/alkylation and copurified with albumin on Cibachron Blue agarose (Sigma, St Louis, MO) and anti-albumin immunoaffinity chromatography . Comparison of different sources and preparations of albumin showed differences in the levels of IL-6-inducing activity; three different lots of commercial fatty acid-free BSA and one lot of polymer-enhanced BSA stimulated IL-6 secretion by more than 100-fold over basal levels whereas other preparations showed more limited activity . A sample of BSA that was active in vitro caused a marked elevation of IL-6 when injected into BALB/c mice, thus demonstrating inflammatory activity in vivo . When the albumin preparations were fractionated by ion exchange and gel filtration chromatography and then analyzed by sodium dodecyl sulfate-gel electrophoresis and Western blot immunoassay, it was found that the IL-6-inducing activity resided in high molecular weight polymers of albumin . The ability of albumin polymers to stimulate IL-6 production represents a novel mechanism for modulation of this cytokine.

DNA Cell Biol, 1993 Nov, 12(9), 777 - 83
Heterologous and homologous protection against influenza A by DNA vaccination: optimization of DNA vectors; Montgomery DL et al.; We have recently shown that direct injection of DNA can be an effective vaccine strategy eliciting both humoral and cell-mediated immune responses . Vectors were designed specifically for vaccination by direct DNA injection and refined to improve plasmid production in Escherichia coli . The vectors consist of a pUC-19 backbone with the cytomegalovirus (CMV) IE1 enhancer, promoter, and intron A transcription regulatory elements and the BGH polyadenylation sequences driving the expression of the reporter gene CAT or influenza A nucleoprotein (NP) or hemagglutinin (HA) . The respective vectors expressed high levels of chloramphenicol acetyltransferase (CAT) and NP in tissue culture, and yielded 14-15 mg of purified plasmid per liter of Escherichia coli culture . Immunization of mice with the NP and HA expression vectors resulted in protection from subsequent lethal challenges of influenza using either heterologous or homologous strains, respectively.

Arch Biochem Biophys, 1993 Nov 1, 306(2), 518 - 20
Effects of paraquat on Escherichia coli: sensitivity to small changes in pH of the medium--a cautionary note; Liochev SI et al.; Uric acid appears to protect Escherichia coli against the growth-inhibiting effect of paraquat, but this is actually due to acidification of the medium and does not occur when the pH of the medium is readjusted to neutrality . Any compound which lowers the pH of the medium will thus diminish the effect of paraquat on E . coli, whether that effect is inhibition of growth or adaptive induction of members of the soxRS regulon.

Arch Biochem Biophys, 1993 Nov 1, 306(2), 443 - 50
Expression of modified cytochrome P450 2C10 (2C9) in Escherichia coli, purification, and reconstitution of catalytic activity; Sandhu P et al.; The human cytochrome P450 (P450) 2C gene family is complex and heterologous expression methods are needed to facilitate the isolation of individual P450 proteins and the elucidation of their catalytic specificities . We prepared a series of constructs of P450 2C10 in the plasmid vector pCW, with modification of the 5' end of the coding sequence of the cDNA . Some were not expressed at all in Escherichia coli; two were expressed at levels of 5-20 nmol membrane-bound P450 (liter culture)-1--one (2C1028) with original codons 2-7 altered by substitution of the 5'-terminal sequence described by Barnes et al . (Barnes, H . J., Arlotto, M . P., and Waterman, M . R., Proc., Natl . Acad . Sci . USA 88, 5597-5601, 1991) and one (2C1029) with original codon 2 modified, codons 3-20 deleted, and alteration of the immediate downstream codons . In both cases the P450 2C10 proteins were found essentially only in the bacterial membranes . These proteins could be purified to a high degree by solubilization and a single DEAE chromatography step . Typical P450 Fe2+.CO absorption spectra were observed in the bacterial membranes and the purified preparations . The P450 2C1029 protein was found to have its N-terminal Met removed and the expected residues 2 (Ala)-24 were identified by amino acid sequence analysis . However, the other P450 (2C1028) was apparently blocked at the N-terminus . Three native P450 2C9/10 preparations isolated from human liver showed the expected sequences (beginning with Met) for at least the first 17 residues . The blocked N-terminus in the P450 2C1028 protein may be the result of the MALLLAVF sequence, which was also used in the expression of P450 3A4 and resulted in a blocked protein . Catalytic activities of P450 2C1028 and P450 2C1029 for tolbutamide hydroxylation were similar to those measured with purified liver P450 C29/10 in the presence of cytochrome b5, although the effect of cytochrome b5 did not always show the same pattern as with the isolated liver enzyme . The recombinant P450 2C10 enzymes did not catalyze (S)-mephenytoin 4'-hydroxylation.

Virology, 1993 Nov, 197(1), 420 - 5
Myristoylation-enhanced binding of the HIV-1 Nef protein to T cell skeletal matrix; Niederman TM et al.; The negative factor, Nef, of HIV-1 was found to associate to an extent of 16-42% with the detergent insoluble cytoskeletal fraction of T lymphocytes . Furthermore, Escherichia coli expressed Nef protein was found to bind during in vitro reactions with the cytoskeletal matrix to an extent of 30-50% . Cytoskeletal association of Nef was significantly enhanced by myristoylation . The specificity of the myristoylation-enhanced binding was demonstrated by the lack of an effect of myristoylation on binding of the HIV-1 Gag protein to the cytoskeleton . Cytoskeletal binding was saturable, and inhibited by high concentrations of sodium chloride, or with SDS or urea . Binding of Nef to the cytoskeletal matrix may be important in mediating its effects on HIV-1 replication.

Res Microbiol, 1993 Nov-Dec, 144(9), 721 - 8
Molecular characterization of enterotoxigenic Escherichia coli (ETEC) isolated in New Caledonia (value of potential protective antigens in oral vaccine candidates); Begaud E et al.; The role of enterotoxigenic Escherichia coli (ETEC) in childhood diarrhoea in New Caledonia was demonstrated in previous epidemiological works . This study was undertaken in order to characterize these strains and to determine whether bacterial components of current vaccine candidates (toxin, colonization factor antigens, O:H antigens) would be useful in our region . A total of 24 ETEC strains were studied: 5 strains produced heat-labile enterotoxin, 17 strains produced heat-stable enterotoxin (9 STp and 8 STh), and 2 strains produced both toxins (1 LT/STp/STh and 1 LT/STh) . E . coli strains were screened for the presence of genes encoding for enterotoxins (DNA dot blot and Southern hybridization assays); results obtained with probes were closely correlated and were in agreement with biological assays . No two ETEC strains possessed similar plasmid profiles, and DNA sequences encoding for enterotoxins were located on plasmids ranging from 58 to 75 MDa . The O:H (O1:H-,O2:H7, O6:H16, O25:H-, O27:H7, O28ab:H9, O52:H10, O64:H5, O70:H-, O78:H12, O88:H25, O99:H6, O101:H-, O126:H12, O166:H30) serotypes are presented (all the strains were typable, but some ETEC serotypes were unusual) . By using antisera against colonization factor antigens (CFA) I and II, results showed that 9 of the 24 ETEC strains expressed CFA (2 CFA/II and 7 CFA/I) . These strains possessed high bacterial surface hydrophobicity . Fifteen ETEC did not possess CFA; among these, 11 did not exhibit high hydrophobicity or show haemagglutination activity . Four of the 15 CFA-negative strains exhibited high hydrophobicity (two O64:H45, one O70:H- and one O88:H25) but no haemagglutination in the presence or absence of mannose.(ABSTRACT TRUNCATED AT 250 WORDS)

Development, 1993 Nov, 119(3), 762 - 72
Spacing ensures autonomous expression of different stripe enhancers in the even-skipped promoter; Small S et al.; The even-skipped (eve) promoter contains a series of enhancers that control the expression of different segmentation stripes in the Drosophila embryo . The stripe 3 enhancer is located 1.7 kb upstream of the stripe 2 enhancer . Here we demonstrate that these enhancers must be physically separated by a minimum distance for proper stripe expression . When they are directly coupled in either orientation, the enhancers generate abnormal patterns of expression in the early embryo . For example, the levels of stripe 2 expression are augmented and there is a posterior expansion of the pattern when the stripe 3 enhancer is positioned immediately upstream of the stripe 2 enhancer . Despite this spacing requirement, the order of the enhancers within the eve promoter can be reversed without affecting the normal expression pattern . These results suggest that spacing maintains the autonomous activities of the stripe enhancers and that interactions between enhancers can generate novel patterns of gene expression.

Development, 1993 Nov, 119(3), 691 - 701
Complex fiber-type-specific expression of fast skeletal muscle troponin I gene constructs in transgenic mice; Hallauer PL et al.; We analyzed, in transgenic mice, the cellular expression pattern of the quail fast skeletal muscle troponin I (TnIfast) gene and of a chimeric reporter construct in which quail TnIfast DNA sequences drive expression of E . coli beta-galactosidase (beta-gal) . Both constructs were actively expressed in skeletal muscle and specifically in fast, as opposed to slow, muscle fibers . Unexpectedly, both constructs showed a marked differential expression among the adult fast fiber subtypes according to the pattern IIB > IIX > IIA . This expression pattern was consistent in multiple lines and differed from the endogenous mouse TnIfast pattern, which shows approximately equal expression in all fast fibers . These observations indicate that distinct regulatory mechanisms contribute to high-level expression of TnIfast in the various fast fiber subtypes and suggest that the outwardly simple pattern of equal expression in all fast fiber types shown by the endogenous mouse TnIfast gene is based on an intricate system of counterbalancing mechanisms . The adult expression pattern of the TnIfast/beta-gal construct emerged in a two-stage developmental process . Differential expression in fast versus slow fibers was evident in neonatal animals, although expression in fast fibers was relatively weak and homogeneous . During the first two weeks of postnatal life, expression in maturing IIB fibers was greatly increased whereas that in IIA/IIX fibers remained weak, giving rise to marked differential expression among fast fiber types . Thus at least two serially acting (pre- and post-natal) fiber-type-specific regulatory mechanisms contribute to high-level gene expression in adult fast muscle fibers . Unexpected similarities between TnIfast transgene expression and that of the myosin heavy chain gene family (which includes differentially expressed IIB-, IIX- and IIA-specific members) suggest that similar mechanisms may regulate adult fast muscle gene expression in a variety of unrelated muscle gene families.

Neurol Neurochir Pol, 1993 Nov-Dec, 27(6), 913 - 7
{A case of transverse myelitis with good outcome}; Milinska B et al.; The study presents a case of transverse myelitis in a 22 year old male . MRI was used to establish the diagnosis . It ought to be mentioned that, according to the medical literature, cases with paraplegia and total sensory loss have a rather poor prognosis . Hence the described case is a rare example of transverse myelitis which was successfully treated.

Biochem Soc Trans, 1993 Nov, 21(4), 1081 - 5
Heterologous expression of cytochrome P-450 in Escherichia coli; Waterman MR; While the bacterial expression system has proved to work well for the expression of functional forms both of microsomal and of mitochondrial P-450, we understand the requirements for expression in bacteria far less clearly than those for expression in eukaryotic cells . Nevertheless, the ability to generate large quantities of P-450 (Table 1) in an inexpensive culture system, the ease of site-directed mutagenesis in bacteria and the ability to purify easily relatively large quantities of P-450s, including human enzymes that are not normally available, make this a particularly useful expression system for P-450 structure/function analysis . Furthermore, the background of P-450 level in E . coli is extremely low . It appears that the minor sequence alterations that have been made at the N-terminus to achieve high expression do not alter the enzymic properties of the recombinant P-450s, and it will not be surprising if vector systems are developed where such changes are not necessary . One concern with bacterial expression of P-450 is whether this system will prove of general use . Preliminary data suggests that certain mutant forms of human P-450c17 that are known to be functional in COS cells are not functional in bacteria, suggesting further that the folding pathways are not the same in bacterial and in eukaryotic cells . This unpredictability of the bacterial system is unsettling and, only as more laboratories adopt this system because of its value for high-level P-450 expression, will we learn more details of the requirements and limitations of this system.(ABSTRACT TRUNCATED AT 250 WORDS)

Chem Res Toxicol, 1993 Nov-Dec, 6(6), 819 - 24
Nucleotide misincorporation on DNA templates containing N-(deoxyguanosin-N2-yl)-2-(acetylamino)fluorene; Shibutani S et al.; An octadecadeoxynucleotide, modified site-specifically with N-(deoxyguanosin-N2-yl)-2-(acetylamino)fluorene (dG-N2-AAF), was prepared by enzymatic synthesis from a comparably modified decamer and then used as a DNA template in primer extension reactions catalyzed by the Klenow fragment of Escherichia coli DNA polymerase I containing (exo+) or lacking (exo-) 3'-->5' exonuclease activity . Using exo- Klenow fragment and all four deoxynucleotide triphosphate (dNTPs), primer extension is blocked one base before and opposite dG-N2-AAF . A small fraction of the reaction product represents translesional synthesis, in which dAMP is incorporated opposite the lesion . Kinetic studies of base insertion and chain extension indicate that the frequency of dAMP insertion opposite dG-N2-AAF is higher than that of other deoxynucleotide monophosphates (dNMPs) and of N-(deoxyguanosin-8-yl)-2-(acetylamino)-fluorene (dG-C8-AAF); however, the rate of extension of dA.dG-N2-AAF from the 3' terminus was much lower than that of dA.dG-C8-AAF . We conclude that dG-N2-AAF is a miscoding lesion and capable of generating G-->T transversion mutations in cells.

Chem Res Toxicol, 1993 Nov-Dec, 6(6), 800 - 7
Regio- and stereoselective oxygenations by adult human liver flavin-containing monooxygenase 3 . Comparison with forms 1 and 2; Lomri N et al.; The cDNA for the adult human liver flavin-containing monooxygenase (form 3) (FMO3) was cloned, sequenced, and expressed in Escherichia coli . The cDNA-expressed FMO3 was used to investigate the regio- and stereoselective N- and S-oxygenation of a number of tertiary amines and sulfides, respectively . For comparison, the N- and S-oxygenation of the same chemicals and drugs were examined with adult human liver microsomes from a normal healthy female donor and FMO1 from pig liver and FMO2 from rabbit lung . Both cDNA-expressed FMO3 and adult human liver microsomes N-oxygenated trifluoperazine or 10-(N,N-dimethylaminoalkyl)-phenothiazines with similar substrate specificities . The substrate specificity for FMO3 differed, however, from that of pig liver FMO1 . Nucleophilic sulfur-containing compounds {i.e., thiobenzamide, (4-bromophenyl)-1,3-oxathiolane, and 2-methyl-1,3-benzodithiole} were efficiently S-oxygenated by cDNA-expressed FMO3 and adult human liver microsomes . Stereoselective S-oxygenation of (+)- and (-)-(4-bromophenyl)-1,3-oxathiolane and 2-methyl-1,3-benzodithiole was therefore investigated . In general, the stereoselectivity observed for S-oxygenation in the presence of FMO3 was similar to that observed in the presence of adult human liver microsomes . In most cases examined, however, the stereoselectivity for S-oxygenation was quite distinct from that observed for pig liver FMO1 . We conclude that FMO3 is the major form of FMO active in adult human liver . Because the stereoselectivity for X-oxygenation and the substrate specificity for tertiary amine N-oxygenation by cDNA-expressed FMO3 are distinct from those of pig liver FMO1, we conclude that the binding channel for each isoform is quite different.(ABSTRACT TRUNCATED AT 250 WORDS)

Mol Biochem Parasitol, 1993 Nov, 62(1), 73 - 82
Cloning and characterization of a Golgi-associated GTP-binding protein homologue from Leishmania major; Cappai R et al.; This paper describes the cloning of a Golgi-associated GTP-binding protein homologue from Leishmania major . The gene was isolated using degenerate oligonucleotides to conserved sequences amongst the small GTP-binding proteins in a polymerase chain reaction on genomic DNA of the L . major cloned line V121 . The reading frame of one clone showed high similarity to the rab/YPT subfamily of small GTP-binding proteins . A full length copy of the gene was isolated from a lambda gt10 V121 genomic library and sequenced . At the amino acid level the gene showed highest similarity to the human/rat rab1 A gene and the mouse/yeast YPT gene and was named LmYPT . The LmYPT gene was present as a single copy gene in both the L . major and L . donovani genomes . Karyotype analysis localized the LmYPT gene to chromosome band 18 in V121, but it was located on a larger chromosome in the different L . major isolate L119 . The LmYPT gene was transcribed as a 3.9-kb transcript in both the promastigote and amastigote forms of the parasite . Western blot analysis, using a polyclonal rabbit antiserum raised against an Escherichia coli expressed portion of the molecule, identified a doublet at 20 and 23 kDa in total promastigote protein . Immunoelectron microscopy in combination with peroxidase staining localized the LmYPT molecule to the Leishmania Golgi apparatus.

J Biochem (Tokyo), 1993 Nov, 114(5), 735 - 9
Characterization of three types of aspartase activated by site-directed mutagenesis, limited proteolysis, and acetylation; Murase S et al.; The activity of aspartase (L-aspartate ammonia-lyase, EC 4.3.1.1) from Escherichia coli is enhanced 2- to 3-fold by three types of modification of the enzyme as reported previously; the replacement of Cys-430 with Trp by site-directed mutagenesis, the truncation of the C-terminal region by limited proteolysis, and the acetylation of amino groups with acetic anhydride . To elucidate the molecular basis of such activation, we have compared the kinetic properties of the modified enzymes in this study . Although the modifications caused very similar changes in the kinetic properties, such as increase in kcat, the half-saturating concentration of substrate, and Hill coefficient values, the modified enzymes differed greatly in sensitivity to the activator L-aspartate and the inhibitor Cl- ions . As a result of the mutation, the binding affinity for the activator was greatly decreased without change in the sensitivity to the inhibitor, whereas after acetylation, the sensitivity to the inhibitor was completely lost without decrease in the binding affinity for the activator . After truncation of the C-terminal region, both a large decrease in the binding affinity for the activator and complete loss of sensitivity to the inhibitor occurred, suggesting that this type of activation is equivalent to the former two types combined.

J Biochem (Tokyo), 1993 Nov, 114(5), 670 - 6
DNaseI footprinting studies of Escherichia coli biotin repressor-operator interactions; Lin KC et al.; Transcription of Escherichia coli biotin operon is repressed by biotin repressor in the presence of biotinyl-5'-adenylate as a corepressor . To determine precisely the site of action of biotin repressor on the operator sequence, DNaseI footprinting experiments were performed on the PCR-produced biotin operator and its mutants . The results indicate that the repressor binds to the wild-type operator as well as mutated operator sequence at +15 position or -15 position, and protects the 39-base region from nucleotide -19 to +20 of the upper strand, and the 40-base region from nucleotide -22 to +18 of the lower strand, with a few hypersensitive sites . This is consistent with previous speculation that the biotin operator is an approximately 40 bp imperfect palindromic DNA sequence capable of binding with two molecules of biotin repressor . However, the protection pattern of the mutant operator which lacks half of the palindromic structure is quite different from the corresponding region of the wild type . Though two repressor monomers bound to the mutant operator, as revealed by parallel binding studies {Lin, Shiuan, and Campbell (1991) Biochim . Biophys . Acta 1090, 317-325}, only 12 to 13 bp on the DNA sequence was protected, suggesting that one monomer of the repressor dimer is hanging near the DNA backbone of the mutant operator even though the biotin repressor is functioning as a dimer.

J Biochem (Tokyo), 1993 Nov, 114(5), 663 - 9
The backbone structure of the major cold-shock protein CS7.4 of Escherichia coli in solution includes extensive beta-sheet structure; Chatterjee S et al.; CS7.4 is the major cold-shock protein specifically expressed to a level as high as 13% of the total cellular protein within the first hour when Escherichia coli cell culture is shifted from 37 to 15 degrees C {Goldstein et al . (1990) Proc . Natl . Acad . Sci . USA 87, 283-287} . It consists of 70 amino acid residues with a very high content of aromatic residues . CS7.4 was overproduced and purified to homogeneity . Its secondary structure was analyzed by examining circular dichroism at both the far and near-UV regions; the results suggest that the protein is largely beta-sheet in conformation . The predominance of beta-sheet structure in the protein was confirmed by using Fourier-transform infrared spectroscopy . A folded compact conformation was also verified by fluorescence emission spectroscopy . We evaluated Tm, delta H, and delta S from the thermal denaturation profile of the protein . Unusual spectral features observed in the far-UV region are attributed to the high content of aromatic residues . The protein is relatively small and contains no disulfide bonds . However, it is surprisingly stable to heat denaturation.

J Biochem (Tokyo), 1993 Nov, 114(5), 627 - 33
Site-directed random mutagenesis of AMP-binding residues in adenylate kinase; Okajima T et al.; Two highly conservative residues, Val67 and Gln101, in adenylate kinase are located in the hydrophobic region putatively involved in binding of the adenine ring of AMP . We have performed polymerase chain reaction-based random mutagenesis of the two residues using recombinant chicken muscle adenylate kinase cDNA as a template . The synthetic oligonucleotide primers contained A,G,C,T-mixed bases in the codons corresponding to those for Val67 and Gln101 . The amplified fragments were ligated with the expression plasmid pKK223-3, and the mutant proteins expressed were identified by immunoblotting . Enzymatically active mutant proteins were selected on the basis of the growth at 45 degrees C of the temperature sensitive Escherichia coli mutant for adenylate kinase . At position 67, various amino acid residues other than Val have been found to restore the growth at 45 degrees C of the ts mutant . In contrast, only Gln, His, and Met could be present at position 101 . These results are compatible with the proposal that Val67 contributes to the AMP binding through hydrophobic interactions and Gln101 by forming a hydrogen bond with the adenine ring . Indeed, several purified Val67 and Gln101 mutant enzymes exhibited markedly high Km values for AMP, whereas the Km values for MgATP were comparable to those of the wild-type enzyme . Substrate specificity for nucleoside monophosphates was changed significantly by the mutagenesis of the two residues.

J Biomol NMR, 1993 Nov, 3(6), 701 - 8
Resolution and sensitivity enhancement of heteronuclear correlation for methylene resonances via 2H enrichment and decoupling; Kushlan DM et al.; Monodeuterated methylene positions exhibit substantially superior spectral characteristics in 1H-13C correlation experiments as compared to diprotio signals . A combination of 2H decoupling and multiplet editing of HMQC and HSQC experiments provides resolution enhancement for both stereoselective and random fractionally deuterated samples . For HMQC experiments with {2-2HR,2-13C}glycine-labeled E . coli thioredoxin (11.7 kDa), 3-fold increases in both 1H and 13C resolution result in a complementary 9-fold enhancement in sensitivity . Owing to a smaller improvement in 13C resolution, the corresponding enhancements for the HSQC experiment are 2-fold less.

Int J Pept Protein Res, 1993 Nov, 42(5), 420 - 31
Prediction of transmembrane beta-strands from hydrophobic characteristics of proteins; Gromiha MM et al.; The assembly of outer-membrane proteins consisting of beta-strands as transmembrane segments is somewhat more complex when compared to the assembly of inner membrane proteins having alpha-helices as transmembrane parts . This is probably due to the difference in the amino acid sequences of the transmembrane part strands and helices . Because of this feature, most predictive schemes which are successful in predicting transmembrane helical segments fail to predict transmembrane strand segments . Here we propose a new predictive scheme for forecasting the transmembrane strand segments in outer-membrane proteins with the use of the general surrounding hydrophobicity scale developed both for the globular and membrane proteins in the preceding article . Two major features of the scheme are (i) that it does not solely depend on the amphipathic character of a sequence segment while identifying it as a transmembrane strand, and it is capable of predicting strands in varied lengths, a facility to reflect the variation in the membrane surfaces . This scheme predicts the transmembrane beta-strands in porin from R . capsulatus at 76% accuracy (giving due weights to over- and under-predictions when compared to X-ray results) . The predicted beta-structure contents in OmpA, porin from E . coli and maltoporin compared with the Raman spectroscopic results at 95% level . These accuracy levels are far superior than the levels obtained from other existing methods . Apart from the above four proteins for which experimental informations are available, ten other outer-membrane proteins, for which there is no information about their secondary structure, are considered in the forecast . The results are analysed and the common features in the folds of the set of fourteen outer-membrane proteins are deduced.

Plant Mol Biol, 1993 Nov, 23(3), 583 - 94
HSP90 homologue from Madagascar periwinkle (Catharanthus roseus): cDNA sequence, regulation of protein expression and location in the endoplasmic reticulum; Schroder G et al.; We describe cDNAs for a HSP90 homologue from Catharanthus roseus and studies on the regulation of expression . The largest cDNA (2670 bp) coded for a protein of 817 amino acids with a calculated size of 93,491 Da and a pI of 4.61 . It contained a eucaryotic secretory signal, the endoplasmic reticulum (ER) targeting and retention signal (Lys-Asp-Glu-Leu), and the HSP90 protein family signature with one conservative exchange (Asn-Lys-Asp-Ile-Phe-Leu instead of Asn-Lys-Glu-Ile-Phe-Leu) . RNA blots revealed a transcript of 2.8-2.9 kb, and genomic DNA blots suggested a single gene . The expression was analysed with antiserum against a fusion protein expressed in Escherichia coli . Immunoblots revealed a protein of 93 +/- 1.5 kDa (often a doublet) only in the membrane fraction, and sucrose density gradients suggested association with the ER . The protein was constitutively expressed in C . roseus cell cultures grown at 25 degrees C, and expression was apparently unaffected by various stress conditions, such as heat, high sucrose, elicitor from Phytophthora megasperma or yeast extract . It was not detectable in young C . roseus plants at room temperature, and heat shock for several hours at 37 degrees C was necessary to obtain detectable expression . In maize (Zea mays), a cross-reacting protein was detectable in cell cultures, but not in young plants . The results suggested that the cloned protein is not a major component in the heat shock response . We propose a chaperone role in the assembly and processing of cell wall components and other secreted proteins, i.e . functions that are very active in cells with a high rate of growth and division.

Infect Immun, 1993 Nov, 61(11), 4902 - 5
P1-antigen-containing avian egg whites as inhibitors of P adhesins among wild-type Escherichia coli strains from patients with urosepsis; Johnson JR et al.; Among 58 Escherichia coli urosepsis isolates, P1-antigen-containing dove and pigeon egg whites were significantly more effective inhibitors of P-adhesin-specific agglutination than were chicken egg whites or globoside . Globoside's inefficacy may have resulted from a proadherence effect of globoside's lipid tail . Adhesin phenotypes determined with dove and pigeon egg whites as as agglutination inhibitors corresponded closely with phenotypes defined by comparative hemagglutination of human P1 and p erythrocytes . These data suggest that avian P1-antigen-containing substances may provide a useful alternative method for P adhesin inhibition among uropathogenic E . coli strains.

Infect Immun, 1993 Nov, 61(11), 4710 - 5
The Escherichia coli heat-stable enterotoxin is a long-lived superagonist of guanylin; Carpick BW et al.; The mechanism by which bacterial heat-stable enterotoxins (ST I STA) cause diarrhea in humans and animals has been linked to the activation of an intestinal membrane-bound guanylate cyclase . Guanylin, a recently discovered rat intestinal peptide, is homologous in structure to ST I and can activate guanylate cyclase present on the human colonic carcinoma cell line T84 . To directly test the mechanistic association of guanylate cyclase activation with diarrhea, we synthesized guanylin and a guanylin analog termed N9P10 guanylin and compared their biological activities with those of a synthetic ST I analog, termed ST Ib(6-18) . We report that guanylin is able to inhibit the binding of a radiolabeled ST I analog to rat intestinal cells but causes diarrhea in infant mice only at doses at least 4 orders of magnitude higher than that of ST Ib(6-18) . In contrast, N9P10 guanylin was enterotoxic in mice at much lower doses than guanylin but proved to be a weaker inhibitor of radiolabeled ST I than guanylin in the receptor binding assay . The pattern of guanylate cyclase activation observed for ST Ib(6-18) and the two guanylin analogs parallels the results observed in the receptor binding assay rather than those observed in the diarrheal assay . Treatment of guanylin with chymotrypsin or lumenal fluid derived from newborn mouse intestines resulted in a rapid loss of binding activity . Together, these results suggest that ST I enterotoxins may represent a class of long-lived superagonists of guanylin.

Bioorg Med Chem, 1993 Nov, 1(5), 349 - 60
Preparation of the pure diastereomeric forms of S-(5'-deoxy-5'-adenosyl)-1-ammonio-4-methylsulfonio-2-cyclopentene and their evaluation as irreversible inhibitors of S-adenosylmethionine decarboxylase from Escherichia coli; Wu YQ et al.; The conformationally restricted S-adenosylmethionine analogue AdoMac {S-(5'-deoxy-5'-adenosyl)-1-ammonio-4-methylsulfonio-2-cyclopenten e} has been shown to act as an enzyme activated, irreversible inhibitor of the Escherichia coli form of the enzyme S-adenosylmethionine decarboxylase . Inactivation of the enzyme is presumably initiated by formation of an imine linkage between the inhibitor and the terminal pyruvate of the enzyme, followed by base-catalyzed elimination of methylthioadenosine and generation of a latent electrophile . Removal of the driving force for the elimination of methylthioadenosine resulted in a reversibly binding inhibitor . Thus, the thioether analogue corresponding to AdoMac, and the corresponding dihydro derivative (H2-AdoMac), reversibly inhibit the enzyme . AdoMac was resolved into its four pure diastereomeric forms, and each diastereomer was evaluated as an irreversible inhibitor of the enzyme . The KI values for the individual diastereomers range between 3.83 and 39.6 microM, with the cis-1S,4R diastereomer being the most potent inhibitor . However, the kinact values for the four diastereomers are not significantly different, suggesting that the binding of each diastereomer to the enzyme is configuration-dependent, while the subsequent inactivation likely proceeds through a single intermediate which is formed from each of the four diastereomers . Since each pure diastereomer represents a distinct conformational mimic exhibiting restricted sidechain rotation, the data suggests that these and related analogues may be useful as conformational probes for the catalytic site of AdoMet-DC.

J Clin Endocrinol Metab, 1993 Nov, 77(5), 1156 - 63
Effects of endotoxin on leucine and glucose kinetics in man: contribution of prostaglandin E2 assessed by a cyclooxygenase inhibitor; Bloesch D et al.; The effects of endotoxin (E) administration on whole body protein and glucose metabolism were studied in normal volunteers . Injection of 4 ng/kg Escherichia coli E iv resulted in a relative increase in leucine flux (1-13C-leucine infusion technique) compared to controls {+0.12 +/- 0.10 vs . -0.45 +/- 0.23 mumol/kg.min after 360 min, P = 0.028, analysis of variance (ANOVA)}, indicating increased proteolysis . Nonoxidative leucine flux was higher after E than after saline administration (0.08 +/- 0.11 vs . -0.47 +/- 0.18 mumol/kg.min, P = 0.007, ANOVA), suggesting increased amino acid incorporation into proteins . E caused a transient decrease of plasma glucose concentration (by 0.5 +/- 0.1 mmol/L after 150 min; P < 0.004 vs . saline controls) due to a relative increase in disappearance compared to appearance of glucose (6,6 D2-glucose infusion technique) . These alterations were associated with increases in plasma concentrations of ACTH, beta-lipoprotein (beta-LPH), GH, cortisol, epinephrine, free fatty acid, beta-hydroxybutyrate, and decreases of plasma insulin . Pretreatment with ibuprofen, a cyclooxygenase inhibitor, blunted the effects of E on whole body leucine flux (P < 0.05 vs . E) and on nonoxidative leucine flux (P < 0.05 vs . E) but enhanced the E-induced decrease of plasma glucose concentration (P < 0.004 vs . E), due to a relative increase in glucose disappearance compared to appearance (P = 0.02) . The increases in counterregulatory hormones (ACTH, beta-LPH, GH, cortisol, epinephrine) were also attenuated by ibuprofen . Thus, acute endotoxinemia results in a redistribution of whole body proteins due to an increase in both protein breakdown and amino acid incorporation into proteins and in decreased plasma glucose concentrations . The ibuprofen data suggested that these effects of E on leucine kinetics, but not on glucose metabolism, were prostaglandin E2-mediated.

Plant Physiol, 1993 Nov, 103(3), 829 - 34
Cloning of tomato (Lycopersicon esculentum Mill.) arginine decarboxylase gene and its expression during fruit ripening; Rastogi R et al.; Arginine decarboxylase (ADC) is the first enzyme in one of the two pathways of putrescine biosynthesis in plants . The genes encoding ADC have previously been cloned from oat and Escherichia coli . Degenerate oligonucleotides corresponding to two conserved regions of ADC were used as primers in polymerase chain reaction amplification of tomato (Lycopersicon esculentum Mill.) genomic DNA, and a 1.05-kb fragment was obtained . This genomic DNA fragment encodes an open reading frame of 350 amino acids showing about 50% identity with the oat ADC protein . Using this fragment as a probe, we isolated several partial ADC cDNA clones from a tomato pericarp cDNA library . The 5' end of the coding region was subsequently obtained from a genomic clone containing the entire ADC gene . The tomato ADC gene contains an open reading frame encoding a polypeptide of 502 amino acids and a predicted molecular mass of about 55 kD . The predicted amino acid sequence exhibits 47 and 38% identify with oat and E . coli ADCs, respectively . Gel blot hybridization experiments show that, in tomato, ADC is encoded by a single gene and is expressed as a transcript of approximately 2.2 kb in the fruit pericarp and leaf tissues . During fruit ripening the amount of ADC transcript appeared to peak at the breaker stage . No significant differences were seen when steady-state ADC mRNA levels were compared between normal versus long-keeping Alcobaca (alc) fruit, although alc fruit contain elevated putrescine levels and ADC activity at the ripe stage . The lack of correlation between ADC activity and steady-state mRNA levels in alc fruit suggests a translational and/or posttranslational regulation of ADC gene expression during tomato fruit ripening.

Plant Physiol, 1993 Nov, 103(3), 771 - 81
Osmoregulation of a pyrroline-5-carboxylate reductase gene in Arabidopsis thaliana; Verbruggen N et al.; In Arabidopsis thaliana (L.) Heynh . proline can account for up to 20% of the free amino acid pool after salt stress . Proline accumulation occurs in plants mainly by de novo synthesis from glutamate . The last step of the proline biosynthetic pathway is catalyzed by pyrroline-5-carboxylate (P5C) reductase . A gene (AT-P5C1) encoding this enzyme in A . thaliana has been cloned and sequenced . Expression of AT-P5C1 in Escherichia coli resulted in the complementation of a proC mutant to prototrophy . A comparison of the AT-P5C1 primary and secondary structures with those of six P5C reductase of other organisms is presented . With the exception of several functionally important amino acid residues, little conservation in the primary structure is seen; much greater similarity exists in the putative secondary structure . The AT-P5C1 protein is probably cytosolic . Under normal growth conditions, the P5C reductase mRNA level was significantly higher in roots and ripening seeds than in green tissue . A salt treatment of A . thaliana plants resulted in a 5-fold induction of the AT-P5C1 transcript, suggesting osmoregulation of the AT-P5C1 promoter region . Moreover, a time-course experiment indicated that this induction precedes proline accumulation.

Microb Pathog, 1993 Nov, 15(5), 399 - 405
Purification and some properties of a Vero toxin 2 (Shiga-like toxin II) variant (SLT-IIva) of Escherichia coli isolated from infantile diarrhea; Ohmura M et al.; A variant of Vero toxin 2 (VT2), or Shiga-like toxin II (SLT-II) was purified from a cloned strain of Escherichia coli carrying a gene that encodes SLT-IIva, which is most closely related to SLT-IIv (VT2vp, VTe) in its nucleotide sequence . The purified SLT-IIva showed a slight difference from the purified SLT-IIv (VT2vp) in mobility on polyacrylamide gel disc electrophoresis and SDS-polyacrylamide gel slab electrophoresis and in pI, but both showed a similar potency in biological activities . Ouchterlony double diffusion test revealed that the purified SLT-IIva and SLT-IIv (VT2vp) formed a spur against anti-SLT-IIva and anti-SLT-IIv (VT2vp) antisera.

J Struct Biol, 1993 Nov-Dec, 111(3), 212 - 21
Direct in situ structural analysis of recombinant outer membrane porins expressed in an OmpA-deficient mutant Escherichia coli strain; Hoenger A et al.; An OmpA-deficient mutant of an OmpF/OmpC-free Escherichia coli B strain was selected using phage K3 . The mutant strain was characterized by SDS-gel electrophoresis, immunoblotting, and electron microscopy . All major outer membrane proteins, including OmpA, were absent . This strain was then transformed with the plasmid pMY222 encoding the K12 OmpF porin or with pBlue-script-derived plasmids, encoding the porins OmpC, PhoE, and maltoporin, respectively . Following SDS extraction of outer membrane sacculi from strains expressing individual porins, crystalline porin arrays that allowed in situ structural analysis to be performed were observed . Furthermore, the absence of endogenous major outer membrane proteins facilitated the purification of native porin-lipopolysaccharide complexes, the functionally active channels, from the sacculi of transformed strains.

Rev Inst Med Trop Sao Paulo, 1993 Nov-Dec, 35(6), 503 - 8
An attempt at reversibility and increase of the virulence of axenic strains of Entamoeba histolytica; Gomes MA et al.; In this study we have tried to verify whether the interaction "in vitro" with bacteria or small pieces of normal hamster liver would modify the pathogenic behavior of axenic strains of E . histolytica: avirulent ones (ICB-32 and ICB-RPS), of attenuated virulence (ICB-CSP and HM1) and of mean virulence (ICB-462) . Every attempt to render virulent, recover or increase the virulence of axenic strains of E . histolytica has failed.

Mol Microbiol, 1993 Nov, 10(3), 675 - 84
Chromosomal supercoiling in Escherichia coli; Miller WG et al.; The Escherichia coli chromosome is compacted into 40-50 negatively supercoiled domains . It has been proposed that these domains differ in superhelical density . Here, we present evidence that this is probably not the case . A modified Tn10 transposable element was inserted at a number of locations around the E . coli chromosome . This element, mTn10-plac-lacZ+, contains the lac operon promoter, plac, whose activity increases with increasing superhelical density, fused to a lacZ+ reporter gene . Although mTn10-plac-lacZ+ fusion expression varies as much as approximately threefold at different insertion sites, the relative levels of expression from these elements are unaffected by replacing plac with the gyrA promoter, pgyrA, which has a reciprocal response to changes in superhelical density . Importantly, topoisomerase mutations and coumermycin, which inhibits DNA gyrase activity, alter mTn10-plac-lacZ+ and mTn10-pgyrA-lacZ+ fusion expression in expected ways, showing that the elements remain responsive to supercoiling and that topoisomerase activity is required for maintaining superhelical density . Fusion expression is not affected by anaerobic growth or osmotic shock, two physiological conditions thought to alter supercoiling . The approximately threefold difference in mTn10-plac-lacZ+ and mTn10-pgyrA-lacZ+ fusion expression observed at different sites may be explained by regional differences in chromosomal copy number that arise from bidirectional replication . Together, these results strongly suggest that the E . coli chromosomal domains do not differ in functional superhelical density.

Mol Microbiol, 1993 Nov, 10(3), 635 - 45
Suppression of temperature-sensitive assembly mutants of heat-labile enterotoxin B subunits; Sandkvist M et al.; Deletions or substitutions of amino acids at the carboxyl-terminus of the heat-labile enterotoxin B subunit (EtxB) affect its assembly into pentamers in a temperature-dependent manner . At 42 degrees C, the mutations prevent the B subunits from achieving their final pentameric structure resulting in membrane association of the monomers . However, mutant B subunits produced at 30 degrees C assemble, in the periplasm, into pentamers that remain stable when transferred to 42 degrees C, indicating that the mutant pentamers are stable under conditions where their formation is inhibited . The mutant pentamers are, similarly to wild-type pentamers, SDS-resistant and stable, in vitro, at temperatures up to 65 degrees C . This suggests that although the C-terminal amino acids are part of the subunit interface, they appear not to contribute significantly to the stability of the final pentameric complex, but are instead essential for the formation or stabilization of an assembly intermediate in the pentamerization process . Single second site mutations suppress the assembly defect of mutant EtxB191.5, which carries substitutions at its C-terminus . The Thr-->Ile replacement at position 75 in the alpha 2-helix probably restores the van der Waals contact between residues 75 and 101, which had been greatly reduced by the Met-->Leu substitution at position 101 in the beta 6-strand of EtxB191.5 . Interaction between the alpha 2-helix and beta 6-strand which contains the C-terminus probably stabilizes a conformation essential for assembly and is therefore required for the formation of pentamers.

Mol Microbiol, 1993 Nov, 10(3), 607 - 13
Transcriptional organization of the Escherichia coli pilus adhesin K99; Inoue OJ et al.; The production of the Escherichia coli pilus adhesin K99 requires the expression of eight unique proteins: FanA-H . The transcriptional organization of the K99 operon was investigated by Northern blot analysis . Four RNAs of 0.54, 1.4, 2.5 and 3.5 kb were detected . When a fanC probe was used all four RNAs were detected while the use of fanD, fanF and fanG probes detected two RNAs each . Using several deletion and TnphoA insertion mutants it was concluded that there were seven unique K99-specific transcripts, several of which were of the same approximate sizes (1.4 and 2.5 kb) . It also was concluded that K99 was comprised of at least three complementation groups, two of which were regulated by catabolite repression.

Mol Microbiol, 1993 Nov, 10(3), 575 - 84
Correlation of gene transcription with the time of initiation of chromosome replication in Escherichia coli; Theisen PW et al.; Transcriptional levels of the Escherichia coli mioC and gidA genes, which flank the chromosomal origin of replication (oriC) and the dnaA gene, were correlated with the time of initiation of chromosome replication . The transcripts were measured either in dnaC2(ts) mutants that had been aligned for initiation of chromosome replication by a temperature shift or in synchronous cultures of cells obtained using the baby machine technique . In both types of experiments, mioC transcription was inhibited prior to initiation of chromosome replication and resumed several minutes after initiation . Conversely, gidA and dnaA transcription were both inhibited after initiation of replication, coincident with the period of hemimethylation of oriC DNA . It is proposed that mioC transcription prevents initiation of chromosome replication, and must terminate before replication can begin . It is further proposed that the eclipse period between rounds of replication, i.e . the minimum interval between successive initiations, encompasses the time required to methylate GATC sequences in newly replicated oriC plus the time required to terminate mioC transcription . Conversely, the active transcription of gidA and dnaA prior to initiation is consistent with their positive effects on initiation, and their shutdown after initiation could serve to limit premature reinitiation.

Mol Microbiol, 1993 Nov, 10(3), 483 - 97
Two distinct ATP-binding domains are needed to promote protein export by Escherichia coli SecA ATPase; Mitchell C et al.; Six putative ATP-binding motifs of SecA protein were altered by oligonucleotide-directed mutagenesis to try to define the ATP-binding regions of this multifunctional protein . The effects of the mutations were analysed by genetic and biochemical assays . The results show that SecA contains two essential ATP-binding domains . One domain is responsible for high-affinity ATP binding and contains motifs A0 and B0, located at amino acid residues 102-109 and 198-210, respectively . A second domain is responsible for low-affinity ATP binding and contains motifs A3 and a predicted B motif located at amino acid residues 503-511 and 631-653, respectively . The ATP-binding properties of both domains were essential for SecA-dependent translocation ATPase and in vitro protein translocation activities . The significance of these findings for the mechanism of SecA-dependent protein translocation is discussed.

Mol Microbiol, 1993 Nov, 10(3), 473 - 81
Topoisomerase mutations affect the relative abundance of many Escherichia coli proteins; Steck TR et al.; The relative abundance of 88 proteins was measured in extracts from three strains of Escherichia coli K-12 that are isogenic except for the topA and gyrB genes . Mutations in these genes slightly raise or lower, respectively, steady-state DNA supercoiling levels but have little effect on growth rate . Altered protein abundances were observed in the mutant strains relative to wild type . Many proteins exhibited minimum abundance at wild-type supercoiling levels, and other proteins exhibited maximal abundance at relaxed levels . A smaller number showed maximal abundance at elevated levels of supercoiling . These data suggest that small, non-lethal changes in DNA supercoiling can have widespread effects on patterns of gene expression.

Mol Microbiol, 1993 Nov, 10(3), 457 - 63
Cell-cycle-specific initiation of replication; Nordstrom K et al.; The following characteristics are relevant when replication of chromosomes and plasmids is discussed in relation to the cell cycle: the timing or replication, the selection of molecules for replication, and the coordination of multiple initiation events within a single cell cycle . Several fundamentally different methods have been used to study these processes: Meselson-Stahl density-shift experiments, experiments with the so-called 'baby machine', sorting of cells according to size, and flow cytometry . The evidence for precise timing and co-ordination of chromosome replication in Escherichia coli is overwhelming . Similarly, the high-copy-number plasmid ColE1 and the low-copy-number plasmids R1/R100 without any doubt replicate randomly throughout the cell cycle . Data about the low-copy-number plasmids F and P1 are conflicting . This calls for new types of experiments and for a better understanding of how these plasmids control their replication and partitioning.

Mol Cell Biochem, 1993 Nov, 127-128, 7 - 18
Expression, purification, characterization, and deletion mutations of phosphorylase kinase gamma subunit: identification of an inhibitory domain in the gamma subunit; Huang CY et al.; A catalytic fragment, gamma 1-298, derived from limited chymotryptic digestion of phosphorylase b kinase (Harris, W.R . et al., J . Biol . Chem., 265: 11740-11745, 1990), is reported to have about six-fold greater specific activity than does the gamma subunit-calmodulin complex . To test whether there is an inhibitory domain located outside the catalytic core of the gamma subunit, full-length wild-type and seven truncated forms of gamma were expressed in E . coli . Recombinant proteins accumulate in the inclusion bodies and can be isolated, solubilized, renatured, and purified further by ammonium sulfate precipitation and Q-Sepharose column . Four out of seven truncated mutants show similar (gamma 1-353 and gamma 1-341) or less (gamma 1-331 and gamma 1-276) specific activity than does the full-length wild-type gamma, gamma 1-386 . Three truncated forms, gamma 1-316, gamma 1-300, and gamma 1-290 have molar specific activities approximately twice as great as those of the full-length wild-type gamma and the nonactivated holoenzyme . All recombinant gamma s exhibit similar Km values for both substrates, i.e., about 18 microM for phosphorylase b and about 75 microM for MgATP . Three truncated gamma s, gamma 1-316, gamma 1-300, and gamma 1-290, have a 1.9- to 2.5-fold greater catalytic efficiency (Vmax/Km) than that of the full-length wild-type gamma and a 3.5- to 4.5-fold greater efficiency than that of the truncated gamma 1-331 . This evidence suggests that there is at least one inhibitory domain in the C-terminal region of gamma, which is located at gamma 301-331 . gamma 1-290, but not gamma 1-276, which contains the highly conserved kinase domain, is the minimum sequence required for the gamma subunit to exhibit phosphotransferase activity . Both gamma 1-290 and gamma 1-300 have several properties similar to full-length wild-type gamma, including metal ion responses (activation by free Mg2+ and inhibition by free Mn2+), pH dependency, and substrate specificities.

Mol Microbiol, 1993 Nov, 10(4), 877 - 84
Interaction of a redox-sensitive DNA-binding factor with the 5'-flanking region of the micF gene in Escherichia coli; Gidrol X et al.; The product of the micF gene is an endogenous antisense RNA which down-regulates the expression of a major outer membrane protein, OmpF, in E . coli . We report here that two DNA-binding factors compete for the same site in the promoter region of the micF gene: RSBF, a high-affinity redox-sensitive DNA-binding factor that responds to an active oxygen species other than hydrogen peroxide or superoxide anions; and HRBF a heat-resistant DNA-binding factor . Both RSBF and HRBF bind to the same DNA sequence, 5'-TTAAAATCAATAACTTATTCTTAA3-', located upstream of the transcription start site of the micF gene . We present evidence that RSBF could be the controlling factor of a novel regulon involved in the response to oxidative stress in E . coli.

Mol Microbiol, 1993 Nov, 10(4), 789 - 97
Interactions between the Escherichia coli cyclic AMP receptor protein and RNA polymerase at class II promoters; West D et al.; The effects of a number of mutations in crp have been measured at different cyclic AMP receptor protein (CRP)-dependent Class II promoters, where the CRP-binding site is centred around 41 1/2 base pairs upstream from the transcription start point . The amino acid substitutions HL159 and TA158 result in reduced CRP-dependent activation, but the reduction varies from one Class II promoter to another . Deletions in the C-terminus of the RNA polymerase alpha subunit suppress the effects of HL159 and TA158 . The role of the C-terminus of alpha at these promoters is assessed . Other changes at E58, K52 and E96 affect CRP activity specifically at Class II promoters and their role is discussed.

Mol Microbiol, 1993 Nov, 10(4), 781 - 7
Loss of activity in the secreted form of Escherichia coli haemolysin caused by an rfaP lesion in core lipopolysaccharide assembly; Stanley PL et al.; A transposon mutant of Escherichia coli 5K was isolated which reduced 10- to 50-fold the secreted extracellular haemolytic activity of cells carrying the complete hlyCABD operon while leaving unaffected the intracellular haemolytic activity and the levels of intracellular and extracellular haemolysin protein, HlyA . The transposon insertion was identified within the rfaP gene (required for attachment of phosphate-containing substituents to the lipopolysaccharide inner core), and extracellular haemolytic activity was restored in trans by the intact rfaP gene . The loss in cytolytic activity of the secreted HlyA protein was not related to the HlyC-directed acylation of the protoxin . Activity of the secreted toxin was restored by chaotropic agents and during rate-zonal centrifugation the mutant-secreted HlyA migrated as a larger species than the wild type . The results indicate that the rfaP mutation affects the aggregation behaviour of the active toxin during or following the signal peptide-independent secretion process.

Mol Microbiol, 1993 Nov, 10(4), 737 - 47
Specific transcriptional requirements for positive regulation of the anaerobically inducible pfl operon by ArcA and FNR; Sawers G; Expression of the pfl operon of Escherichia coli is induced 12- to 15-fold by anaerobiosis and transcription is mediated by seven co-ordinately regulated promoters . The 5' non-translated regulatory region of the operon is approximately 450bp in length and contains two of the seven promoters, termed promoter 6 and promoter 7 . Site-directed mutagenesis was used to aid the identification of DNA sequences important in directing transcription from the two promoters and to examine the effects such mutations had on the regulation of anaerobic pfl operon expression . Introduction of chromosomal mutations either in the FNR-binding site or -10 region of promoter 6 blocked transcription from this promoter, as determined by primer extension . Similarly, mutation of the -10 region or the putative FNR half-site located at -50 relative to the transcription start site of promoter 7 severely reduced transcription from that promoter . Prevention of transcription from promoter 6 by the -10 box mutation had no influence on promoter 7 transcription . Surprisingly, however, alteration of the FNR-binding site at promoter 6 did reduce transcription from promoter 7 . Thus, a cis mutation located 280 bp downstream on the DNA had a profound effect on promoter 7 transcription . This effect would be commensurate with this mutation disrupting an important interaction between proteins bound at promoter 7 with those bound at promoter 6 . Primer extension demonstrated that the promoter 7 mutations had no apparent influence on promoter 6 transcription . By using pfl-lacZ gene fusions it could be shown that the FNR-binding site and -10 region mutations at promoter 6 abolished FNR-dependent anaerobic regulation of pfl operon expression . The equivalent mutations at promoter 7 caused a 25% reduction in anaerobic expression . The residual anaerobic expression in such constructs was FNR-, but no longer ArcA-dependent . A construct in which the -10 region of both promoters 6 and 7 was mutated showed no anaerobic induction of pfl operon expression . This indicates that transcription from both promoters is required for maximal anaerobic regulation by ArcA and FNR.

Mol Microbiol, 1993 Nov, 10(4), 713 - 35
Light-induced carotenogenesis in Myxococcus xanthus: DNA sequence analysis of the carR region; McGowan SJ et al.; The carR region encodes a light-inducible promoter, a negative regulator of the promoter and a trans-acting activator that controls the light-inducible Myxococcus xanthus carotenoid biosynthesis regulon . DNA sequence analysis revealed, downstream of the promoter, three translationally coupled genes, carQ, carR and carS . Sequencing of mutations demonstrated that carR encoded the negative regulator and was an integral membrane protein . Mutant construction and sequencing revealed that carS was the trans-acting activator and that carQ was a positive regulator of the promoter . Neither gene encodes proteins with known sequence-specific DNA-binding motifs . The sequence of the light-inducible promoter region, identified by primer extension analysis, showed similarity to the consensus sequence of the Escherichia coli stress response ('heat-shock') promoters.

J Bacteriol, 1993 Nov, 175(22), 7391 - 403
Characterization of fimbriae produced by enteropathogenic Escherichia coli; Giron JA et al.; Enteropathogenic Escherichia coli (EPEC) express rope-like bundles of filaments, termed bundle-forming pili (BFP) (J . A . Giron, A . S . Y . Ho, and G . K . Schoolnik, Science 254:710-713, 1991) . Expression of BFP is associated with localized adherence to HEp-2 cells and the presence of the EPEC adherence factor plasmid . In this study, we describe the identification of rod-like fimbriae and fibrillae expressed simultaneously on the bacterial surface of three prototype EPEC strains . Upon fimbrial extraction from EPEC B171 (O111:NM), three fimbrial subunits with masses of 16.5, 15.5, and 14.7 kDa were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis . Their N-terminal amino acid sequence showed homology with F9 and F7(2) fimbriae of uropathogenic E . coli and F1845 of diffuse-adhering E . coli, respectively . The mixture of fimbrial subunits (called FB171) exhibited mannose-resistant agglutination of human erythrocytes only, and this activity was not inhibited by alpha-D-Gal(1-4)-beta-Gal disaccharide or any other described receptor analogs for P, S, F, M, G, and Dr hemagglutinins of uropathogenic E . coli, which suggests a different receptor specificity . Hemagglutination was inhibited by extracellular matrix glycoproteins, i.e., collagen type IV, laminin, and fibronectin, and to a lesser extent by gangliosides, fetuin, and asialofetuin . Scanning electron microscopic studies performed on clusters of bacteria adhering to HEp-2 cells revealed the presence of structures resembling BFP and rod-like fimbriae linking bacteria to bacteria and bacteria to the eukaryotic cell membrane . We suggest a role of these surface appendages in the interaction of EPEC with eukaryotic cells as well as in the overall pathogenesis of intestinal disease caused by EPEC.

EMBO J, 1993 Nov, 12(11), 4137 - 44
DnaK, DnaJ and GrpE form a cellular chaperone machinery capable of repairing heat-induced protein damage; Schroder H et al.; Members of the conserved Hsp70 chaperone family are assumed to constitute a main cellular system for the prevention and the amelioration of stress-induced protein damage, though little direct evidence exists for this function . We investigated the roles of the DnaK (Hsp70), DnaJ and GrpE chaperones of Escherichia coli in prevention and repair of thermally induced protein damage using firefly luciferase as a test substrate . In vivo, luciferase was rapidly inactivated at 42 degrees C, but was efficiently reactivated to 50% of its initial activity during subsequent incubation at 30 degrees C . DnaK, DnaJ and GrpE did not prevent luciferase inactivation, but were essential for its reactivation . In vitro, reactivation of heat-inactivated luciferase to 80% of its initial activity required the combined activity of DnaK, DnaJ and GrpE as well as ATP, but not GroEL and GroES . DnaJ associated with denatured luciferase, targeted DnaK to the substrate and co-operated with DnaK to prevent luciferase aggregation at 42 degrees C, an activity that was required for subsequent reactivation . The protein repair function of DnaK, GrpE and, in particular, DnaJ is likely to be part of the role of these proteins in regulation of the heat shock response.

Biosci Biotechnol Biochem, 1993 Nov, 57(11), 1960 - 1
Expression of the cellulase (FI-CMCase) gene of Aspergillus aculeatus in Escherichia coli; Ooi T et al.; FI-CMCase cDNA of Aspergillus aculeatus was expressed in Escherichia coli by using the tac promoter of E . coli . Transformants of E . coli harboring a plasmid pHEM06 containing mature form FI-CMCase cDNA produced FI-CMCase in the cytoplasm of the cells . The enzyme from E . coli cells was purified to yield 56% and it was immunological identical to that of FI-CMCase purified from A . aculeatus.

Biosci Biotechnol Biochem, 1993 Nov, 57(11), 1955 - 7
Availability of dihydrofolate reductase affinity handle in expressing human prolactin as a soluble fusion protein; Iwakura M et al.; Human prolactin (PRL) cDNA was successfully expressed in Escherichia coli cells with the aid of a dihydrofolate reductase (DHFR) affinity handle . The formed DHFR-PRL fusion protein was accumulated in E . coli cells as a soluble protein with DHFR activity at 30 degrees C . The fusion protein was highly purified with monitored the DHFR activity by methotrexate-bound affinity chromatography, suggesting the usefulness of the handle even in expressing a large polypeptide as a fusion protein.

Proc Natl Acad Sci U S A, 1993 Nov 1, 90(21), 10046 - 50
Diversified sequences of peptide epitope for same-RNA recognition; Kim S et al.; We replaced an essential RNA-binding, 30-amino acid helix-loop in an Escherichia coli tRNA synthetase with an inactive and simplified "generic" sequence having 23 of the 30 amino acids as alanine and serine . Wild-type residues were restored in random combinations to generate a library with a sequence complexity of about 1.9 x 10(7) . Active molecules were obtained by genetic selection at a frequency of approximately 1% and contained variants with as many as 11 alanine/serine replacements and a total of 17 alanine/serine residues . These variants have activities which are thermodynamically competitive with that of the native protein and therefore are functionally and, most likely, conformationally equivalent.

J Bacteriol, 1993 Nov, 175(22), 7228 - 35
Cloning and characterization of btr, a Bordetella pertussis gene encoding an FNR-like transcriptional regulator; Bannan JD et al.; To determine whether hemolytic factors other than the bifunctional hemolysin-adenylate cyclase toxin (cyclolysin) are expressed by Bordetella pertussis, a gene library was constructed from a virulent strain of B . pertussis, BP504, transformed into nonhemolytic Escherichia coli, and screened on blood agar plates . A strongly hemolytic colony which contained the plasmid pHLY1A was isolated . Nucleotide sequencing of pHLY1A revealed an open reading frame that could encode a 27-kDa protein . No similarity was detected between the deduced amino acid sequence of this open reading frame and those of any known bacterial cytolysins . However, significant homology was detected with FNR of E . coli and several other transcriptional regulators including HylX from Actinobacillus pleuropneumoniae, which can also confer a hemolytic phenotype on E . coli . An fnr mutant of E . coli, JRG1728, could be complemented by pHLY1A . Thus, the B . pertussis transcriptional regulator-like gene and the protein which it encoded were named btr and BTR, respectively . A BTR-deficient B . pertussis strain, BJB1, was constructed . The btr::kan mutation had no effect on the expression of hemolytic activity or on phase variation . Northern (RNA) blotting revealed that btr expression was not regulated by the BvgAS two-component sensor-regulator . On the basis of sequence similarity to FNR-like transcriptional regulators and the ability to complement an anaerobically deficient E . coli strain (JRG1728) in growing anaerobically, BTR may regulate B . pertussis gene expression in response to changes in oxygen levels or to changes in the redox potential of the bacterial environment . Its role in virulence remains to be determined.

Hepatology, 1993 Nov, 18(5), 1055 - 60
Peripheral-blood mononuclear cell responses to recombinant hepatitis C virus antigens in patients with chronic hepatitis C; Schupper H et al.; Peripheral blood mononuclear cell proliferative responses in vitro to recombinant yeast or Escherichia coli hepatitis C virus fusion proteins were evaluated in 20 patients with chronic hepatitis C who were reactive for antibody to hepatitis C virus (on enzyme immunoassay, version 2.0, and a four-antigen recombinant immunoblot assay) . Twenty age-matched, healthy individuals negative for antibody to hepatitis C virus were used as a control group . Peripheral-blood mononuclear cells from all chronic hepatitis C patients with antibodies to hepatitis C virus antigens c22 and c100-3 proliferated in vitro in response to the corresponding recombinant hepatitis C virus fusion protein . Peripheral-blood mononuclear cells from 75% of patients infected with hepatitis C virus proliferated in response to cytidine monophosphate-keto-3-deoxyoctulosonic acid-core recombinant antigen but there was no proliferative response to cytidine monophosphate-keto-3-deoxyoctulosonic acid-EF (derived from the NS5 region) . All hepatitis C virus-infected patients had 33c antibody, but peripheral-blood mononuclear cells from only 9 of 14 (64%) proliferated in vitro in response to 33c . Ninety-five percent of all hepatitis C virus-infected patients had peripheral-blood mononuclear cells that proliferated in response to at least one recombinant hepatitis C virus fusion protein . The numbers and percentages of CD3 T cells, CD19 B cells and natural killer cells from patients with chronic hepatitis C virus infection did not differ from those in the healthy control group . However, the number of non-major histocompatibility complex-restricted cytotoxic T cells (CD3-positive, CD56-positive, CD16-positive) was increased in patients with chronic hepatitis C virus infection (p < 0.05).

EMBO J, 1993 Nov, 12(11), 4297 - 303
Autogenous regulation of the EcoRII methylase gene at the transcriptional level: effect of 5-azacytidine; Som S et al.; mRNA of the EcoRII methylase (M.EcoRII), a type II modification enzyme, was induced when Escherichia coli carrying a cloned M.EcoRII gene was exposed to the bacteriocidal drug 5-azacytidine . Induction occurred only when transcription was initiated from its own promoter . When the 5' promoter sequences were deleted or replaced with the lac promoter sequences, no induction occurred . The induction was independent of the template DNA level, but the presence of an intact M.EcoRII protein was a requirement . The drug is incorporated into DNA which then inhibits M.EcoRII by binding tightly to the enzyme . A deletion within the M.EcoRII coding region caused a marked increase in the basal level of mRNA transcribed from the M.EcoRII promoter, but no induction occurred upon 5-azacytidine treatment . The level could be reduced to normal by M.EcoRII in trans . In vitro, the enzyme bound to the sequences upstream of the transcription start sites and inhibited the initiation of transcription . These experiments indicate that expression of the M.EcoRII gene was autogenously regulated at the transcriptional level . Similar regulation is also noted in another DNA (cytosine-5) methylase, M.MspI.

C R Acad Sci III, 1993 Nov, 316(11), 1283 - 5
Somatostatin specifically binds p86 subunit of the autoantigen Ku; Le Romancer M et al.; The heterodimer Ku, first described as a nuclear autoantigen, is a regulatory factor of DNA replication and transcription . We have expressed the p86 subunit of Ku in Escherichia coli as a fusion protein with glutathione-S-transferase, using the vector pGEX-2T . After splitting up by thrombin, p86 was isolated by Sephacryl S200 gel filtration . The recombinant protein was found to have the same electrophoretic migration and to react with the same monoclonal antibody as the somatostatin-binding protein we recently isolated from the human gastric tumor cell HGT1 {7} . Furthermore, using the analog {125I}Tyr-11 somatostatin-14 as a tracer, we found that, like the HGT1 cell-purified protein, recombinant p86 specifically bound somatostatin with high affinity (KD = 2.3 +/- 0.3 nM) and large capacity (10,300 +/- 1,700 pmol/mg protein) . These findings suggest that p86 subunit of Ku stands for the protein we previously isolated from the HGT1 cell . It could represent a new somatostatin receptor subtype perhaps involved in the antimitogenic effect of this peptide.

Khirurgiia (Mosk), 1993 Nov, (11), 7 - 10
{The effect of absorbent materials with drugs on wound healing}; Buianov VM et al.; Absorptive hydrophilic materials (MAH) containing dioxydin and terrilytin as drug components were studied in experiments on guinea pigs infected with K-11 strain of E . coli . The results showed that the wounds in animals who did not receive treatment were cleaned by the 14th day, while the wounds in animals treated by applications of MAH with dioxydin and terrilytin became clean on the 3rd day, i . e . 5 times quicker . Absorptive hydrophilic materials containing dioxydin and teriilytin were easily handled, easily applied and removed, they did not injure the granulation tissue, and intensified growth of peripheral epithelialization . Thus, experimental study showed absorptive hydrophilic materials containing dioxydin and terrilytin to be highly effective in the treatment of purulent wounds, which allows them to be recommended for clinical use.

Protein Eng, 1993 Nov, 6(8), 965 - 70
Structural analysis of the CD2 T lymphocyte antigen by site-directed mutagenesis to introduce a disulphide bond into domain 1; Gray F et al.; Many proteins have been predicted to contain domains with immunoglobulin-like folds and hence to be members of the immunoglobulin superfamily (IgSF) . However, several members lack the Cys residues capable of forming the disulphide bond that forms a characteristic bridge between the beta sheets in the Ig fold, e.g . domain 1 of the lymphocyte antigen CD2 . The assignment of beta strands in CD2 by sequence analysis was tested by attempting to introduce a disulphide bridge between the beta sheets by mutating two residues in the relevant positions to Cys . Mutant, soluble forms of CD2 were expressed in Chinese hamster ovary cell lines and amino acid sequencing showed that a disulphide bond was formed as predicted, but not in the control where one Cys residue was misplaced by four residues . Evidence that both mutated molecules folded correctly is given by the indistinguishable binding of three monoclonal antibodies recognising different epitopes on CD2 . The 3-D structure of rat CD2 domain 1 has been determined by NMR spectroscopy and X-ray crystallography, confirming the predictions from the sequence . Applications of this method of insertion of disulphide residues for probing protein structures are discussed, together with other structures of IgSF domains lacking the typical inter-sheet disulphide bond.

Mol Biol Cell, 1993 Nov, 4(11), 1189 - 204
The Gly/Arg-rich (GAR) domain of Xenopus nucleolin facilitates in vitro nucleic acid binding and in vivo nucleolar localization; Heine MA et al.; Epitope-tagged Xenopus nucleolin was expressed in Escherichia coli cells and in Xenopus oocytes either as a full-length wild-type protein or as a truncation that lacked the distinctive carboxy glycine/arginine-rich (GAR) domain . Both full-length and truncated versions of nucleolin were tagged at their amino termini with five tandem human c-myc epitopes . Whether produced in E . coli or in Xenopus, epitope-tagged full-length nucleolin bound nucleic acid probes in in vitro filter binding assays . Conversely, the E . coli-expressed GAR truncation failed to bind the nucleic acid probes, whereas the Xenopus-expressed truncation maintained slight binding activity . Indirect immunofluorescence staining showed that myc-tagged full-length nucleolin properly localized to the dense fibrillar regions within the multiple nucleoli of Xenopus oocyte nuclei . The epitope-tagged GAR truncation also translocated to the oocyte nuclei, but it failed to efficiently localize to the nucleoli . Our results show that the carboxy GAR domain must be present for nucleolin to efficiently bind nucleic acids in vitro and to associate with nucleoli in vivo.

Br J Pharmacol, 1993 Nov, 110(3), 1189 - 95
The induction of nitric oxide synthase and intestinal vascular permeability by endotoxin in the rat; Boughton-Smith NK et al.; 1 . The effect of endotoxin (E . coli lipopolysaccharide) on the induction of nitric oxide synthase (NOS) and the changes in vascular permeability in the colon and jejunum over a 5 h period have been investigated in the rat . 2 . Under resting conditions, a calcium-dependent constitutive NOS, determined by the conversion of radiolabelled L-arginine to citrulline, was detected in homogenates of both colonic and jejunal tissue . 3 . Administration of endotoxin (3 mg kg-1, i.v.) led, after a 2 h lag period, to the appearance of calcium-independent NOS activity in the colon and jejunum ex vivo, characteristic of the inducible NOS enzyme . 4 . Administration of endotoxin led to an increase in colonic and jejunal vascular permeability after a lag period of 3 h, determined by the leakage of radiolabelled albumin . 5 . Pretreatment with dexamethasone (1 mg kg-1 s.c., 2 h prior to challenge) inhibited both the induction of NOS and the vascular leakage induced by endotoxin . 6 . Administration of the NO synthase inhibitor NG-monomethyl-L-arginine (12.5-50 mg kg-1, s.c.) 3 h after endotoxin injection, dose-dependently reduced the subsequent increase in vascular permeability in jejunum and colon, an effect reversed by L-arginine (300 mg kg-1, s.c.) . 7 . These findings suggest that induction of NOS is associated with the vascular injury induced by endotoxin in the rat colon and jejunum.

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