Escherichia coli

Plant Mol Biol, 1999 Jul, 40(4), 679 - 86
Altering the 3 UTR endonucleolytic cleavage site of a Chlamydomonas chloroplast mRNA affects 3-end maturation in vitro but not in vivo; Rott R et al.; The 3' ends of chloroplast mRNAs are produced by the processing of longer precursors . The 3' ends of most plastid mRNAs are located at, or several nucleotides downstream of, stem-loop structures, which act as 3'-end-processing signals and RNA stability elements . In chloroplasts of the green alga Chlamydomonas reinhardtii, 3'-end maturation of atpB mRNA involves endonucleolytic cleavage of the pre-mRNA at an AU-rich site located about 10 nucleotides downstream of the stem-loop structure . This cleavage is followed by exonucleolytic resection to generate the mature 3' end . In order to define critical nucleotides of the endonucleolytic cleavage site, we mutated its sequence . Incubation of synthetic atpB pre-RNAs containing these mutations in a chloroplast protein extract resulted in the accumulation of 3'-end-processed products . However, in two cases where the AU-rich sequence of this site was replaced with a GC-rich one, the 3' end of the stable processing product differed from that of the wild-type product . To examine whether these mutations affected atpB mRNA processing or accumulation in vivo, the endogenous 3' UTR was replaced with mutated sequences by biolistic transformation of Chlamydomonas chloroplasts . Analysis of the resulting strains revealed that the accumulation of atpB mRNA was approximately equal to that of wild-type cells, and that a wild-type atpB 3' end was generated . These results imply that Chlamydomonas atpB 3' processing parallels the situation with other endonucleases such as Escherichia coli RNAse E, where specific sequences are required for correct in vitro processing, but in vivo these mutations can be overcome.

Plant Mol Biol, 1999 Jul, 40(4), 555 - 65
Secondary xylem-specific expression of caffeoyl-coenzyme A 3-O-methyltransferase plays an important role in the methylation pathway associated with lignin biosynthesis in loblolly pine; Li L et al.; Two types of structurally distinct O-methyltransferases mediate the methylation of hydroxylated monomeric lignin precursors in angiosperms . Caffeate 3-O-methyltransferase (COMT; EC 2.1.1.68) methylates the free acids and caffeoyl CoA 3-O-methyltransferase (CCoAOMT; EC 2.1.1.104) methylates coenzyme A esters . Recently, we reported a novel hydroxycinnamic acid/hydroxycinnamoyl CoA ester O-methyltransferase (AEOMT) from loblolly pine differentiating xylem that was capable of methylating both acid and ester precursors with similar efficiency . In order to determine the possible existence and role of CCoAOMT in lignin biosynthesis in gymnosperms, a 1.3 kb CCoAOMT cDNA was isolated from loblolly pine that showed 79-82% amino acid sequence identity with many angiosperm CCoAOMTs . The recombinant CCoAOMT expressed in Escherichia coli exhibited a significant methylating activity with hydroxycinnamoyl CoA esters whereas activity with hydroxycinnamic acids was insignificant . Moreover, 3.2 times higher catalytic efficiency for methylating caffeoyl CoA over 5-hydroxyferuloyl CoA was observed which could serve as a driving force towards synthesis of guaiacyl lignin . The secondary xylem-specific expression of CCoAOMT was demonstrated using RNA blot analysis, western blot analysis, and O-methyltransferase enzyme assays . In addition, Southern blot analysis indicated that CCoAOMT may exist as a single-copy gene in loblolly pine genome . The transgenic tobacco plants carrying loblolly pine CCoAOMT promoter-GUS fusion localized the site of GUS activity at the secondary xylem tissues . These data suggest that CCoAOMT, in addition to AEOMT, plays an important role in the methylation pathway associated with lignin biosynthesis in loblolly pine.

J Chromatogr A, 1999 Aug 6, 852(1), 151 - 9
Affinity purification of Schistosoma japonicum glutathione-S-transferase and its site-directed mutants with glutathione affinity chromatography and immobilized metal affinity chromatography; Chen HM et al.; A C-terminally polyhistidine-tagged protein of Schistosoma japonicum glutathione-S-transferase, named as SjGST/His, and its Cys85-->Ser, Cys138-->Ser, and Cys178-->Ser site-directed mutants were prepared and highly expressed in Escherichia coli . Both immobilized metal affinity chromatography (IMAC) and glutathione (GSH) affinity chromatography were used to purify these four enzymes . All of them were purified with equal efficiency by Ni2+-chelated nitrilotriacetic acid agarose gel, but not by GSH Sepharose 4B gel . The protein amounts of wild-type and Cys85-->Ser enzymes purified by the latter gel were three to seven-fold greater than those of the other two enzymes purified by the same gel, while their specific activities were two-fold lower, presumably because of the occurrence of noncovalent aggregation . Both purification methods yielded highly pure enzymes, while there were minor amounts of inter- and intra-disulfide forms in the IMAC purified enzymes except for the Cys85-->Ser mutant . Addition of dithiothreitol to GSH-affinity purified enzymes shifted all of their mass spectra of matrix-assisted laser desorption/ionization-time of flight mass spectrometry toward low molecular-mass regions, while addition of GSH to IMAC purified enzymes shifted the spectra toward high molecular-mass regions . The shift values of wild-type enzyme were larger than those of the three mutants, indicating that the Cys85, Cys138, and Cys178 residues were S-thiolated by GSH during the GSH-affinity purification . This result was confirmed by isoelectric focusing . These findings suggest that IMAC is more efficient than the conventional GSH-affinity system for the purification of SjGST/His enzyme, especially for its mutants and fusion proteins.

J Chromatogr A, 1999 Aug 6, 852(1), 117 - 28
Histidines in affinity tags and surface clusters for immobilized metal-ion affinity chromatography of trimeric tumor necrosis factor alpha; Gaberc-Porekar V et al.; In order to achieve efficient IMAC (immobilized metal-ion affinity chromatography) purification of tumor necrosis factor alpha (TNF-alpha) and its analogs by a common chromatographic procedure, we tested four histidine-rich affinity tags attached to the N-termini of the trimeric TNF-alpha molecule . Using low cultivation temperature and appropriate protease deficient E . coli strains, it was possible to obtain intact, full-length proteins with NHis2Xa and HisArg tags, which could be purified to over 95% purity in a single step . However, in comparison to model proteins bearing a surface histidine cluster, accumulation of the histidine-tagged proteins in E . coli was significantly reduced, even in protease deficient strains . In addition, the histidine tagged TNF-alpha proteins never displayed good chromatographic behavior, which was otherwise easily achieved with model proteins . Although the most popular hexa-histidine tag is generally recognized as very convenient for single step isolation of monomeric proteins, our results with trimeric TNF-alpha indicate that oligomeric proteins may require further optimization of the tag, with respect to its length, composition, and location . Histidines, relatively rigidly inserted in the structure, as in our model proteins, display superior chromatographic characteristics vis a vis flexible tags with the same total number of histidines.

J Dairy Res, 1999 Aug, 66(3), 431 - 9
Novel angiotensin-I-converting enzyme inhibitory peptides derived from recombinant human alpha s1-casein expressed in Escherichia coli; Kim YK et al.; Recombinant human alpha s1-casein expressed in Escherichia coli was purified and digested with trypsin in an attempt to find peptides with angiotensin-I-converting enzyme (ACE) inhibitory activity . Three novel ACE inhibitory peptides, A-II, B-II and C, were isolated and their amino acid sequences identified as Tyr-Pro-Glu-Arg (residues 8-11), Tyr-Tyr-Pro-Gln-Ile-Met-Gln-Tyr (residues 136-143) and Asn-Asn-Val-Met-Leu-Gln-Trp (residues 164-170) respectively . ACE inhibitory activities were measured for the corresponding synthetic peptides, and the ACE IC50 (the amount of peptide causing 50% inhibition of ACE activity) values of A-II, B-II and C estimated to be 132.5, 24.8 and 41.0 mumol/l respectively . Peptides A-II and C were resistant to further digestion by pepsin, whereas peptide B-II was hydrolysed . All three peptides were resistant to digestion by chymotrypsin . These ACE inhibitory peptides may prove useful for oral administration in the treatment of hypertension.

J Dairy Res, 1999 Aug, 66(3), 375 - 83
Respiratory burst activity in activated and unstimulated isolated bovine blood neutrophils during experimentally induced Escherichia coli mastitis; Van Oostveldt K et al.; The respiratory burst activity, measured as H2O2 production, of isolated bovine polymorphonuclear leucocytes (PMN) was evaluated during experimentally induced Escherichia coli mastitis by means of flow cytometry in cells activated by phorbol 12-myristate 13-acetate (PMA) and in unstimulated cells . As expected, a significantly reduced respiratory burst activity was observed in PMA-activated PMN 18 h after intramammary inoculation with Escherichia coli . At this time only 75% of the PMA-activated PMN showed a respiratory burst, but with a higher intensity than that measured before and later after infection with Esch . coli . In addition, an increase in the respiratory burst activity was observed in unstimulated blood PMN during a short period at 18 h after infection, when up to 30% of the unstimulated PMN had a respiratory burst activity . The increase in the respiratory burst intensity of PMA-activated PMN and the spontaneously augmented production of reduced oxygen species by the unstimulated PMN during infection with Esch . coli might indicate the production of a natural stimulator of burst activity in circulation, most probably originating from the inflamed udder.

J Struct Biol, 1999 Aug, 127(1), 88 - 91
Crystallization and preliminary X-ray crystallographic analysis of pyridoxine 5'-phosphate oxidase complexed with flavin mononucleotide; Musayev FN et al.; Pyridoxine 5'-phosphate oxidase (PNP Ox) catalyzes the terminal step in the biosynthesis of pyridoxal 5'-phosphate . The 53-kDa homodimeric enzyme contains a noncovalently bound flavin mononucleotide (FMN) on each monomer . Three crystal forms of Escherichia coli PNP Ox complexed with FMN have been obtained at room temperature . The first crystal form belongs to trigonal space group P3(1)21 or P3(2)21 with unit cell dimensions a = b = 64.67A, c = 125.64A, and has one molecule of the complex (PNP Ox-FMN) per asymmetric unit . These crystals grow very slowly to their maximum size in about 2 to 4 months and diffract to about 2.3 A . The second crystal form belongs to tetragonal space group P4(1) or P4(3) with unit cell dimensions a = b = 54.92A, c = 167.65A, and has two molecules of the complex per asymmetric unit . The crystals reach their maximum size in about 5 weeks and diffract to 2.8 A . A third crystal form with a rod-like morphology grows faster and slightly larger than the other two forms, but diffracts poorly and could not be characterized by X-ray analysis . The search for heavy-atom derivatives for the first two crystal forms to solve the structure is in progress.

J Struct Biol, 1999 Aug, 127(1), 72 - 5
Preliminary crystallographic studies of human mitochondrial NAD(P)(+)-dependent malic enzyme; Bhargava G et al.; Human mitochondrial NAD(P)(+)-dependent malic enzyme was overexpressed in Escherichia coli and purified by anion-exchange, ATP affinity, and gel filtration chromatography . The protein was crystallized with the hanging-drop vapor diffusion method . Many different crystal forms were observed, five of which were characterized in some detail . A 2.5-A multiple-wavelength anomalous diffraction data set and a 2.1-A native data set were collected using synchrotron radiation on crystals containing selenomethionyl residues . These crystals belong to space group B2, with a = 204.4 A, b = 107.0 A, c = 59.2 A, and gamma = 101.9 degrees . Self-rotation functions demonstrated that the tetramer of this enzyme obeys 222 symmetry.

J Infect Dis, 1999 Oct, 180(4), 1374 - 7
Induction of glomerular lesions in the kidneys of mice infected with vero toxin-producing Escherichia coli by lipopolysaccharide injection; Shimizu K et al.; Lipopolysaccharide was injected into germ-free mice after they had been infected with Vero toxin-producing Escherichia coli . Microscopic examination of the kidneys of these mice showed an increased number of mesangial cells and a vacancy in the glomerular capillary lumen . A significant elevation in the expression level of interferon (IFN)-gamma in the kidney may have played a key role in the induction of glomerular lesions, because the administration of neutralizing antibody to IFN-gamma markedly alleviated such lesions.

Biosci Biotechnol Biochem, 1999 Jul, 63(7), 1291 - 4
Expression of mature pokeweed antiviral protein with or without C-terminal extrapeptide in Escherichia coli as a fusion with maltose-binding protein; Honjo E et al.; Genomic clones encoding the mature pokeweed antiviral protein with or without C-terminal extrapeptide (PAPMC and PAPM), which have been reported to be highly toxic to E . coli cells, were inserted into the expression vector pMAL-p2 . The recombinant PAPs (rPAPMC and rPAPM) were successfully expressed in E . coli at 25 degrees C, being exported to the periplasm as soluble fusions with maltose-binding protein (MBP) . The rPAPs were cleaved from MBP by treatment with factor Xa and subsequently purified with final yields of 4.0 mg/liter (rPAPMC) and 5.5 mg/liter (rPAPM) . rPAPM was resistant to protease digestion, but the C-terminal extrapeptide appeared to be susceptible and was partially digested by some protease in E . coli . Both rPAPMC and rPAPM were as active as the native PAPM from pokeweed leaves in depurinating rat liver and E . coli ribosomes, while the activities of rPAPMC on both ribosomes were decreased at least 60-fold by fusion with MBP.

Blood, 1999 Sep 1, 94(5), 1717 - 26
Suppression of interleukin-12 production by human monocytes after preincubation with lipopolysaccharide; Wittmann M et al.; Interleukin-12 (IL-12) is a potent proinflammatory and immunoregulatory cytokine skewing T lymphocytes to express a type 1 cytokine pattern . Optimal expression of IL-12 mRNA and bioactivity in vitro requires specific priming of monocytes by interferon-gamma (IFN-gamma) or granulocyte-macrophage colony-stimulating factor (GM-CSF) before lipopolysaccharide (LPS) stimulation . We show here for the first time that the production of IL-12 by IFN-gamma- or GM-CSF-primed human monocytes can be completely suppressed by preincubation with LPS (from Escherichia coli Serotype 055:B5) for 6 to 24 hours before the priming procedure . A dose-dependent suppression of IL-12p70 was measured on the levels of intracellular cytokine production and cytokine secretion . mRNA studies on the expression of p40 and p35 showed an LPS-induced downregulation of both subunits . The results of several different experimental approaches suggest that IL-12 downregulation was not due to endogenous IL-10, IL-4, prostaglandin E(2) (PGE(2)), tumor necrosis factor-alpha (TNF-alpha), or nitric oxide (NO) production induced by LPS . Moreover, preincubation of monocytes with LPS did not lead to a downregulation of the CD14 antigen, which is an LPS receptor . LPS preincubation in this experimental setting did not result in a general hyporesponsiveness of the monocytes, as IL-6 production as well as IFN-gamma-induced upregulation of CD54 did not decline . Downregulation of IL-12 was not due to changes in mRNA stability . These findings show that the immunoregulatory important cytokine, IL-12, underlies itself a complex regulation.

J Vasc Surg, 1999 Sep, 30(3), 542 - 50
In vivo adenovirus-mediated gene transfer and expression in ischemic rabbit spinal cord; Sakurai M et al.; PURPOSE: In an attempt to study whether ischemic spinal cord expresses a foreign gene in vivo, a replication-defective adenoviral vector containing the Escherichia coli lacZ gene was directly injected into the ischemic spinal cord of rabbits, and temporal and spatial profiles of the exogenous gene expression were compared with that of the control spinal cord . METHODS: Thirty-nine Japanese domesticated white rabbits weighing 2 to 3 kg were used in this study and were divided into two subgroups, a 15-minute ischemia group and a sham control group . The adenoviral vector was directly injected into lumbar spinal cord by a needle from dorsal spine just after the infrarenal aortic occlusion in the case of ischemia . Animals were allowed to recover at ambient temperature and were killed at 1, 2, 4, and 7 days after reperfusion (n = 3 at each time point) . RESULTS: In the control rabbit, adenoviral vector was transferred into the spinal cord, and the lacZ gene was expressed at dorsal astroglia and anterior motor neurons at 1 to 7 days of reperfusion . After 15 minutes of ischemia, the lacZ gene was expressed at 2 and 4 days of reperfusion in dorsal astroglia and anterior motor neurons, which were positive for Fas antigen . CONCLUSION: This result suggests that it is possible to transfer and express the lacZ gene in ischemic motor neurons, which eventually show apoptotic change with induction of Fas antigen, and also suggests a great potential of gene therapy for paraplegic patients in the future.

Mutat Res, 1999 Jul 21, 444(1), 123 - 31
Molecular analysis of mutations induced by a benzene metabolite, p-benzoquinone, in mouse cells using a novel shuttle vector plasmid; Nakayama A et al.; Human population has been continually exposed to benzene which is present in our environment as an essential component of petroleum . p-Benzoquinone (p-BQ) is one of the benzene metabolites and is thought to be an ultimate toxic or carcinogenic substance . For molecular analysis of carcinogen-induced mutations in mouse cells, we constructed a new shuttle vector plasmid pNY200 that has supF gene as a target of the mutations and replicates in mouse and in Escherichia coli cells . In p-BQ-treated pNY200 propagated in mouse cells, base substitutions were induced predominantly at G:C sites, and the major mutation was G:C-->A:T transition . Many tandem base substitutions were also induced at CC:GG sequences . By a postlabeling analysis and a polymerase stop assay, we confirmed that p-BQ adducts formed in DNA and mutation sites roughly correspond to the sites where the adducts were formed . Comparing data of pNY200 in mouse cells with those of the similar shuttle vector plasmid pMY189 in human cells should be important for extrapolation of data from mouse to human, because carcinogenicity of chemicals is tested in mice.

Biochem J, 1999 Sep 15, 342 Pt 3, 715 - 9
Editing of non-cognate aminoacyl adenylates by peptide synthetases; Pavela-Vrancic M et al.; Non-ribosomally formed peptides display both highly conserved and variable amino acid positions, the variations leading to a wide range of peptide families . Activation of the amino acid substrate proceeds in analogy to the ribosomal biosynthetic mechanism generating aminoacyl adenylate and acyl intermediates . To approach the mechanism of fidelity of amino acid selection, the stability of the aminoacyl adenylates was studied by employing a continuous coupled spectrophotometric assay . The apo-form of tyrocidine synthetase 1 (apo-TY1) was used, generating an l-phenylalanyl-adenylate intermediate stabilized by the interaction of two structural subdomains of the adenylation domain . Adenylates of substrate analogues have shown variable and reduced degrees of stability, thus leading to an enhanced generation of pyrophosphate due to hydrolysis and continuous adenylate formation . Discrimination of the non-aromatic amino acids l-Leu and l-Met, or l-Phe analogues such as p-amino- and p-chloro-l-Phe derivatives, as well as the stereospecific selection of l-Phe, is supported by less-stable adenylate intermediates exhibiting elevated susceptibility to hydrolysis . Breakdown of the l-phenylalanyl intermediate utilizing 2'-deoxy-ATP as the nucleotide substrate was significantly enhanced compared with the natural analogue . Apo-TY1 engineered at positions involved in adenylate formation showed variable protection against hydrolysis . The results imply that stability of the aminoacyl intermediates may act as an essential factor in substrate selection and fidelity of non-ribosomal-peptide-forming systems.

Biochem J, 1999 Sep 15, 342 Pt 3, 647 - 53
Interaction with amylopectin influences the ability of granule-bound starch synthase I to elongate malto-oligosaccharides; Denyer K et al.; This paper examines the properties in soluble form of two isoforms of starch synthase . One of these, granule-bound starch synthase I (GBSSI), is responsible for the synthesis of amylose inside the amylopectin matrix of the starch granule in vivo . The other, starch synthase II (SSII), is involved in amylopectin synthesis . Both isoforms can use amylopectin and malto-oligosaccharide as substrates in vitro . As well as acting as a substrate for GBSSI, amylopectin acts as an effector of this isoform, increasing the rate at which it elongates malto-oligosaccharides and promoting a processive rather than distributive mode of elongation of these compounds . The affinity of GBSSI for amylopectin as an effector is greater than its affinity for amylopectin as a substrate . The rate and mode of elongation of malto-oligosaccharides by SSII are not influenced by amylopectin . These results suggest that specific interaction with amylopectin in the matrix of the starch granule is a unique property of GBSSI and is critical in determining the nature of its products.

Biochem J, 1999 Sep 15, 342 Pt 3, 625 - 32
Production and functional activity of a recombinant von Willebrand factor-A domain from human complement factor B; Williams SC et al.; Factor B is a five-domain 90 kDa serine protease proenzyme which is part of the human serum complement system . It binds to other complement proteins C3b and properdin, and is activated by the protease factor D . The fourth domain of factor B is homologous to the type A domain of von Willebrand Factor (vWF-A) . A full-length human factor B cDNA clone was used to amplify the region encoding the vWF-A domain (amino acids 229-444 of factor B) . A fusion protein expression system was then used to generate it in high yield in Escherichia coli, where thrombin cleavage was used to separate the vWF-A domain from its fusion protein partner . A second vWF-A domain with improved stability and solubility was created using a Cys(267)-->Ser mutation and a four-residue C-terminal extension of the first vWF-A domain . The recombinant domains were investigated by analytical gel filtration, sucrose density centrifugation and analytical ultracentrifugation, in order to show that both domains were monomeric and possessed compact structures that were consistent with known vWF-A crystal structures . This expression system and its characterization permitted the first investigation of the function of the isolated vWF-A domain . It was able to inhibit substantially the binding of (125)I-labelled factor B to immobilized C3b . This demonstrated both the presence of a C3b binding site in this portion of factor B and a ligand-binding property of the vWF-A domain . The site at which factor D cleaves factor B is close to the N-terminus of both recombinant vWF-A domains . Factor D was shown to cleave the vWF-A domain in the presence or absence of C3b, whereas the cleavage of intact factor B under the same conditions occurs only in the presence of C3b.

Mol Biochem Parasitol, 1999 Jul 30, 102(1), 21 - 33
Molecular and biochemical characterization of a protein kinase B from Trypanosoma cruzi; Pascuccelli V et al.; A Trypanosoma cruzi gene, PKB, coding for a putative protein kinase was cloned and sequenced . Analysis of the sequence showed that the encoded protein (called PKB) corresponds to a relatively novel subgroup of Ser/Thr protein kinases denominated protein kinases B (PKB), related to A and C protein kinases (RAC), or protein kinases of the transforming retrovirus AKT8 (Akt) in which the catalytic domains show similarity to corresponding domains of protein kinases A and protein kinases C . Unlike mammalian enzymes belonging to the same subgroup, PKB did not have a pleckstrin (PH)-homologous domain . PKB was expressed in Escherichia coli and the recombinant protein was found to be a Thr-specific protein kinase that required Mn2+ for activity and used ATP as phosphate donor (Km = 1.8 microM) . Classical protein kinase A and protein kinase C modulators and inhibitors were found to have only marginal or no effect on PKB activity . Antisera raised against the recombinant protein recognized PKB in Western blotting analysis of cell extracts as a membrane bound protein . Evidence was obtained suggesting the presence of a Cys-linked acyl anchor . Northern and Western blotting analysis showed that PKB was constitutively expressed in the lag, exponential and stationary phases of T . cruzi epimastigote growth, as well as in the amastigote and metacyclic trypomastigote stages of differentiation . This is the first description of the existence of a protein kinase B in trypanosomatid protozoa.

Plant J, 1999 Aug, 19(3), 353 - 61
A synthetic gene coding for the green fluorescent protein (GFP) is a versatile reporter in Chlamydomonas reinhardtii; Fuhrmann M et al.; The use of Chlamydomonas reinhardtii as a model system has been hindered by difficulties encountered in expressing foreign genes . We have synthesised a gene encoding green fluorescent protein (GFP) adapted to the codon usage of C . reinhardtii (cgfp) . After verifying the gene was functional in Escherichia coli, the cgfp was fused in frame to the phleomycin resistance gene ble from Streptoalloteichus hindustanus and expressed in C . reinhardtii under control of the rbcS2 promoter and intron sequences . The GFP-fluorescence was seen only in the nucleus demonstrating the nuclear accumulation of the Ble-GFP fusion protein . The cgfp was also fused to the chlamyopsin gene, cop, and expressed in C . reinhardtii under control of the cop promoter . The eyespot became fluorescent indicating that the opsin-GFP fusion protein was correctly directed into the eyespot along with the endogenous unmodified opsin . We conclude that cgfp provides a useful tool to visualize protein synthesis and localisation in vivo in C . reinhardtii and possibly in related green algal species.

Plant J, 1999 Jul, 19(2), 195 - 201
Cloning and expression of a cDNA encoding homospermidine synthase from Senecio vulgaris (Asteraceae) in Escherichia coli; Kaiser A; The enzyme homospermidine synthase catalyzes the NAD+-dependent conversion of 2 mol putrescine into homospermidine . Instead of putrescine, spermidine can substitute for the first putrescine moiety in plants, in which case diaminopropane instead of ammonia is released . The enzyme facilitates the formation of the 'uncommon' polyamine homospermidine which is an important precursor in the biosynthesis of pyrrolizidine alkaloids . The first plant homospermidine synthase was purified to apparent chemical homogenity from the root tissue culture Senecio vernalis (Asteraceae) (Bottcher et al . 1994, Can . J . Chem . 72, 80-85; Ober 1997, Dissertation) . Four endopeptidase LysC fragments were sequenced from the purified protein . With the aid of degenerate primers against these peptides, a cDNA encoding homospermidine synthase was now cloned and characterized from Senecio vulgaris . The nucleotide sequence of the cloned cDNA revealed an open reading frame of 1155-base pairs containing 385 amino acids with a predicted Mr of 44500 . GenBank research revealed that the deduced amino acid sequence shows 59% identity to human deoxyhypusine synthase . The homospermidine synthase encoding cDNA was subcloned into the expression vector pet15b and overexpressed in E . coli . The recombinant enzyme formed upon expression catalyzed homospermidine synthesis.

Plant J, 1999 Jul, 19(2), 183 - 93
Flavonoid hydroxylase from Catharanthus roseus: cDNA, heterologous expression, enzyme properties and cell-type specific expression in plants; Kaltenbach M et al.; We investigated the P450 dependent flavonoid hydroxylase from the ornamental plant Catharanthus roseus . cDNAs were obtained by heterologous screening with the CYP75 Hf1 cDNA from Petunia hybrida . The C . roseus protein shared 68-78% identity with other CYP75s, and genomic blots suggested one or two genes . The protein was expressed in Escherichia coli as translational fusion with the P450 reductase from C . roseus . Enzyme assays showed that it was a flavonoid 3', 5'-hydroxylase, but 3'-hydroxylated products were also detected . The substrate specificity was investigated with the C . roseus enzyme and a fusion protein of the Petunia hybrida CYP75 with the C . roseus P450 reductase . Both enzymes accepted flavanones as well as flavones, dihydroflavonols and flavonols, and both performed 3'- as well as 3'5'-hydroxylation . Kinetics with C . roseus cultures on the level of enzyme activity, protein and RNA showed that the F3'5'H was present in dark-grown cells and was induced by irradiation . The same results were obtained for cinnamic acid 4-hydroxylase and flavanone 3beta-hydroxylase . In contrast, CHS expression was strictly dependent on light, although CHS is necessary in the synthesis of the F3'5'H substrates . Immunohistochemical localization of F3'5'H had not been performed before . A comparison of CHS and F3'5'H in cotyledons and flower buds from C . roseus identified CHS expression preferentially in the epidermis, while F3'5'H was only detected in the phloem . The cell-type specific expression suggests that intercellular transport may play an important role in the compartmentation of the pathways to the different flavonoids.

Plant J, 1999 Jul, 19(2), 173 - 181
Transcription activation mediated by the bZIP factor SPA on the endosperm box is modulated by ESBF-1 in vitro; Conlan RS et al.; A modified in vitro transcription system has been used to study the function of the cloned bZIP transcription factor SPA and the binding activity ESBF I in activating transcription from the bifactorial endosperm box region of the wheat prolamin LMWG-1D1 gene . Recombinant SPA expressed in Escherichia coli activated transcription from the endosperm box motif, and this was dependent upon the binding of the nuclear protein ESBF I . ESBF I did not activate transcription independently, but potentiated SPA-mediated transcriptional activation . ESBF I is likely to be the equivalent of, or contain the recently characterised DOF class of, Zn-finger protein called WPBF . These data provide new information about the interplay of members of the bZIP and DOF transcription factor families in regulating expression from bifactorial sites found in a variety of plant promoters.

Parasite Immunol, 1999 Sep, 21(9), 439 - 50
A hookworm allergen which strongly resembles calreticulin; Pritchard DI et al.; Immmoglobulin E-rich plasma from patients from Papua New Guinea infected with Necator americanus has been used to probe an adult N . americanus cDNA library for the presence of hookworm allergens . Using this approach, one hookworm allergen has been identified as calreticulin, which was subsequently expressed in Escherichia coli . Little serological cross reactivity was seen between the recombinant calreticulins of this hookworm and its host . Prospective roles for hookworm calreticulin in the host-parasite relationship are discussed in depth.

Mol Microbiol, 1999 Sep, 33(5), 1081 - 92
Expression and heat-responsive regulation of a TFIIB homologue from the archaeon Haloferax volcanii; Thompson DK et al.; Multiple divergent genes encoding the eukaryal-like TFIIB (TFB) transcription initiation factor have been identified in the archaeon Haloferax volcanii . Expression of one of these TFB-encoding genes, referred to here as tfb2, was induced specifically in response to heat shock at the transcription level . A time course for tfb2 induction demonstrated that mRNA levels increased as much as eightfold after 15 min at 60 degrees C . A transcription fusion of the tfb2 promoter region with a stable RNA reporter gene confirmed the heat responsiveness of the tfb2 core promoter, and immunoblot analysis using antibodies generated against a recombinant His-tagged TFB2 showed that the protein levels of one TFB increased slightly in response to elevated temperatures . An archaeal consensus TATA element (5'-TTTATA-3') was located 110 bp upstream of the translation start site and appeared to be used for both basal and heat shock-induced expression . The long DNA leader region (79 bp) preceding the predicted AUG translation start codon for TFB2 contained a T-rich sequence element located 22 bp downstream of the transcription start site . Using an in vivo transcription termination assay, we demonstrated that this T-rich element can function as a sequence-dependent transcription terminator, which may serve to downregulate expression of the tfb2 gene under both non-heat shock and heat shock conditions.

Mol Microbiol, 1999 Sep, 33(5), 1059 - 68
Tn5053 family transposons are res site hunters sensing plasmidal res sites occupied by cognate resolvases; Minakhina S et al.; DNA sequence database search revealed that most of Tn5053/Tn402 family transposons inserted into natural plasmids were located in putative res regions upstream of genes encoding various resolvase-like proteins . Some of these resolvase genes belonged to Tn3 family transposons and were closely related to the tnpR genes of Tn1721 and a recently detected Tn5044 . Using recombinant plasmids containing fragments of Tn1721 or Tn5044 as targets in transposition experiments, we have demonstrated that Tn5053 displays striking insertional preference for the res regions of these transposons: more than 70% of Tn5053 insertion events occur in clusters inside the target res regions, while most remaining insertion events occur no further than 200 base pairs away from both sides of the res regions . We demonstrate that Tn5053 insertions (both into and outside a res region of the target plasmid) require the presence of a functional cognate resolvase gene either in cis or in trans . To our knowledge, this is the first case when a site-specific recombination system outside a transposon has been shown to be involved in transposition.

Mol Microbiol, 1999 Sep, 33(5), 1027 - 36
Norfloxacin-induced DNA cleavage occurs at the dif resolvase locus in Escherichia coli and is the result of interaction with topoisomerase IV; Hojgaard A et al.; The dif locus is a site-specific recombination site located within the terminus region of the chromosome of Escherichia coli . Recombination at dif resolves circular dimer chromosomes to monomers, and this recombination requires the XerC, XerD and FtsK proteins, as well as cell division . In order to characterize other enzymes that interact at dif, we tested whether quinolone-induced cleavage occurs at this site . Quinolone drugs, such as norfloxacin, inhibit the type 2 topoisomerases, DNA gyrase and topoisomerase IV, and can cleave DNA at sites where these enzymes interact with the chromosome . Using strains in which either DNA gyrase or topoisomerase IV, or both, were resistant to norfloxacin, we determined that specific interactions between dif and topoisomerase IV caused cleavage at that site . This interaction required XerC and XerD, but did not require the C-terminal region of FtsK or cell division.

Mol Microbiol, 1999 Sep, 33(5), 1004 - 14
Sequence-selective interactions with RNA by CspB, CspC and CspE, members of the CspA family of Escherichia coli; Phadtare S et al.; The CspA family of Escherichia coli comprises nine homologous proteins, CspA to CspI . CspA, the major cold shock protein, binds RNA with low sequence specificity and low binding affinity . This is considered to be important for its proposed function as an RNA chaperone to prevent the formation of secondary structures in RNA molecules, thus facilitating translation at low temperature . The cellular functions of other Csp proteins are yet to be fully elucidated, and their sequence specific binding capabilities have not been identified . As a step towards identification of the target genes of Csp proteins, we investigated the RNA binding specificities of CspB, CspC and CspE by an in vitro selection approach (SELEX) . In the present study, we show that these proteins are able to bind preferentially to specific RNA/single-stranded DNA sequences . The consensus sequences for CspB, CspC and CspE are U/T stretches, AGGGAGGGA and AU/AT-rich regions, especially AAAUUU, respectively . CspE and CspB have Kd values in the range 0.23-0.9 x 10(-6) M, while CspC has 10-fold lower binding affinity . Consistent with our recent findings of transcriptional regulation of cspA by CspE, we have identified a motif identical to the CspE consensus . This motif is the putative CspE-mediated transcription pause recognition site in a 5'-untranslated region of the cspA mRNA.

Mol Microbiol, 1999 Sep, 33(5), 982 - 93
Stimulation of transposition of the Mycobacterium tuberculosis insertion sequence IS6110 by exposure to a microaerobic environment; Ghanekar K et al.; The Mycobacterium tuberculosis-specific insertion sequence IS6110/986 has been widely used as a probe because of the multiple polymorphism observed among different strains . To investigate transposition of IS6110, a series of artificially constructed composite transposons containing IS6110 and a kanamycin resistance marker were constructed . The composite transposons were inserted into a conditionally replicating, thermosensitive, Escherichia coli-mycobacterial shuttle vector and introduced into M . smegmatis mc2155 . Lawns of transformants were grown at the permissive temperature on kanamycin-supplemented agar and subsequently prevented from further growth by shifting to the non-permissive temperature . Under normal atmospheric conditions, kanamycin-resistant papillae appeared after only about 5-6 weeks of incubation . However, these events were not associated with transposon mobilization . In contrast, lawns that were exposed to a 48 h microaerobic shock generated kanamycin-resistant papillae after only 6-14 days . These events were generated by conservative transposition of the IS6110 composite transposon into the M . smegmatis chromosome, with loss of the shuttle vector . In common with other IS3 family elements, transposition of IS6110 is thought to be controlled by translational frameshifting . However, we were unable to detect any significant frameshifting within the putative frameshifting site of IS6110, and the level of frameshifting was not affected by microaerobic incubation . The finding that transposition of IS6110 is stimulated by incubation at reduced oxygen tensions may be relevant to transposition of IS6110 in M . tuberculosis harboured within TB lesions.

Mol Microbiol, 1999 Sep, 33(5), 959 - 70
Delayed nucleoid segregation in Escherichia coli; Huls PG et al.; To study the role of cell division in the process of nucleoid segregation, we measured the DNA content of individual nucleoids in isogenic Escherichia coli cell division mutants by image cytometry . In pbpB(Ts) and ftsZ strains growing as filaments at 42 degrees C, nucleoids contained, on average, more than two chromosome equivalents compared with 1.6 in wild-type cells . Because similar results were obtained with a pbpB recA strain, the increased DNA content cannot be ascribed to the occurrence of chromosome dimers . From the determination of the amount of DNA per cell and per individual nucleoid after rifampicin inhibition, we estimated the C and D periods (duration of a round of replication and time between termination and cell division respectively), as well as the D' period (time between termination and nucleoid separation) . Compared with the parent strain and in contrast to ftsQ, ftsA and ftsZ mutants, pbpB(Ts) cells growing at the permissive temperature (28 degrees C) showed a long D' period (42 min versus 18 min in the parent) indicative of an extended segregation time . The results indicate that a defective cell division protein such as PbpB not only affects the division process but also plays a role in the last stage of DNA segregation . We propose that PbpB is involved in the assembly of the divisome and that this structure enhances nucleoid segregation.

Mol Microbiol, 1999 Sep, 33(5), 933 - 45
Cloning of the mspA gene encoding a porin from Mycobacterium smegmatis; Niederweis M et al.; Porins form channels in the mycolic acid layer of mycobacteria and thereby control access of hydrophilic molecules to the cell . We purified a 100 kDa protein from Mycobacterium smegmatis and demonstrated its channel-forming activity by reconstitution in planar lipid bilayers . The mspA gene encodes a mature protein of 184 amino acids and an N-terminal signal sequence . MALDI mass spectrometry of the purified porin revealed a mass of 19 406 Da, in agreement with the predicted mass of mature MspA . Dissociation of the porin by boiling in 80% dimethyl sulphoxide yielded the MspA monomer, which did not form channels any more . Escherichia coli cells expressing the mspA gene produced the MspA monomer and a 100 kDa protein, which had the same channel-forming activity as whole-cell extracts of M . smegmatis with organic solvents . These proteins were specifically detected by a polyclonal antiserum that was raised to purified MspA of M . smegmatis . These results demonstrate that the mspA gene encodes a protein of M . smegmatis, which assembles to an extremely stable oligomer with high channel-forming activity . Database searches did not reveal significant similarities to any other known protein . Southern blots showed that the chromosomes of fast-growing mycobacterial species contain homologous sequences to mspA, whereas no hybridization could be detected with DNA from slow growing mycobacteria . These results suggest that MspA is the prototype of a new class of channel-forming proteins.

Anesth Analg, 1999 Sep, 89(3), 665 - 9
Ketamine suppresses proinflammatory cytokine production in human whole blood in vitro; Kawasaki T et al.; The production of proinflammatory cytokines, such as tumor necrosis factor (TNF) a, interleukin (IL)-6, and IL-8, increases in patients with sepsis; marked production causes organ failure and septic shock . We previously reported that ketamine suppressed lipopolysaccharide (LPS)-induced TNF-alpha production in mice . However, there are no reports on the effect of ketamine on cytokine production in human whole blood . Therefore, in this study, we investigated the efficacy of ketamine on LPS-induced TNF-alpha, IL-6, and IL-8 production and recombinant human (rh) TNF-a-induced IL-6 and IL-8 production in human whole blood . After adding different doses of ketamine to whole blood, the blood was stimulated with LPS or rhTNF . After incubation, the plasma TNF-alpha activity and IL-6 and IL-8 concentrations were measured using the L929 cell cytotoxic assay or an enzyme-linked immunoassay . Ketamine significantly suppressed LPS-induced TNF-alpha production at concentrations >20 microg/mL . At concentrations >100 microg/mL, ketamine also significantly suppressed both LPS-induced and rhTNF-induced IL-6 and IL-8 production . In this study, we demonstrated that ketamine directly inhibits the production of proinflammatory cytokines such as TNF-alpha, IL-6, and IL-8 in human whole blood . IMPLICATIONS: We found that ketamine suppressed lipopolysaccharide-induced tumor necrosis factor alpha, interleukin (IL)-6, and IL-8 production and recombinant human tumor necrosis factor-induced IL-6 and IL-8 production in human whole blood . Ketamine directly suppresses proinflammatory cytokine production.

Clin Chem Lab Med, 1999 Jun, 37(6), 631 - 7
Characterization of monoclonal antibodies to human protein 1/Clara cell 10 kilodalton protein; Yamaguchi T et al.; Human protein 1/Clara cell Mr 10,000 protein consists of two identical subunits of seventy amino acid residues each . In the present study, eight clones of monoclonal antibodies against native protein 1 were prepared and their respective epitopes were immunochemically and immunohistochemically characterized using native protein 1, truncated recombinant protein 1 and synthesized peptides . Among the clones, three designated as TY-5, TY-7 and TY-8 recognized amino acid residues 7-16, residues 19-28, and residues 39-46, respectively, all of which comprise the hydrophobic cavity of protein 1, possibly associated with chemical binding function . With the exception of TY-4, the remaining clones recognized residues 61-68 which are exposed to solvent . The epitope of TY-4 remains undetermined . Proper selection and combination of clones and recombinant protein 1 may be useful for fundamental and clinical studies of protein 1.

Yakugaku Zasshi, 1999 Aug, 119(8), 612 - 23
{Molecular identification of cytokinin-specific binding protein}; Fujimoto Y et al.; Synthetic phenylurea derivatives such as N-phenyl-N'-(4-pyridyl)urea (4PU) and N-(2-chloro-4-pyridyl)-N'-phenylurea (4PU30) have strong cytokinin activities . Using tritiated 4PU30 as a probe, we found the presence of a cytokinin-specific binding protein (CSBP) with high affinity for 4PU30 (Ka for 4PU30 = 4 x 10(10) M-1) in the soluble fraction of etiolated mung bean seedlings . We purified CSBP by the use of 4PU-Sepharose 4B, an affinity gel ligated with 4PU . Analysis of its cDNA revealed that CSBP was a novel member of a major pollen allergen/pathogenesis-related protein family with a calculated molecular weight of 17 kDa . Recombinant CSBP was expressed in Escherichia coli was confirmed to bind specifically to cytokinins.

Mutagenesis, 1999 Jan, 14(1), 129 - 34
Effect of Tn10/Tn5 transposons on the survival and mutation frequency of halogen light-irradiated AB1157 Escherichia coli K-12; Wojcik A et al.; We show here that the Tn10/Tn5 transposon when inserted into the chromosome of strain AB1157 makes the bacteria more sensitive to and less mutable by halogen light irradiation . These effects are most probably caused by depletion of UmuD and UmuC proteins since: (i) transformation of the transposon-bearing bacteria with plasmids harbouring umuD'C (or umuDC) leads to recovery of the original survival and mutation frequencies; (ii) insertion of Tn10/Tn5 into chromosomal DNA has no effect on the level of mutation induced by ethyl methane-sulphonate treatment, a mutagen whose activity is umuDC-independent; (iii) the decline in survival is in about the same range for Tn10-bearing bacteria as for bacteria with deleted umuDC . However, whereas transformation of bacteria deleted in umuDC with plasmids carrying umuD'C/umuDC leads to full recovery of halogen light-induced mutability, recovery of survival is poor . This suggests that the mechanisms leading to umuDC-dependent mutagenesis and umuDC-dependent protection of cell survival are different . None of these effects occurs in bacteria bearing the Tn9 transposon in their DNA.

Mutagenesis, 1999 Jan, 14(1), 95 - 102
Hydrogen peroxide and coffee induce G:C-->T:A transversions in the lacI gene of catalase-defective Escherichia coli; Ruiz-Laguna J et al.; The mutagenicity of hydrogen peroxide (H2O2) was compared with that of coffee, a complex mixture which generates H2O2 . An Escherichia coli strain defective in catalase activity (katG katE double mutant) and carrying a single copy mucAB (pRW144) plasmid was constructed to enhance the mutagenic response to oxidants . The ability of the mucAB genes to influence the type, frequency and distribution of H2O2-induced mutations was also investigated in isogenic bacteria lacking pRW144 . Induced mutational spectra were characterized and compared with that of spontaneous mutagenesis . A total of 444 independent forward mutations affecting the first 210 bp of the lacI gene were identified by DNA sequence analysis . The spontaneous mutation spectrum showed no bias (P = 0.52) for substitutions at G:C base pairs . In contrast, in the H2O2-induced spectrum substitutions occurred preferentially at G:C base pairs (P < 0.0001) with a preponderance of G:C-->T:A transversions (43.4% of H2O2-induced mutants versus 17.3% of spontaneous mutants) . These data support the view that 7,8-dihydro-8-oxoguanine is the main premutagenic lesion induced by H2O2 and that catalase-defective bacteria have elevated levels of 8-oxoguanine in chromosome DNA after H2O2 exposure . Coffee produced a similar distribution of mutational events as H2O2 (P > 0.05), suggesting that this compound may be the main cause of the coffee-induced mutagenesis . The presence of plasmid pRW144 did not affect the frequency of H2O2-induced G:C-->T:A transversions, but caused an increase in A:T-->T:A transversions and a decrease in -1 base frameshifts . Although the frequencies of G:C-->T:A transversions were similar in all three induced spectra (H2O2 and coffee +/- pRW144), differences were observed in location of mutations throughout the target gene.

Nucleosides Nucleotides, 1999 Jun-Jul, 18(6-7), 1575 - 6
Thermodynamics of site-specific variant tRNA(Ala) acceptor stem microhairpins; Biala E et al.; Thermal denaturation studies were carried out on a set of site-specific variants of a 22mer RNA hairpin comprising the aminoacyl acceptor stem sequence of E . coli tRNA(Ala) . The pairing thermodynamics were calculated from the melting profiles.

FEMS Microbiol Lett, 1999 Aug 15, 177(2), 327 - 34
Heat-inactivation of plasmid-encoded CI857 repressor induces gene expression from Ind- lambda prophage in recombinant Escherichia coli; Hoffmann F et al.; We have observed significant cell lysis upon temperature up-shift of recombinant Escherichia coli cultures harboring CI857-repressed lambda-based expression vectors . This event, that becomes evident about 30-40 min after the heat shock, takes place when using the lambda promoter system in Ind- lysogenic strains, but not in others commonly employed for recombinant gene expression . These results strongly suggest that the thermosensitive CI857 repressor, encoded by the expression vector, competes with CI Ind- molecules for binding to the prophage operator region, allowing for expression of lytic genes from the integrated Ind- viral genome upon temperature up-shift . Transcription of viral lytic genes does not include unspecific expression of a reporter sulA::lacZ gene fusion carried in the prophage genome . These results prompt, however, to carefully evaluate the limitations of expression systems based on pL/pR-CI857 in bacterial strains modified through lambda Ind- gene transfer vehicles.

FEMS Microbiol Lett, 1999 Aug 15, 177(2), 271 - 7
Identification of genetic factors altering the SOS induction of DNA damage-inducible yebG gene in Escherichia coli; Oh TJ et al.; The yebG gene of Escherichia coli is a novel SOS regulon gene, but details of its regulation mechanism and biological function are not yet known . To characterize the regulation of yebG gene as a SOS gene, we identified the genetic factors affecting the SOS induction of yebG gene using yebG-lacZ operon fusion plasmid . We found that the SOS induction of yebG occurs as the cells enter into the stationary growth phase, but its induction is not observed in LB medium in the presence of 1% glucose . A stationary phase SOS induction of the yebG gene does not require the global regulator of stationary phase-specific genes, rpoS, or gyrA functions, but requires cya encoding the adenylate cyclase and hns encoding the histone-like protein H-NS functions . Our results demonstrated that the induction of a DNA damage-inducible yebG gene of E . coli is dependent on cyclic AMP and H-NS.

FEMS Microbiol Lett, 1999 Aug 15, 177(2), 191 - 7
The Tol proteins of Escherichia coli and their involvement in the uptake of biomolecules and outer membrane stability; Lazzaroni JC et al.; The Tol proteins of Escherichia coli are involved in outer membrane stability . They are also required for the uptake of the group A colicins and the translocation of filamentous phage DNA into the cytoplasm . The tol-pal genes constitute two operons in the E . coli genome, orfltolQRA and tolBpalorf2 . The TolQ TolR TolA proteins form a complex in the cytoplasmic membrane, while TolB and Pal interact near the outer membrane . Most of the amino acid residues of TolA, TolB, TolR and Pal are localized in the periplasm . Recent advances in the knowledge of interactions of Tol-Pal proteins with other envelope components, or with group A colicins, are presented, together with current hypotheses about the role of the Tol proteins in outer membrane stability.

Eur J Endocrinol, 1999 Sep, 141(3), 272 - 8
T-cell mediated autoimmunity to the insulinoma-associated protein 2 islet tyrosine phosphatase in type 1 diabetes mellitus; Dotta F et al.; The target molecules of the T-cell response in type 1 diabetes, despite their pathogenic importance, remain largely uncharacterized, especially in humans . Interestingly, molecules such as insulin and glutamic acid decarboxylase (GAD) have been shown to be a target not only of autoantibodies, but also of autoreactive T-lymphocytes both in man and in the non-obese diabetic (NOD) mouse . In the present study we aimed to determine the existence of a specific T-cell response towards the insulinoma-associated protein 2 (IA-2) islet tyrosine phosphatase, a recently identified autoantigen which is the target of autoantibodies strongly associated with diabetes development . Human recombinant IA-2 produced in Escherichia coli, was tested for its reactivity with peripheral blood lymphocytes obtained from 16 newly diagnosed type 1 diabetic patients and from 25 normal controls, 15 of whom were HLA-DR-matched . A T-cell proliferation assay was performed in triplicate employing freshly isolated cells in the absence or in the presence of the antigen to be tested (at two different concentrations: 2 microg/ml and 10 microg/ml) . A specific T-cell proliferation (defined as a stimulation index (S.I.) >/=3) was observed against IA-2 used at a concentration of 10 microg/ml (but not of 2 microg/ml) in 8/16 diabetic patients, in 1/15 HLA-DR-matched control subjects (P<0.01 by Fisher exact test) and in 0/10 of the remaining normal individuals . A statistically significant difference (P<0.003 by Mann-Whitney U test) was also observed in S.I . values between patients (3.1+/-1.4) and HLA-DR-matched controls (1.7+/-0.54) employing IA-2 at a concentration of 10 microg/ml . However, when IA-2 was used at a concentration of 2 microg/ml, the difference in S . I . between patients (1.65+/-0.8) and controls (1.0+/-0.3) did not reach statistical significance . In conclusion, these data show the presence of a specific, dose-dependent T-lymphocyte response against the IA-2 islet tyrosine phosphatase at the onset of type 1 diabetes . Consequently, this molecule appears to be a target not only at the B-lymphocyte but also at the T-lymphocyte level, reinforcing the potential pathogenic role of this autoantigen in the islet destructive process.

Int Arch Allergy Immunol, 1999 Aug, 119(4), 275 - 81
Identification and characterisation of two allergens from the dust mite Acarus siro, homologous with fatty acid-binding proteins; Eriksson TL et al.; BACKGROUND: Dust mites are a major cause of allergic disease worldwide . The dust mite Acarus siro is an inducer of occupational allergy among farmers, but sensitisation has also been found in non-farming populations . METHODS: A degenerate primer was designed to the N-terminal amino acid sequence of a 15-kD IgE-binding protein in A . siro extract . The cDNA sequence was obtained by using reverse transcriptase polymerase chain reaction, standard cloning and sequencing techniques . The protein was expressed in Escherichia coli with a 6-histidine tag at its C-terminus . Immunoblotting of the recombinant protein and whole extract was performed using patient sera . RESULTS AND CONCLUSION: 15 and 17-kD allergens were identified in a fraction of A . siro extract . The cDNA of the 15-kD allergen was isolated, cloned and sequenced and the allergen was expressed as a recombinant protein . The calculated molecular weight of the cDNA-encoded protein is 14.2 kD . The predicted amino acid sequence has one potential N-glycosylation site at position 4-6 and a cytosolic fatty acid-binding protein signature at position 5-22 . The protein has 64% sequence identity with Blo t 13, an allergen from the dust mite Blomia tropicalis, as well as homology with several other fatty acid-binding proteins (FABPs) from different organisms . The allergen was named Aca s 13 and was recognised strongly by 3 of 13 (23%) of the subjects investigated . The amino acid sequence of the 17-kD protein was partly determined and it also showed high sequence homology with Blo t 13 and FABPs.

J Biol Chem, 1999 Sep 10, 274(37), 26185 - 91
Enzyme I(Ntr) from Escherichia coli . A novel enzyme of the phosphoenolpyruvate-dependent phosphotransferase system exhibiting strict specificity for its phosphoryl acceptor, NPr; Rabus R et al.; The phosphoenolpyruvate (PEP)-dependent phosphotransferase system (PTS) phosphorylates sugars and regulates cellular metabolic processes using a phosphoryl transfer chain including the general energy coupling proteins, Enzyme I (EI) and HPr as well as the sugar-specific Enzyme II complexes . Analysis of the Escherichia coli genome has revealed the presence of 5 paralogues of EI and 5 paralogues of HPr, most of unknown function . The ptsP gene encodes an EI paralogue designated Enzyme I(nitrogen) (EI(Ntr)), and two genes located in the rpoN operon encode PTS protein paralogues, NPr and IIA(Ntr), both implicated in the regulation of sigma(54) activity . The ptsP gene was polymerase chain reaction amplified from the E . coli chromosome and cloned into an overexpression vector allowing the overproduction and purification of EI(Ntr) . EI(Ntr) was shown to phosphorylate NPr in vitro using either a {(32)P}PEP-dependent protein phosphorylation assay or a quantitative sugar phosphorylation assay . EI(Ntr) phosphorylated NPr but not HPr, whereas Enzyme I exhibited a strong preference for HPr . These two pairs of proteins (EI(Ntr)/NPr and EI/HPr) thus exhibit little cross-reactivity . Phosphoryl transfer from PEP to NPr catalyzed by EI(Ntr) has a pH optimum of 8.0, is dependent on Mg(2+), is stimulated by high ionic strength, and exhibits two K(m) values for NPr (2 and 10 microM) possibly because of negative cooperativity . The results suggest that E . coli possesses at least two distinct PTS phosphoryl transfer chains, EI(Ntr) --> NPr --> IIA(Ntr) and EI --> HPr --> IIA(sugar) . Sequence comparisons allow prediction of residues likely to be important for specificity . This is the first report demonstrating specificity at the level of the energy coupling proteins of the PTS.

Clin Diagn Lab Immunol, 1999 Sep, 6(5), 745 - 50
Cloning and expression of an immunogenic membrane-associated protein of Helicobacter hepaticus for use in an enzyme-linked immunosorbent assay; Livingston RS et al.; Helicobacter hepaticus is a bacterial pathogen that causes chronic active hepatitis and inflammatory bowel disease in mice . The purpose of this study was to develop a recombinant antigen-based enzyme-linked immunosorbent assay (ELISA) to detect H . hepaticus-infected mice . A genomic library of H . hepaticus was constructed and was screened with sera from H . hepaticus-infected mice . A 459-bp open reading frame that coded for an 18-kDa immunoreactive protein, MAP18, was identified . The gene had high identity with genes coding for outer membrane proteins of other bacteria, and the predicted amino acid sequence of MAP18 had a putative membrane-trafficking signal sequence and a putative signal peptidase II cleavage site . The recombinant protein was expressed in Escherichia coli as a glutathione S-transferase (GST) fusion protein, GST-MAP18, and purified by affinity chromatography . The 44-kDa fusion protein was detected on Western blots probed with sera from H . hepaticus-infected mice but was not detected on blots probed with sera from mice infected with Helicobacter muridarum or Helicobacter bilis or with sera from mice free of Helicobacter infection . The GST-MAP18 fusion protein was used as an antigen in an ELISA to detect anti-H . hepaticus antibodies in sera from infected mice . This ELISA was compared to an H . hepaticus-specific ELISA that uses a detergent extract of H . hepaticus as the antigen . Sera from mice naturally and experimentally infected with H . hepaticus, H . bilis, or H . muridarum and sera from mice free of Helicobacter infection were evaluated . Both ELISAs performed with a high specificity (98%); however, the detergent extract-based ELISA performed with a higher sensitivity (89%) than the recombinant protein-based ELISA (sensitivity, 66%) . These data indicate that H . hepaticus carries a gene that encodes an immunogenic 18-kDa membrane-associated protein; however, antibodies to this protein are not detected in all infected mice.

J Biol Chem, 1999 Sep 10, 274(37), 26572 - 8
Ribonuclease III processing of coaxially stacked RNA helices; Franch T et al.; The RNase III family of endoribonucleases participates in maturation and decay of cellular and viral transcripts by processing of double-stranded RNA . RNase III degradation is inherent to most antisense RNA-regulated gene systems in Escherichia coli . In the hok/sok system from plasmid R1, Sok antisense RNA targets the hok mRNA for RNase III-mediated degradation . An intermediate in the pairing reaction between Sok RNA and hok mRNA forms a three-way junction . A complex between a chimeric antisense RNA and hok mRNA that mimics the three-way junction was cleaved by RNase III both in vivo and in vitro . Footprinting using E117A RNase III binding to partially complementary RNAs showed protection of the 13 base pairs of interstrand duplex and of the bottom part of the transcriptional terminator hairpin of the antisense RNA . This suggests that the 13 base pairs of RNA duplex are coaxially stacked on the antisense RNA terminator stem-loop and that each stem forms a monomer half-site, allowing symmetrical binding of the RNase III dimer . This processing scheme shows an unanticipated diversity in RNase III substrates and may have a more general implication for RNA metabolism.

J Biol Chem, 1999 Sep 10, 274(37), 26272 - 8
Adherence of Borrelia burgdorferi . Identification of critical lysine residues in DbpA required for decorin binding; Brown EL et al.; Borrelia burgdorferi, the causative agent of Lyme disease, expresses on its surface two decorin binding adhesins, DbpA and DbpB . Previous studies have demonstrated that vaccination of mice with DbpA provided protection against challenge with heterologous Borrelia strains despite considerable sequence variability among DbpA in these strains . We have now examined the importance of individual amino acid residues in DbpA for decorin binding . We demonstrated that chemical modification of lysine residues resulted in loss of ligand binding activity . Of the 27 lysine residues in native DbpA from strain 297, 6 are present in most and 5 are conserved in all 30 DbpA sequences examined so far . Analysis of recombinant DbpA in which individual lysine residues have been mutated to alanine suggested that three of the conserved residues distributed throughout the DbpA sequence are required for decorin binding . These mutants lost their ability to bind decorin in Western ligand blot assay and bound reduced amounts of decorin in an ELISA . Furthermore, these mutant DbpA proteins did not inhibit the adherence of B . burgdorferi to a decorin substrata, and they did not recognize decorin in an extracellular matrix established by human fibroblast cultures . We conclude that the three lysine residues Lys-82, Lys-163, and Lys-170 are crucial for the binding of DbpA to decorin.

J Biol Chem, 1999 Sep 10, 274(37), 26225 - 32
Dissecting the role of a conserved motif (the second region of homology) in the AAA family of ATPases . Site-directed mutagenesis of the ATP-dependent protease FtsH; Karata K et al.; Escherichia coli FtsH is an ATP-dependent protease that belongs to the AAA protein family . The second region of homology (SRH) is a highly conserved motif among AAA family members and distinguishes these proteins in part from the wider family of Walker-type ATPases . Despite its conservation across the AAA family of proteins, very little is known concerning the function of the SRH . To address this question, we introduced point mutations systematically into the SRH of FtsH and studied the activities of the mutant proteins . Highly conserved amino acid residues within the SRH were found to be critical for the function of FtsH, with mutations at these positions leading to decreased or abolished ATPase activity . The effects of the mutations on the protease activity of FtsH correlated strikingly with their effects on the ATPase activity . The ATPase-deficient SRH mutants underwent an ATP-induced conformational change similar to wild type FtsH, suggesting an important role for the SRH in ATP hydrolysis but not ATP binding . Analysis of the data in the light of the crystal structure of the hexamerization domain of N-ethylmaleimide-sensitive fusion protein suggests a plausible mechanism of ATP hydrolysis by the AAA ATPases, which invokes an intermolecular catalytic role for the SRH.

J Biol Chem, 1999 Sep 10, 274(37), 26157 - 64
An Escherichia coli mutant quinol:fumarate reductase contains an EPR-detectable semiquinone stabilized at the proximal quinone-binding site; Hagerhall C et al.; The EPR and thermodynamic properties of semiquinone (SQ) species stabilized by mammalian succinate:quinone reductase (SQR) in situ in the mitochondrial membrane and in the isolated enzyme have been well documented . The equivalent semiquinones in bacterial membranes have not yet been characterized, either in SQR or quinol:fumarate reductase (QFR) in situ . In this work, we describe an EPR-detectable QFR semiquinone using Escherichia coli mutant QFR (FrdC E29L) and the wild-type enzyme . The SQ exhibits a g = 2.005 signal with a peak-to-peak line width of approximately 1.1 milliteslas at 150 K, has a midpoint potential (E(m(pH 7.2))) of -56.6 mV, and has a stability constant of approximately 1.2 x 10(-2) at pH 7.2 . It shows extremely fast spin relaxation behavior with a P(1/2) value of >>500 milliwatts at 150 K, which closely resembles the previously described SQ species (SQ(s)) in mitochondrial SQR . This SQ species seems to be present also in wild-type QFR, but its stability constant is much lower, and its signal intensity is near the EPR detection limit around neutral pH . In contrast to mammalian SQR, the membrane anchor of E . coli QFR lacks heme; thus, this prosthetic group can be excluded as a spin relaxation enhancer . The trinuclear iron-sulfur cluster FR3 in the {3Fe-4S}(1+) state is suggested as the dominant spin relaxation enhancer of the SQ(FR) spins in this enzyme . E . coli QFR activity and the fast relaxing SQ species observed in the mutant enzyme are sensitive to the inhibitor 2-n-heptyl-4-hydroxyquinoline N-oxide (HQNO) . In wild-type E . coli QFR, HQNO causes EPR spectral line shape perturbations of the iron-sulfur cluster FR3 . Similar spectral line shape changes of FR3 are caused by the FrdC E29L mutation, without addition of HQNO . This indicates that the SQ and the inhibitor-binding sites are located in close proximity to the trinuclear iron-sulfur cluster FR3 . The data further suggest that this site corresponds to the proximal quinone-binding site in E . coli QFR.

J Biol Chem, 1999 Sep 10, 274(37), 26065 - 70
Energetics and topology of CzcA, a cation/proton antiporter of the resistance-nodulation-cell division protein family; Goldberg M et al.; The membrane-bound CzcA protein, a member of the resistance-nodulation-cell division (RND) permease superfamily, is part of the CzcCB(2)A complex that mediates heavy metal resistance in Ralstonia sp . CH34 by an active cation efflux mechanism driven by cation/proton antiport . CzcA was purified to homogeneity after expression in Escherichia coli, reconstituted into proteoliposomes, and the kinetics of heavy metal transport by CzcA was determined . CzcA is composed of 12 transmembrane alpha-helices and two large periplasmic domains . Two conserved aspartate and a glutamate residue in one of these transmembrane spans are essential for heavy metal resistance and proton/cation antiport but not for facilitated diffusion of cations . Generalization of the resulting model for the function of CzcA as a two-channel pump might help to explain the functions of other RND proteins in bacteria and eukaryotes.

J Biol Chem, 1999 Sep 10, 274(37), 26027 - 32
Oxidative stress defense and deterioration of growth-arrested Escherichia coli cells; Dukan S et al.; Analysis of protein carbonylation demonstrates that the stasis-induced catalases and cytoplasmic superoxide dismutases (SOD) have a role in preventing accelerated protein oxidation during growth arrest of Escherichia coli cells . A larger number of proteins are carbonylated in cells lacking cytoplasmic SOD compared with cells lacking catalases, OxyR, or RpoS which, in turn, exhibit a larger number of oxidized proteins than the wild-type parent . Proteins exclusively oxidized during stasis in mutants lacking cytoplasmic SOD include GroEL, EF-G, and the acidic isoform of H-NS indicating that these mutants experience problems in peptide elongation and maintaining protein and DNA architecture . These mutants also survive stasis poorly . Likewise, but to a much lesser extent, mutations in oxyR, an oxidative stress regulator, shorten the life-span of stationary phase cells . The low plating efficiency of cells lacking OxyR is the result of their inability to grow on standard culture plates unless plating is performed anaerobically or with high concentration of catalase . In contrast, cells lacking cytoplasmic SOD appear to die prior to plating . Our data points to the importance of oxidative stress defense in stasis survival, and we also demonstrate that the life-span of growth-arrested wild-type E . coli cells can be significantly extended by omitting oxygen.

J Biol Chem, 1999 Sep 10, 274(37), 26008 - 14
An archaebacterial ATPase, homologous to ATPases in the eukaryotic 26 S proteasome, activates protein breakdown by 20 S proteasomes; Zwickl P et al.; In eukaryotes, the 20 S proteasome is the proteolytic core of the 26 S proteasome, which degrades ubiquitinated proteins in an ATP-dependent process . Archaebacteria lack ubiquitin and 26 S proteasomes but do contain 20 S proteasomes . Many archaebacteria, such as Methanococcus jannaschii, also contain a gene (S4) that is highly homologous to the six ATPases in the 19 S (PA700) component of the eukaryotic 26 S proteasome . To test if this putative ATPase may regulate proteasome function, we expressed it in Escherichia coli and purified the 50-kDa product as a 650-kDa complex with ATPase activity . When mixed with the well characterized 20 S proteasomes from Thermoplasma acidophilum and ATP, this complex stimulated degradation of several unfolded proteins 8-25-fold . It also stimulated proteolysis by 20 S proteasomes from another archaebacterium and mammals . This effect required ATP hydrolysis since ADP and the nonhydrolyzable analog, 5'-adenylyl beta, gamma-imidophosphate, were ineffective . CTP and to a lesser extent GTP and UTP were also hydrolyzed and also stimulated proteolysis . We therefore named this complex PAN for proteasome-activating nucleotidase . However, PAN did not promote the degradation of small peptides, which, unlike proteins, should readily diffuse into the proteasome . This ATPase complex appears to have been the evolutionary precursor of the eukaryotic 19 S complex, before the coupling of proteasome function to ubiquitination.

J Biol Chem, 1999 Sep 10, 274(37), 26003 - 7
Transport function and regulation of mitochondrial uncoupling proteins 2 and 3; Jaburek M et al.; Uncoupling protein 1 (UCP1) dissipates energy and generates heat by catalyzing back-flux of protons into the mitochondrial matrix, probably by a fatty acid cycling mechanism . If the newly discovered UCP2 and UCP3 function similarly, they will enhance peripheral energy expenditure and are potential molecular targets for the treatment of obesity . We expressed UCP2 and UCP3 in Escherichia coli and reconstituted the detergent-extracted proteins into liposomes . Ion flux studies show that purified UCP2 and UCP3 behave identically to UCP1 . They catalyze electrophoretic flux of protons and alkylsulfonates, and proton flux exhibits an obligatory requirement for fatty acids . Proton flux is inhibited by purine nucleotides but with much lower affinity than observed with UCP1 . These findings are consistent with the hypothesis that UCP2 and UCP3 behave as uncoupling proteins in the cell.

J Biol Chem, 1999 Sep 10, 274(37), 25979 - 82
Differential rates of NTP hydrolysis by the mutant {S69G}RecA protein . Evidence for a coupling of NTP turnover to DNA strand exchange; Nayak S et al.; The x-ray crystal structure of the Escherichia coli RecA protein indicates that the phosphate groups of the nucleotide cofactor are bound by a loop whose amino acid sequence ((66)GPESSGKT(73)) corresponds to a consensus phosphate binding loop sequence (GXXXXGK{T/S}) found in many NTP-binding proteins . As part of an investigation of the role of the P-loop in ATP hydrolysis, we prepared a mutant RecA protein in which serine 69 was replaced by a glycine residue . We have found that the {S69G}RecA mutation has a differential effect on the hydrolysis of various nucleoside triphosphates . The {S69G}RecA protein catalyzes the single-stranded DNA-dependent hydrolysis of rATP, ddATP, and dATP with turnover numbers of 10, 20, and 36 min(-1), respectively . The wild type RecA protein, in contrast, hydrolyzes each of these nucleoside triphosphates with similar turnover numbers of 20-24 min(-1) . Significantly, the {S69G}RecA protein promotes strand exchange with all three nucleoside triphosphates, and the rate of strand exchange is directly proportional to the rate of hydrolysis of each of the nucleotide cofactors . These findings with the {S69G}RecA protein provide support for the existence of a mechanistic coupling between NTP hydrolysis and DNA strand exchange.

J Biol Chem, 1999 Sep 10, 274(37), 25975 - 8
Abasic sites induce triplet-repeat expansion during DNA replication in vitro; Lyons-Darden T et al.; The occurrence of triplet-repeat expansion (TRE) during transmission of genetic information is involved in many neurological and neuromuscular diseases including Fragile X syndrome and myotonic dystrophy . DNA slippage during replicative synthesis appears to cause TRE . The causes of DNA slippage, however, remain mostly unknown . We investigated the effects of abasic sites on the occurrence of TRE during DNA replication in vitro using Escherichia coli Klenow polymerase I as the model polymerase . Here we show that a single abasic site analog, synthesized in the triplet-repeat tract at the 5' end of the template strand, induced dramatic TRE during DNA synthesis . The amount of TRE induced decreased when the abasic site was moved to the middle of the repeat tract, consistent with effectively decreasing the length of the repeat tract . Placing the abasic site in the primer did not induce TRE . TRE was sequence-dependent . The damage-induced increase in growing strand TRE depended on the sequence of the growing strand repeat as AAT approximately ATT > CAG > CTG . The expansions required replication from a primer complementary to the repeat tract . The expanded tracts were sequenced and contained multiple additions of the original repeat . The results imply that DNA damage can play a significant role in generating TRE in vivo.

Clin Diagn Lab Immunol, 1999 Sep, 6(5), 734 - 40
A new sensitive serological assay for detection of lentivirus infections in small ruminants; Saman E et al.; Lentivirus infections in small ruminants represent an economic problem affecting several European countries with important sheep-breeding industries . Programs for control and eradication of these infections are being initiated and require reliable screening assays . This communication describes the construction and evaluation of a new serological screening enzyme-linked immunosorbent assay (ELISA) for the detection of antibodies to maedi-visna virus (MVV) in sheep and to caprine arthritis encephalitis virus (CAEV) in goats . The solid phase is sensitized with a combination of the major core protein p25 of MVV produced in Escherichia coli and a peptide derived from the immunodominant region of the viral transmembrane protein gp46 . The peptide carries an N-terminal biotin residue and is complexed with streptavidin prior to being coated . The new assay was evaluated with 2,336 sheep serum samples from different European countries with large differences in the levels of prevalence of MVV infections, and the results have been compared to those of the standard agar gel immunodiffusion test . Discrepant samples were analyzed by Western blotting with viral lysate, and most sera could be classified unambiguously . The estimated overall sensitivity of the new ELISA was 99.4% (95% confidence interval {CI}, 98.4 to 99 . 8%) and the specificity was 99.3% (95% CI, 98.7 to 99.6%) . A limited set of goat sera (n = 212) was also analyzed, with similar results . These data indicate that the new assay is a reliable tool that can be used in control and eradication programs for small ruminant lentivirus infections.

Clin Diagn Lab Immunol, 1999 Sep, 6(5), 701 - 4
A reverse-sandwich enzyme-linked immunosorbent assay for verocytotoxin 1 and 2 antibodies in human and bovine sera; Miyazawa H et al.; A reverse-sandwich enzyme-linked immunosorbent assay (ELISA), in which an antibody is sandwiched by antigens, was established for the titration of antibodies to verocytotoxins (VT) in human and animal sera . This assay has two advantages over a conventional indirect ELISA: (i) higher specificity and sensitivity and (ii) the ability to comparably titrate antibodies from different species . The VT1 (Shiga-like toxin 1) antibody-positive rates were 5% in 202 normal adult humans and 99% in 93 normal cattle at a dairy farm . This ELISA is most suitable for seroepidemiologic studies of infections with VT-producing Escherichia coli in humans and various animal species.

Appl Environ Microbiol, 1999 Sep, 65(9), 3955 - 63
Cloning of the gene encoding a novel thermostable alpha-galactosidase from Thermus brockianus ITI360; Fridjonsson O et al.; An alpha-galactosidase gene from Thermus brockianus ITI360 was cloned, sequenced, and expressed in Escherichia coli, and the recombinant protein was purified . The gene, designated agaT, codes for a 476-residue polypeptide with a calculated molecular mass of 53, 810 Da . The native structure of the recombinant enzyme (AgaT) was estimated to be a tetramer . AgaT displays amino acid sequence similarity to the alpha-galactosidases of Thermotoga neapolitana and Thermotoga maritima and a low-level sequence similarity to alpha-galactosidases of family 36 in the classification of glycosyl hydrolases . The enzyme is thermostable, with a temperature optimum of activity at 93 degrees C with para-nitrophenyl-alpha-galactopyranoside as a substrate . Half-lives of inactivation at 92 and 80 degrees C are 100 min and 17 h, respectively . The pH optimum is between 5.5 and 6.5 . The enzyme displayed high affinity for oligomeric substrates . The K(m)s for melibiose and raffinose at 80 degrees C were determined as 4.1 and 11.0 mM, respectively . The alpha-galactosidase gene in T . brockianus ITI360 was inactivated by integrational mutagenesis . Consequently, no alpha-galactosidase activity was detectable in crude extracts of the mutant strain, and it was unable to use melibiose or raffinose as a single carbohydrate source.

Appl Environ Microbiol, 1999 Sep, 65(9), 3901 - 7
Construction of environmental DNA libraries in Escherichia coli and screening for the presence of genes conferring utilization of 4-hydroxybutyrate; Henne A et al.; Environmental DNA libraries from three different soil samples were constructed . The average insert size was 5 to 8 kb and the percentage of plasmids with inserts was approximately 80% . The recombinant Escherichia coli strains (approximately 930,000) were screened for 4-hydroxybutyrate utilization . Thirty-six positive E . coli clones were obtained during the initial screen, and five of them contained a recombinant plasmid (pAH1 to pAH5) which conferred a stable 4-hydroxybutyrate-positive phenotype . These E . coli clones were studied further . All five were able to grow with 4-hydroxybutyrate as sole carbon and energy source and exhibited 4-hydroxybutyrate dehydrogenase activity in crude extracts . Sequencing of pAH5 revealed a gene homologous to the gbd gene of Ralstonia eutropha, which encodes a 4-hydroxybutyrate dehydrogenase . Two other genes (orf1 and orf6) conferring utilization of 4-hydroxybutyrate were identified during subcloning and sequencing of the inserts of pAH1 and pAH3 . The deduced orf1 gene product showed similarities to members of the DedA family of proteins . The sequence of the deduced orf6 gene product harbors the fingerprint pattern of enoyl-coenzyme A hydratases/isomerases . The other sequenced inserts of the plasmids recovered from the positive clones revealed no significant similarity to any other gene or gene product whose sequence is available in the National Center for Biotechnology Information databases.

J Ethnopharmacol, 1999 Sep, 66(3), 301 - 9
Apoptosis-associated generation of reactive oxygen intermediates and release of pro-inflammatory cytokines in human lymphocytes and granulocytes by extracts from the seeds of Acalypha wilkesiana; Bussing A et al.; Seeds from Acalypha wilkesiana (Euphorbiaceae) are an essential component of a complex plant mixture used empirically by traditional healers in Southwest Nigeria to treat breast tumours and inflammation . To investigate their biological properties, we incubated human lymphocytes and granulocytes with aqueous and ethanolic extracts of A . wilkesiana seeds (AWS) . In lymphocytes, we observed an induction of apoptosis and generation of reactive oxygen intermediates (ROI), as measured by the oxidation of hydroethidine, within 2 h, while in granulocytes, an aqueous seed extract induced the oxidative burst and enhanced phagocytosis of Escherichia coli within 10-20 min . In the supernatants of 72-h cultured lymphocytes, AWS induced the release of the pro-inflammatory cytokines tumour necrosis factor-alpha and interleukin-6, and also T-cell-associated cytokines interleukin-5 and interferon-gamma . These preliminary results encourage further investigations of this drug with both cytotoxic and immunomodulating properties.

Clin Cancer Res, 1999 Aug, 5(8), 2178 - 84
Chlorambucil induction of HsRad51 in B-cell chronic lymphocytic leukemia; Christodoulopoulos G et al.; Our previous studies with B-cell chronic lymphocytic leukemia (B-CLL) have suggested that one of the mechanisms of nitrogen mustard (NM) drug resistance is increased repair of drug-induced damage . We have postulated that recombination may play a crucial role in this process . The human homologue of Rad51, (HsRad51), has homology to the RecA protein in Escherichia coli, which is implicated in recombination repair and induction of DNA repair enzymes . In this report, we have examined the expression and distribution of HsRad51 protein in lymphocytes from patients with B-CLL to see whether the expression of HsRad51 is associated with NM damage to the malignant B lymphocytes, specifically chlorambucil (CLB), which is the standard alkylating agent used to treat patients with B-CLL . We have analyzed the intracellular distribution of HsRad51 protein in these lymphocytes before and after treatment with CLB by immunofluorescence . In vitro CLB treatment induces Rad51 expression, as measured by increased immunopositive staining in all CLL samples . In the CLB-resistant CLL lymphocytes, there was a linear correlation between induction of Rad51 protein at 5.4 microM CLB and the in vitro LD50 dose of CLB . Surprisingly, although it has been reported that Rad51 is induced in S phase and only 10% of cells from cell lines expressed positive immunostaining for Rad51, our CLL lymphocytes, which were not subjected to in vitro drug exposure, were 90% positive for Rad51, despite their nonproliferative state, which suggests that there is chronic activation of the protein . Our results suggest that CLB activates HsRad51-directed recombination repair and that this process may be important in NM drug-induced cytotoxicity.

Klin Padiatr, 1999 Jul-Aug, 211(4), 201 - 4
Bleeding and thrombosis in children with acute lymphoblastic leukaemia, treated according to the ALL-BFM-90 protocol; Sutor AH et al.; A multi-center retrospective survey was conducted to evaluate the incidence and types of hemostatic complications occurring in children with acute lymphoblastic leukemia (ALL) during treatment according to the ALL-BFM-90 treatment protocol . All of the BFM-treatment centers (n = 77) were approached and a 95% response rate with information on 1100 patients was obtained . Thrombotic or bleeding episodes occurred in 31 patients (2.8%), 19 of whom had thrombosis and 12 bleeding complications, involving the central nervous system (42%), the subclavian vein (29%), the gastro-intestinal tract, skin, lower extremities or pelvis (29%) . Recovery was noted in 28 of 31 patients, while 3 died as a result of hemostatic complications . Bleeding or thrombosis occurred in patients receiving prophylactic substitution with plasma or plasma-derived concentrates (n = 16) as well as in those without substitution (n = 13) . The majority of hemostatic complications (90%) occurred during the induction therapy of the treatment protocol, in particular during the period which included simultaneous administration of glucocorticoids and E . coli L-asparaginase . The concurrent administration of E . coli L-asparaginase and glucocorticoids may be an additional risk factor for thromboembolic events during therapy according to the ALL-BFM-90 protocol.

Biomed Pharmacother, 1999 Aug, 53(7), 323 - 8
Induction of cellular immunosuppression by the human papillomavirus type 16 E7 oncogenic protein; Le Buanec H et al.; The human papillomavirus type 16 (HPV-16) E7 oncogenic protein is found in the culture supernatant of SiHa cells, a cervical carcinoma cell line . Extracellular E7 protein, acting as a viral toxin in human immune cells, induces the overproduction of the immune suppressive IFN alpha cytokine by APCs, and inhibits the T-cell response to recall and allogenic antigens . These effects should be taken into account for the design of anti-human cervical carcinoma vaccines.

FEBS Lett, 1999 Sep 3, 457(3), 393 - 6
High level expression of Thermococcus litoralis 4-alpha-glucanotransferase in a soluble form in Escherichia coli with a novel expression system involving minor arginine tRNAs and GroELS; Imamura H et al.; The Thermococcus litoralis 4-alpha-glucanotransferase (GTase) gene has a high content of AGA and AGG codons for arginine, which are extremely rare in Escherichia coli . Expression of the GTase gene in E . coli resulted in low protein production and the accumulation of inclusion bodies . However, simultaneous expression of GTase with tRNA(AGA), tRNA(AGG) and GroELS affected both the production and solubility of GTase, and production of soluble GTase increasing about 5-fold . This new E . coli expression system should be applicable to the expression of not only archaeal but also eukaryotic genes, which usually contain a large number of AGA and AGG codons.

FEBS Lett, 1999 Sep 3, 457(3), 327 - 32
Comparison of DeltapH- and Delta***φ***-driven ATP synthesis catalyzed by the H(+)-ATPases from Escherichia coli or chloroplasts reconstituted into liposomes; Fischer S et al.; The H(+)-ATPases from Escherichia coli, EF(0)F(1), and from chloroplasts, CF(0)F(1), were reconstituted in liposomes from phosphatidylcholine/phosphatidic acid . The proteoliposomes were energized by an acid-base transition and a K(+)/valinomycin diffusion potential and the initial rate of ATP synthesis was measured as a function of the transmembrane pH difference, DeltapH, and the electric potential difference, Deltaφ . With EF(0)F(1), a rate of 80 s(-1) is observed at DeltapH=4.1 and Deltaφ approximately 140 mV . The rate decreases sigmoidally with Deltaφ and at Deltaφ approximately 0 mV, the rate is about 1 s(-1) although DeltapH is still 4.1 . Under the same conditions with CF(0)F(1), a rate of 280 s(-1) is observed which decreases to 190 s(-1) when Deltaφ is abolished, i.e . ATP synthesis catalyzed by EF(0)F(1) and CF(0)F(1) depends in a different way on DeltapH and Deltaφ . EF(0)F(1)-catalyzed ATP synthesis was measured as a function of DeltapH at a constant Deltaφ . The rate depends sigmoidally on DeltapH reaching a maximal rate which cannot be further increased by increasing DeltapH . However, this maximal rate depends on Deltaφ, i.e . DeltapH and Deltaφ are not kinetically equivalent in driving ATP synthesis . We assume that EF(0)F(1) must be converted into a metastable, active state before it catalyzes proton transport-coupled ATP synthesis . For EF(0)F(1), this activation step depends only on Deltaφ, whereas for CF(0)F(1), the activation depends on DeltapH and Deltaφ.

FEBS Lett, 1999 Aug 27, 457(2), 271 - 6
Interleukin 2-Bax: a novel prototype of human chimeric proteins for targeted therapy; Aqeilan R et al.; During the past few years many chimeric proteins have been developed to target and kill cells expressing specific surface molecules . Generally, these molecules carry a bacterial or plant toxin that destroys the unwanted cells . The major obstacle in the clinical application of such chimeras is their immunogenicity and non-specific toxicity . We have developed a new generation of chimeric proteins, taking advantage of apoptosis-inducing proteins, such as the human Bax protein, as novel killing components . The first prototype chimeric protein, IL2-Bax, directed toward IL2R-expressing cells, was constructed, expressed in Escherichia coli and partially purified . IL2-Bax increased the population of apoptotic cells in a variety of target T cell lines, as well as in human fresh PHA-activated lymphocytes, in a dose-dependent manner and had no effect on cells lacking IL2R expression . The IL2-Bax chimera represents an innovative approach for constructing chimeric proteins comprising a molecule that binds a specific cell type and an apoptosis-inducing protein . Such new chimeric proteins could be used for targeted treatment of human diseases.

FEBS Lett, 1999 Aug 27, 457(2), 223 - 6
Role of a bound ubiquinone on reactions of the Escherichia coli cytochrome bo with ubiquinol and dioxygen; Mogi T et al.; To probe the functional role of a bound ubiquinone-8 in cytochrome bo-type ubiquinol oxidase from Escherichia coli, we examined reactions with ubiquinol-1 and dioxygen . Stopped-flow studies showed that anaerobic reduction of the wild-type and the bound ubiquinone-free (DeltaUbiA) enzymes with ubiquinol-1 immediately takes place with four kinetic phases . Replacement of the bound ubiquinone with 2,6-dibromo-4-cyanophenol (PC32) suppressed the anaerobic reduction of the hemes with ubiquinol-1 by eliminating the fast phase . Flow-flash studies in the reaction of the fully reduced enzyme with dioxygen showed that the heme b-to-heme o electron transfer occurs with a rate constant of approximately 1x10(4) s(-1) in all three preparations . These results support our previous proposal that the bound ubiquinone is involved in facile oxidation of substrates in subunit II and subsequent intramolecular electron transfer to low-spin heme b in subunit I.

Nucleic Acids Res . 1999 Sep 15;27(18):e22.
Tandem arrayed ligation of expressed sequence tags (TALEST): a new method for generating global gene expression profiles; Spinella DG et al.; We have developed a new and simple method for quantitatively analyzing global gene expression profiles from cells or tissues . The process, called TALEST, or tandem arrayed ligation of expressed sequence tags, employs an oligonucleotide adapter containing a type IIs restriction enzyme site to facilitate the generation of short (16 bp) ESTs of fixed position in the mRNA . These ESTs are flanked by GC-clamped punctuation sequences which render them resistant to thermal denaturation, allowing their concatenation into long arrays and subsequent recognition and analysis by high-throughput DNA sequencing . A major advantage of the TALEST technique is the avoidance of PCR in all stages of the process and hence the attendant sequence-specific amplification biases that are inherent in other gene expression profiling methods such as SAGE, Differential Display, AFLP, etc . which rely on PCR.

Nucleic Acids Res . 1999 Sep 15;27(18):e20.
Selective detection of ribose-methylated nucleotides in RNA by a mass spectrometry-based method; Qiu F et al.; Post-transcriptional methylation of ribose at position O-2' is one of the most common and conserved types of RNA modification . Details of the functional roles of these methylations are far from clear, although in tRNA they are involved at position 34 in regulation of codon recognition and in eukaryotic rRNAs they are required for subunit assembly . Experimental difficulties in the mapping of ribose methylations increase with RNA molecular size and the complexity of mixtures resulting from nuclease digestion . A new and relatively rapid approach based on tandem mass spectrometry is described in which any of four ion reaction pathways occurring in the mass spectrometer can be monitored which are highly specific for the presence of 2'-O -methylribose residues . These pathways emanate from further dissociation of ribose-methylated mononucleotide (Nmp) ions formed in the electrospray ionization region of the mass spectrometer to then form the base, methylribose phosphate or PO(3)(-)anions . The mass spectrometer can be set for detection of generic ribose methylation (Nm) in oligonucleotides, selectively for each of the common methylated nucleo-sides Cm, Gm, Am or Um or for specific cases in which the base or sugar is further modified . By direct combination of mass spectrometry with liquid chromatography the method can be applied to analysis of complex mixtures of oligonucleotides, as for instance from synthetic or in vitro reaction mixtures or from nuclease digests of RNA . An example is given in which the single ribose-methylated nucleoside in Escherichia coli 16S rRNA (1542 nt), N(4),O-2'-dimethylcytidine, is detected in 25 pmol of a RNase T1 digest and localized to the fragment 1402-CCCGp-1405 in a single 45 min analysis.

Nucleic Acids Res . 1999 Sep 15;27(18):e18.
Recombination and chimeragenesis by in vitro heteroduplex formation and in vivo repair; Volkov AA et al.; We describe a simple method for creating libraries of chimeric DNA sequences derived from homologous parental sequences . A heteroduplex formed in vitro is used to transform bacterial cells where repair of regions of non-identity in the heteroduplex creates a library of new, recombined sequences composed of elements from each parent . Heteroduplex recombination provides a convenient addition to existing DNA recombination methods ('DNA shuffling') and should be particularly useful for recombining large genes or entire operons . This method can be used to create libraries of chimeric polynucleotides and proteins for directed evolution to improve their properties or to study structure-function relationships . We also describe a simple test system for evaluating the performance of DNA recombination methods in which recombination of genes encoding truncated green fluorescent protein (GFP) reconstructs the full-length gene and restores its characteristic fluorescence . Comprising seven truncated GFP constructs, this system can be used to evaluate the efficiency of recombination between mismatches separated by as few as 24 bp and as many as 463 bp . The optimized heteroduplex recombination protocol is quite efficient, generating nearly 30% fluorescent colonies for recombination between two genes containing stop codons 463 bp apart (compared to a theoretical limit of 50%).

Nucleic Acids Res, 1999 Sep 15, 27(18), 3762 - 9
Drosophila and human RecQ5 exist in different isoforms generated by alternative splicing; Sekelsky JJ et al.; Members of the RecQ helicase superfamily have been implicated in DNA repair, recombination and replication . Although the genome of the budding yeast Saccharomyces cerevisiae encodes only a single member of this family, there are at least five human RecQ-related genes: RecQL, BLM, WRN, RecQ4 and RecQ5 . Mutations in at least three of these are associated with diseases involving a predisposition to malignancies and a cellular phenotype that includes increased chromosome instability . Metazoan RecQ helicases are defined by a core region with characteristic helicase motifs and sequence similarity to Escherichia coli RecQ protein . This core region is typically flanked by extensive, highly charged regions, of largely unknown function . The recently reported human RecQ5, however, has only the core RecQ-homologous region . We describe here the identification of the Drosophila RecQ5 gene . We recovered cDNAs corresponding to three alternative splice forms of the RecQ5 transcript . Two of these generate nearly identical 54 kDa proteins that, like human RecQ5, consist of the helicase core only . The third splice variant encodes a 121 kDa isoform that, like other family members, has a C-terminal extension rich in charged residues . A combination of RACE and cDNA analysis of human RECQ5 demonstrates extensive alternative splicing for this gene also, including some forms lacking helicase motifs and other conserved regions.

Nucleic Acids Res, 1999 Sep 15, 27(18), 3702 - 11
Tolerance of 5-fluorodeoxyuridine resistant human thymidylate synthases to alterations in active site residues; Landis DM et al.; Fluoropyrimidines, such as 5-fluorouracil (5-FU), are used extensively in cancer therapy . In the cell, 5-FU is metabolized to 5-fluorodeoxyuridylate (5-FdUMP), a tight binding covalent inhibitor of thymidylate synthase (TS) . In order to create 5-FdUMP resistant enzymes to protect chemosensitive normal cells and further understand mechanisms of 5-FdUMP resistance, we have randomized four residues within the active site of TS . Our previous studies identified alterations in residues which produce active TS with enhanced resistance to 5-fluorouridine (5-FdUR) . By remutagenizing a subset of the 13 previously targeted residues (A197, L198, C199 and V204), an unbiased random library can be created allowing for extensive testing of all possible amino acid substitutions at each of the sites . Using genetic complementation and selection in Escherichia coli, we identified the spectrum of substitutions that yield active TS as well as those that resulted in 5-FdUR resistant mutants of TS . The 5-FdUR resistant TS were found to share several structural features including hydrophobic substitutions at residue 197, retention of the wild-type leucine 198, the alteration C199L (present in 64% of the drug-resistant library), and polar alterations of valine 204 . The catalytic activity of mutants with these features was approximately equal to that of the wild-type TS.

Nucleic Acids Res, 1999 Sep 15, 27(18), 3690 - 5
Repression of IS200 transposase synthesis by RNA secondary structures; Beuzon CR et al.; The IS 200 transposase, a 16 kDa polypeptide encoded by the single open reading frame (ORF) of the insertion element, has been identified using an expression system based on T7 RNA polymerase . In wild-type IS 200, two sets of internal inverted repeats that generate RNA secondary structures provide two independent mechanisms for repression of transposase synthesis . The inverted repeat located near the left end of IS 200 is a transcriptional terminator that terminates read-through transcripts before they reach the IS 200 ORF . The terminator is functional in both directions and may terminate >80% of transcripts . Another control operates at the translational level: transposase synthesis is inhibited by occlusion of the ribosome-binding site (RBS) of the IS 200 ORF . The RBS (5'-AGGGG-3') is occluded by formation of a mRNA stem-loop structure whose 3' end is located only 3 nt upstream of the start codon . This mechanism reduces transposase synthesis approximately 10-fold . Primer extension experiments with AMV reverse transcriptase have provided evidence that this stem-loop RNA structure is actually formed . Tight repression of transposase synthesis, achieved through synergistic mechanisms of negative control, may explain the unusually low transposition frequency of IS 200.

Nucleic Acids Res, 1999 Sep 15, 27(18), 3667 - 75
An NMR and mutational analysis of an RNA pseudoknot of Escherichia coli tmRNA involved in trans-translation; Nameki N et al.; Transfer-messenger RNA (tmRNA) is a unique molecule that combines properties from both tRNA and mRNA, and facilitates a novel translation reaction termed trans -translation . According to phylogenetic sequence analysis among various bacteria and chemical probing analysis, the secondary structure of the 350-400 nt RNA is commonly characterized by a tRNA-like structure, and four pseudoknots with different sizes . A mutational analysis using a number of Escherichia coli tmRNA variants as well as a chemical probing analysis has recently demonstrated not only the presence of the smallest pseudoknot, PK1, upstream of the internal coding region, but also its direct implication in trans -translation . Here, NMR methods were used to investigate the structure of the 31 nt pseudoknot PK1 and its 11 mutants in which nucleotide substitutions are introduced into each of two stems or the linking loops . NMR results provide evidence that the PK1 RNA is folded into a pseudoknot structure in the presence of Mg(2+) . Imino proton resonances were observed consistent with formation of two helical stem regions and these stems stacked to each other as often seen in pseudoknot structures, in spite of the existence of three intervening nucleo-tides, loop 3, between the stems . Structural instability of the pseudoknot structure, even in the presence of Mg(2+), was found in the PK1 mutants except in the loop 3 mutants which still maintained the pseudoknot folding . These results together with their biological activities indicate that trans -translation requires the pseudoknot structure stabilized by Mg(2+)and specific residues G61 and G62 in loop 3.

Nucleic Acids Res, 1999 Sep 15, 27(18), 3645 - 52
Escherichia coli RNA polymerase translocation is accompanied by periodic bending of the DNA; Zaychikov E et al.; RNA polymerase was halted in consecutive registers of RNA synthesis ranging from registers 11 to 68 . Non-denaturing gel electrophoresis shows that the mobility of the complexes varies (up to 15%), indicating that halted complexes differ in their conformation . The electrophoretic mobility changes with an approximate 10-register periodicity . The change of the mobility can be attributed to relative changes of RNA polymerase-induced bending angle . We suggest that the periodicity of the bending angle reflects periodic changes of the conformation of the halted complexes that might have relevance for the translocation mechanism.

Nucleic Acids Res, 1999 Sep 15, 27(18), 3638 - 44
Differential subcellular localization of human MutY homolog (hMYH) and the functional activity of adenine:8-oxoguanine DNA glycosylase; Takao M et al.; The post-replicative adenine:8-oxo-7,8-dihydroguanine (GO) mismatch is crucial for G:C to T:A transversion . This mismatch is corrected by Escherichia coli MutY which excises the adenine from A:GO . A candidate gene coding for the human counterpart of MutY has been cloned as hMYH . However, the function and enzyme activities of the gene product have not been identified . We previously demonstrated that an epitope-tagged hMYH protein behaves as a mitochondrial protein . In the present study, we have identified an alternative hMYH transcript, termed type 2, which differs in the exon 1 sequence of the known transcript (type 1) . A nuclear localization for the type 2 protein was revealed by detection of epitope-tagged protein in COS-7 cells . Expression of both type 1 and type 2 transcripts was reduced in post-mitotic tissues . hMYH cDNA suppressed the mutator phenotype of E.coli mutY . In vitro expressed hMYH showed adenine DNA glycosylase activity toward the A:GO substrate . The protein can bind to A:GO, and to T:GO and G:GO without apparent catalysis . These results represent the first demonstration of the function of the hMYH gene product which is differentially transported into the nucleus or the mitochondria by alternative splicing

Nucleic Acids Res, 1999 Sep 15, 27(18), 3631 - 7
Transfer RNA identity contributes to transition state stabilization during aminoacyl-tRNA synthesis; Ibba M et al.; Sequence-specific interactions between aminoacyl-tRNA synthetases and their cognate tRNAs ensure both accurate RNA recognition and the efficient catalysis of aminoacylation . The effects of tRNA(Trp)variants on the aminoacylation reaction catalyzed by wild-type Escherichia coli tryptophanyl-tRNA synthe-tase (TrpRS) have now been investigated by stopped-flow fluorimetry, which allowed a pre-steady-state analysis to be undertaken . This showed that tRNA(Trp)identity has some effect on the ability of tRNA to bind the reaction intermediate TrpRS-tryptophanyl-adenylate, but predominantly affects the rate at which trypto-phan is transferred from TrpRS-tryptophanyl adenylate to tRNA . Use of the binding ( K (tRNA)) and rate constants ( k (4)) to determine the energetic levels of the various species in the aminoacylation reaction showed a difference of approximately 2 kcal mol(-1)in the barrier to transition state formation compared to wild-type for both tRNA(Trp)A-->C73 and . These results directly show that tRNA identity contributes to the degree of complementarity to the transition state for tRNA charging in the active site of an aminoacyl-tRNA synthetase:aminoacyl-adenylate:tRNA complex.

Genetics, 1999 Sep, 153(1), 5 - 12
Mutational adaptation of Escherichia coli to glucose limitation involves distinct evolutionary pathways in aerobic and oxygen-limited environments; Manch K et al.; Mutational adaptations leading to improved glucose transport were followed with Escherichia coli K-12 growing in glucose-limited continuous cultures . When populations were oxygen limited as well as glucose limited, all bacteria within 280 generations contained mutations in a single codon of the ptsG gene . V12F and V12G replacements in the enzyme IIBC(Glc) component of the glucose phosphotransferase system were responsible for improved transport . In stark contrast, ptsG mutations were uncommon in fully aerobic glucose-limited cultures, in which polygenic mutations in mgl, mlc, and malT (regulating an alternate high-affinity Mgl/LamB uptake pathway) spread through the adapted population . Hence the same organism adapted to the same selection (glucose limitation) by different evolutionary pathways depending on a secondary environmental factor . The clonal diversity in the adapted populations was also significantly different . The PtsG V12F substitution under O(2) limitation contributed to a universal "winner clone" whereas polygenic, multiallelic changes led to considerable polymorphism in aerobic cultures . Why the difference in adaptive outcomes? E . coli physiology prevented scavenging by the LamB/Mgl system under O(2) limitation; hence, ptsG mutations provided the only adaptive pathway . But ptsG mutations in aerobic cultures are overtaken by mgl, mlc, and malT adaptations with better glucose-scavenging ability . Indeed, when an mglA::Tn10 mutant with an inactivated Mgl/LamB pathway was introduced into two independent aerobic chemostats, adaptation of the Mgl(-) strain involved the identical ptsG mutation found under O(2)-limited conditions with wild-type or Mgl(-) bacteria.

Antimicrob Agents Chemother, 1999 Sep, 43(9), 2273 - 7
Inhibition of Escherichia coli-induced meningitis by carboxyfullerence; Tsao N et al.; The effect of a water-soluble malonic acid derivative of carboxyfullerence (C60) against Escherichia coli-induced meningitis was tested . C60 can protect the mice from E . coli-induced death in a dose-dependent manner . C60 administered intraperitoneally as late as 9 h after E . coli injection was still protective . The C60-treated mice had less tumor necrosis factor alpha and interleukin-1beta production by staining of brain tissue compared to the levels of production for nontreated mice . The E . coli-induced increases in blood-brain barrier permeability and inflammatory neutrophilic infiltration were also inhibited . These data suggest that C60 is a potentially therapeutic agent for bacterial meningitis.

Trends Biochem Sci, 1999 Sep, 24(9), 359 - 63
The enzymatic biotinylation of proteins: a post-translational modification of exceptional specificity; Chapman-Smith A et al.; Biotin is a coenzyme essential to all life forms . The vitamin has biological activity only when covalently attached to certain key metabolic enzymes . Most organisms have only one enzyme for attachment of biotin to other proteins and the sequences of these proteins and their substrate proteins are strongly conserved throughout nature . Structures of both the biotin ligase and the biotin carrier protein domain from Escherichia coli have been determined . These, together with mutational analyses of biotinylated proteins, are beginning to elucidate the exceptional specificity of this protein modification.

Biochem Biophys Res Commun, 1999 Sep 7, 262(3), 739 - 43
Expression of bone morphogenetic protein-2 via adenoviral vector in C2C12 myoblasts induces differentiation into the osteoblast lineage; Okubo Y et al.; To examine the effectiveness of a gene transfer of bone morphogenetic protein (BMP)-2 into C2C12 myoblasts, we constructed a human BMP-2-expressing replication-deficient adenoviral vector, AxCAOBMP-2 . C2C12 cells were infected in vitro with either this viral vector or an Escherichia coli LacZ gene-expressing control adenovirus vector . An efficient gene transfer to the C2C12 cells was confirmed with the LacZ gene-expressing vector by X-gal staining . Abundant BMP-2 expression in C2C12 cells infected with this viral vector was confirmed by immunofluorescence and Western blot analysis . C2C12 cells transferred with the BMP-2 gene by this vector produced alkaline phosphatase in the cells and also produced and secreted osteocalcin in the culture medium, demonstrating that a gene transfer of BMP-2 into C2C12 cells in vitro could convert these cells from myoblast to osteoblast lineage .

Biochemistry, 1999 Aug 31, 38(35), 11349 - 58
Sequence and functional-group specificity for cleavage of DNA junctions by RuvC of Escherichia coli; Fogg JM et al.; RuvC is the DNA junction-resolving enzyme of Escherichia coli . While the enzyme binds to DNA junctions independently of base sequence, it exhibits considerable sequence selectivity for the phosphodiester cleavage reaction . We have analyzed the sequence specificity using a panel of DNA junctions, measuring the rate of cleavage of each under single-turnover conditions . We have found that the optimal sequence for cleavage can be described by (A approximately T)TT downward arrow(C>G approximately A), where downward arrow denotes the position of backbone scission . Cleavage is fastest when the cleaved phosphodiester linkage is located at the point of strand exchange . However, cleavage is possible one nucleotide 3' of this position when directed by the sequence, with a rate that is 1 order of magnitude slower than the optimal . The maximum sequence discrimination occurs at the central TT in the tetranucleotide site, where any alteration of sequence results in a rate reduction of at least 100-fold and cleavage is undetectable for some changes . However, certain sequences in the outer nucleotides are strongly inhibitory to cleavage . Introduction of base analogues around the cleavage site reveals a number of important functional groups and suggests that major-groove contacts in the center of the tetranucleotide are important for the cleavage process . Since RuvC binds to all the variant junctions with very similar affinity, any contacts affecting the rate of cleavage must be primarily important in the transition state . Introduction of the optimal cleavage sequence into a three-way DNA junction led to relatively efficient cleavage by RuvC, at a rate only 3-fold slower than the optimal four-way junction . This is consistent with a protein-induced alteration in the conformation of the DNA.

Pharmacogenetics, 1999 Jun, 9(3), 295 - 306
Human CYP2B6: expression, inducibility and catalytic activities; Gervot L et al.; Human cytochrome (CYP)2B6 cDNA was cloned and expressed in bacteria and in yeast . Its expression in Saccharomyces cerevisiae enabled us to obtain, at a high level, an active yeast-expressed CYP2B6 protein, so as to assess its role in the metabolism of ethoxyresorufin, pentoxyresorufin, benzyloxyresorufin, ethoxycoumarin, testosterone and cyclophosphamide . Kinetic analysis showed that human CYP2B6 preferentially metabolized benzyloxyresorufin and pentoxyresorufin, although other CYPs also metabolized these substrates in human liver microsomes . CYP2B6 also manifested a strong 4-hydroxycyclophosphamide activity . Its expression in Escherichia coli enabled us to produce a very specific anti-human CYP2B6 antibody . No cross reactivity of this antibody was observed with CYPs1A1, 1A2, 3A4, 3A5, 2C8, 2C9, 2C18, 2C19, 2D6 or 2E1 . This antibody enabled us to study the hepatic and extrahepatic expression of CYP2B6 in man, as well as its expression and inducibility in primary cultured human hepatocytes and in different human cell lines . Immunoblot analysis revealed that the CYP2B6 protein was expressed in 43 of the 48 human liver samples tested, with levels ranging from 0.4 to 8 pmol/mg of microsomal protein with a mean of 1.7 pmol/mg protein . CYP2B was also expressed in human brain, intestine and kidney, and at a lower level in the lung . CYP2B mRNA was detected in human liver, kidney, lung, trachea and intestine . We also found that CYP2B6 is induced at protein and mRNA levels by phenobarbital (2 mM) and cyclophosphamide (1 mM), an anticancer drug known to be metabolized by CYP2B6 . No expression or inducibility of CYP2B6 was observed in any of the human cell lines tested.

J Virol Methods, 1999 Jul, 80(2), 129 - 36
An efficient in vivo recombination cloning procedure for modifying and combining HSV-1 cosmids; Kong Y et al.; A helper virus-free herpes simplex virus type-1 (HSV-1) plasmid vector system developed recently may have applications in gene therapy and basic physiological studies . This system might be improved by mutating specific HSV-1 genes in the packaging system and by creating large vectors . An in vivo recombination cloning procedure is reported that supports the routine manipulation of relatively large DNAs such as the five cosmids that comprise this helper virus-free HSV-1 packaging system . In vivo recombination cloning is carried out by transforming overlapping DNA fragments into a specific RecA+ Escherichia coli, BJ5183 . The cloning efficiency was improved by using a modified version of the Hanahan transformation procedure, and the background was lowered by either using vectors with different combinations of ends (5' overhangs, 3' overhangs, blunt ends) or by treating the vector with calf intestinal phosphatase . The range of usable overlap sizes is from 251 bp to 18 kb with 500 bp to 5 kb preferred . This procedure supports the routine construction and mutation of HSV-1 cosmids, by use of up to six different DNA fragments, and the construction of plasmids up to 65 kb in size . This procedure may also have applications to other vector systems and to studies on large viruses.

Adv Enzyme Regul, 1999, 39, 263 - 73
Folylpoly-gamma-glutamate synthetase: generation of isozymes and the role in one carbon metabolism and antifolate cytotoxicity; Qi H et al.; A single human gene encodes both mitochondrial and cytosolic isoforms of the enzyme . The major mRNA species in human cells encodes the mitochondrial isoform but alternate translation initiation at a downstream in-frame ATG also generates the cytosolic isoform . Cytosolic FPGS may also be generated by use of alternate transcription initiation start sites 3' to the start ATG of the mitochondrial FPGS . Three additional human FPGS mRNAs differing in exon 1 have been identified . One of these is a major species in HEP-G2 cells and other tissue culture cells, and can encode a protein lacking the first 8 amino acids of cytosolic FPGS . A protein of the predicted size is observed in coupled transcription/translation systems . However, expression of this protein in E . coli does not generate an active enzyme . Mutagenesis studies indicate that Tyr-3 of the missing N terminal residues is required for enzyme activity . The major cellular folate pools are in the cytosol and mitochondria and FPGS activity is normally distributed in both compartments . Mitochondrial FPGS activity is required for mitochondrial folate accumulation, and cells lacking this isozyme are auxotrophic for glycine . Overexpression of cytosolic FPGS does not complement the lack of mitochondrial activity . Cells expressing FPGS activity solely in the mitochondria are glycine prototrophs, but also possess cytosolic folylpolyglutamates and are prototrophic for thymidine and purines, products of cytosolic one carbon metabolism . Although cytosolic folylpolyglutamates cannot enter the mitochondrion, mitochondrial folylpolyglutamates are released intact into the cytosolic compartment . Cellular accumulation of some antifolates and their cytotoxic efficacy is highly responsive to the level of FPGS activity . Polyglutamylation of methotrexate (MTX) has little affect on its affinity for dihydrofolate reductase, its target enzyme, but does affect the cellular accumulation of the drug . The sensitivity of model cells, expressing a range of FPGS activities similar to that observed in leukemia blasts, to MTX varied over four orders of magnitude . MTX toxicity was dependent on cytosolic FPGS activity as this drug does not enter the mitochondria, and cells expressing very high levels of FPGS solely in the mitochondria were resistant to MTX . The cytotoxic efficacy of other folate antagonists that are transported into the mitochondria was enhanced by mitochondrial FPGS activity, even when their loci of inhibition was a cytosolic enzyme . Mitochondrial metabolism of these drugs increased cytosolic drug levels . Compartmentalization of antifolate metabolism has to be considered in evaluating mechanisms for increased drug cytotoxicity and for the development of acquired resistance to these agents.

EMBO J, 1999 Sep 1, 18(17), 4889 - 901
Strand opening by the UvrA(2)B complex allows dynamic recognition of DNA damage; Zou Y et al.; Repair proteins alter the local DNA structure during nucleotide excision repair (NER) . However, the precise role of DNA melting remains unknown . A series of DNA substrates containing a unique site-specific BPDE-guanine adduct in a region of non-complementary bases were examined for incision by the Escherichia coli UvrBC endonuclease in the presence or absence of UvrA . UvrBC formed a pre-incision intermediate with a DNA substrate containing a 6-base bubble structure with 2 unpaired bases 5' and 3 unpaired bases 3' to the adduct . Formation of this bubble served as a dynamic recognition step in damage processing . UvrB or UvrBC may form one of three stable repair intermediates with DNA substrates, depending upon the state of the DNA surrounding the modified base . The dual incisions were strongly determined by the distance between the adduct and the double-stranded-single-stranded DNA junction of the bubble, and required homologous double-stranded DNA at both incision sites . Remarkably, in the absence of UvrA, UvrBC nuclease can make both 3' and 5' incisions on substrates with bubbles of 3-6 nucleotides, and an uncoupled 5' incision on bubbles of >/=>/=10 nucleotides . These data support the hypothesis that the E.coli and human NER systems recognize and process DNA damage in a highly conserved manner.

EMBO J, 1999 Sep 1, 18(17), 4882 - 8
Escherichia coli SeqA protein affects DNA topology and inhibits open complex formation at oriC; Torheim NK et al.; Chromosome replication in Escherichia coli is initiated by the DnaA protein . Binding of DnaA to the origin, oriC, followed by formation of an open complex are the first steps in the initiation process . Based on in vivo studies the SeqA protein has been suggested to function negatively in the initiation of replication, possibly by inhibiting open complex formation . In vitro studies have shown that SeqA inhibits oriC-dependent replication . Here we show by KMnO(4) probing that SeqA inhibits open complex formation . The inhibition was not caused by prevention of DnaA binding to the oriC plasmids, indicating that SeqA prevented strand separation in oriC either directly, by interacting with the AT-rich region, or indirectly, by changing the topology of the oriC plasmids . SeqA was found to restrain the negative supercoils of the oriC plasmid . In comparison with the effect of HU on plasmid topology, SeqA seemed to act more cooperatively . It is likely that the inhibition of open complex formation is caused by the effect of SeqA on the topology of the plasmids . SeqA also restrained the negative supercoils of unmethylated oriC plasmids, which do not bind SeqA specifically, suggesting that the effect on topology is not dependent on binding of SeqA to a specific sequence in oriC.

EMBO J, 1999 Sep 1, 18(17), 4804 - 15
Control of glycosylation of MHC class II-associated invariant chain by translocon-associated RAMP4; Schroder K et al.; Protein translocation across the membrane of the endoplasmic reticulum (ER) proceeds through a proteinaceous translocation machinery, the translocon . To identify components that may regulate translocation by interacting with nascent polypeptides in the translocon, we used site-specific photo-crosslinking . We found that a region C-terminal of the two N-glycosylation sites of the MHC class II-associated invariant chain (Ii) interacts specifically with the ribosome-associated membrane protein 4 (RAMP4) . RAMP4 is a small, tail-anchored protein of 66 amino acid residues that is homologous to the yeast YSY6 protein . YSY6 suppresses a secretion defect of a secY mutant in Escherichia coli . The interaction of RAMP4 with Ii occurred when nascent Ii chains reached a length of 170 amino acid residues and persisted until Ii chain completion, suggesting translocational pausing . Site-directed mutagenesis revealed that the region of Ii interacting with RAMP4 contains essential hydrophobic amino acid residues . Exchange of these residues for serines led to a reduced interaction with RAMP4 and inefficient N-glycosylation . We propose that RAMP4 controls modification of Ii and possibly also of other secretory and membrane proteins containing specific RAMP4-interacting sequences . Efficient or variable glycosylation of Ii may contribute to its capacity to modulate antigen presentation by MHC class II molecules.

EMBO J, 1999 Sep 1, 18(17), 4619 - 32
Phosphorylase recognition and phosphorolysis of its oligosaccharide substrate: answers to a long outstanding question; Watson KA et al.; Phosphorylases are key enzymes of carbohydrate metabolism . Structural studies have provided explanations for almost all features of control and substrate recognition of phosphorylase but one question remains unanswered . How does phosphorylase recognize and cleave an oligosaccharide substrate? To answer this question we turned to the Escherichia coli maltodextrin phosphorylase (MalP), a non-regulatory phosphorylase that shares similar kinetic and catalytic properties with the mammalian glycogen phosphorylase . The crystal structures of three MalP-oligosaccharide complexes are reported: the binary complex of MalP with the natural substrate, maltopentaose (G5); the binary complex with the thio-oligosaccharide, 4-S-alpha-D-glucopyranosyl-4-thiomaltotetraose (GSG4), both at 2.9 A resolution; and the 2.1 A resolution ternary complex of MalP with thio-oligosaccharide and phosphate (GSG4-P) . The results show a pentasaccharide bound across the catalytic site of MalP with sugars occupying sub-sites -1 to +4 . Binding of GSG4 is identical to the natural pentasaccharide, indicating that the inactive thio compound is a close mimic of the natural substrate . The ternary MalP-GSG4-P complex shows the phosphate group poised to attack the glycosidic bond and promote phosphorolysis . In all three complexes the pentasaccharide exhibits an altered conformation across sub-sites -1 and +1, the site of catalysis, from the preferred conformation for alpha(1-4)-linked glucosyl polymers.

Curr Biol, 1999 Aug 26, 9(16), 907 - 10
Mutator phenotypes of common polymorphisms and missense mutations in MSH2; Drotschmann K et al.; Hereditary non-polyposis colorectal cancer (HNPCC) is associated with germline mutations in the DNA mismatch repair gene hMSH2 {1}, the human homologue of the Escherichia coli MutS gene . These are mostly nonsense, frameshift or deletion mutations that result in loss of intact protein and complete inactivation of DNA mismatch repair . However, cancer is also associated with hMSH2 missense mutations that are merely inferred to be deleterious because they result in non-conservative substitutions of amino acids that are highly conserved among MutS family proteins . Moreover, sequence polymorphisms exist in hMSH2 that also change conserved amino acids but whose functional consequences and relationship to cancer are uncertain . Here, we show that yeast strains harboring putative equivalents of three hMSH2 polymorphisms have elevated mutation rates . Mutator effects were also observed for yeast equivalents of hMSH2 missense mutations found in HNPCC families and in an early onset colon tumor . Several distinct phenotypes were observed, indicating that these missense mutations have differential effects on MSH2 function(s) . The results suggest that cancer may be associated with even partial loss of hMSH2 function and they are consistent with the hypothesis that polymorphisms in hMSH2 might predispose humans to disease.

Anal Biochem, 1999 Sep 10, 273(2), 298 - 304
A direct spectrophotometric assay for peptide deformylase; Guo XC et al.; A direct UV-VIS spectrophotometric assay has been developed for peptide deformylase . This assay employs a novel class of peptide mimetics as deformylase substrates which, upon enzymatic removal of the N-terminal formyl group, rapidly release free thiols . The released thiols are quantitated using Ellman's reagent . A variety of peptide analogues that contain beta-thiaphenylalanine or beta-thiamethionine as the N-terminal residue were synthesized and found to be excellent substrates of the peptide deformylase from Escherichia coli (k(cat)/K(M) = 6.9 x 10(5) M(-1) s(-1) for the most reactive substrate) . The deformylase reaction is conveniently monitored on a UV-VIS spectrophotometer in a continuous fashion . The versatility of the assay has been demonstrated by its application to kinetic characterization of the deformylase, pH profile studies, and enzyme inhibition assays . The assay can also be performed in an end-point fashion . The results demonstrate that this assay is a simple, highly sensitive, and rapid method to study kinetic properties of deformylases without the use of any coupling enzymes .

J Biochem (Tokyo), 1999 Sep, 126(3), 584 - 90
Construction of a screening system for selecting lysozyme mutants unable to form a stable structure from random mutants; Kunichika K et al.; To collect folding information, we screened and analyzed the recombinant hen lysozyme mutants which were not secreted from yeast . As model mutants, Leu8Arg, Ala10Gly, and Met12Arg were prepared by site-directed mutagenesis and analyzed as to whether they were secreted from yeast or not . Consequently, Ala10Gly was found to be secreted from yeast, but Leu8Arg and Met12Arg were not . Next, these mutants were expressed in Escherichia coli and refolded in vitro . As a result, Ala10Gly folded as the wild-type did . Leu8Arg efficiently refolded in renaturation buffer containing glycerol . Met12Arg did not refold even in the presence of glycerol . These results show that the Ala10Gly mutation does not affect folding or stability, that Leu8Arg is too unstable to be secreted from yeast, and that Met12Arg may be very unstable or the mutation affects the folding pathway . We screened the mutants that were not secreted by yeast from a randomly mutated lysozyme library, and obtained Asp18His/Leu25Arg and Ala42Val/Ser50Ile/Leu56Gln . These two mutants were expressed in E . coli and then refolded in the presence of urea or glycerol . These mutants were refolded only in the presence of glycerol . Each single mutant of Asp18His/Leu25Arg and Ala42Val/Ser50Ile/Leu56Gln was independently prepared and folded in vitro . The results showed that Leu25Arg and Leu56Gln were the dominant mutations, respectively, which cause destabilization . These results show that the mutant lysozymes which were not secreted from yeast may be unstable or have a defect in the folding pathway . Thus, we established a screening system for selecting mutants which are unable to form a stable structure from random mutants.

J Biochem (Tokyo), 1999 Sep, 126(3), 510 - 9
Azide- and cyanide-binding to the Escherichia coli bd-type ubiquinol oxidase studied by visible absorption, EPR and FTIR spectroscopies; Tsubaki M et al.; Cytochrome bd-type ubiquinol oxidase contains two hemes b (b(558) and b(595)) and one heme d as the redox metal centers . To clarify the structure of the reaction center, we analyzed Escherichia coli cytochrome bd by visible absorption, EPR and FTIR spectroscopies using azide and cyanide as monitoring probes for the exogenous ligand binding site . Azide-binding caused the appearance of a new EPR low-spin signal characteristic of ferric iron-chlorin-azide species and a new visible absorption band at 647 nm . However, the bound azide ((14)N(3)) anti-symmetric stretching infrared band (2, 010.5 cm(-1)) showed anomalies upon (15)N-substitutions, indicating interactions with surrounding protein residues or heme b(595) in close proximity . The spectral changes upon cyanide-binding in the visible region were typical of those observed for ferric iron-chlorin species with diol substituents in macrocycles . However, we found no indication of a low-spin EPR signal corresponding to the ferric iron-chlorin-cyanide complexes . Instead, derivative-shaped signals at g = 3.19 and g = 7.15, which could arise from the heme d(Fe(3+))-CN-heme b(595)(Fe(3+)) moiety, were observed . Further, after the addition of cyanide, a part of ferric heme d showed the rhombic high-spin signal that coexisted with the g(z) = 2.85 signal ascribed to the minor heme b(595)-CN species . This indicates strong steric hindrance of cyanide-binding to ferric heme d with the bound cyanide at ferric heme b(595).

Insect Mol Biol, 1999 Aug, 8(3), 369 - 80
Molecular cloning and expression in Escherichia coli of an aquaporin-like gene from adult buffalo fly (Haematobia irritans exigua); Elvin CM et al.; A gene fragment encoding a putative member of the aquaporin gene family was amplified using cDNA prepared from unfed adult buffalo fly poly(A)+ RNA and degenerate PCR primers designed from highly conserved regions of amino acids found in all members of the aquaporin gene family . This PCR product was labelled with digoxigenin-dUTP and used as a probe to screen a lambdagt-11 cDNA library constructed from unfed adult buffalo fly . One positively hybridizing clone (AqpBF1), contained an insert of 1878 bp, and DNA sequence analysis revealed an open reading frame of 753 bp encoding a polypeptide of predicted Mr = 26 163 Da . Comparison of the AqpBF1 deduced protein sequence with the GenBank database revealed significant homology to many aquaporin genes, including 72% identity with a partial DNA sequence encoding a member (DRIP) of the MIP protein family isolated from Drosophila melanogaster . The most closely related, full-length, GenBank sequence was an aquaporin gene isolated from the digestive tract of the sap-sucking insect Cicadella viridis, which was 53% identical to the buffalo fly AqpBF1 protein sequence . The full-length coding sequence of AqpBF1 was cloned into the (His)6-fusion vector, pQE10, and the recombinant protein was expressed in Escherichia coli following induction by IPTG . The recombinant (His)6-fusion protein was localized predominantly in the membrane fraction of E . coli . The protein was solubilized from E . coli membranes with n-octyl beta-D-glucopyranoside and purified by affinity chromatography on a Ni++-sepharose column in the presence of detergent.

Genes Cells, 1999 Jul, 4(7), 415 - 24
Identification of the core domain and the secondary structure of the transcriptional coactivator MBF1; Ozaki J et al.; BACKGROUND: Multiprotein bridging factor 1 (MBF1) is a transcriptional coactivator necessary for transcriptional activation caused by DNA binding activators, such as FTZ-F1 and GCN4 . MBF1 bridges the DNA-binding regions of these activators and the TATA-box binding protein (TBP), suggesting that MBF1 functions by recruiting TBP to promoters where the activators are bound . In addition, MBF1 stimulates DNA binding activities of the activators to their recognition sites . To date, little is known about structures of coactivators that bind to TBP . RESULTS: The two-dimensional (2D) 1H-15N correlation spectrum of 15N labeled MBF1 indicated that MBF1 consists of both flexible and well structured parts . Limited digestion of MBF1 by alpha-chymotrypsin yielded a approximately 9 kDa fragment . N-terminal sequence analysis and NMR measurements revealed that this fragment originates from the C-terminal 80 residues of MBF1 and form a well structured C-terminal domain of MBF1, MBF1CTD . As previous deletion analyses have shown that MBF1CTD is capable of binding to TBP, it is suggested that MBF1CTD is the TBP binding domain of MBF1 . Sequential assignments have been obtained by means of three-dimensional (3D) and four dimensional (4D) heteronuclear correlation spectroscopies, and then the secondary structure of MBF1CTD was determined . As a result, MBF1CTD was shown to contain four amphipathic helices and a conserved C-terminal region . Asp106 which is assumed to be responsible for the binding to TBP is located at the hydrophilic side of the third helix . CONCLUSIONS: Structural analyses revealed that MBF1 consists of two structurally different domains . A N-terminal region is indispensable for the binding to activators, and does not form a well defined structure . In contrast, the C-terminal 80 residues, which is capable of binding to TBP by itself, form a well-structured domain, MBF1CTD . MBF1CTD is made up of four amphipathic helices and a conserved C-terminal tail . A putative TBP binding residue is located on the hydrophilic surface of the third helix.

Genes Cells, 1999 Jul, 4(7), 391 - 9
Negative regulation of the pts operon by Mlc: mechanism underlying glucose induction in Escherichia coli; Tanaka Y et al.; BACKGROUND: The pts operon of Escherichia coli consists of three genes ptsH, ptsI and crr, each encoding for central components of the phosphoenolpyruvate: carbohydrate phosphotransferase system, HPr, enzyme I and IIAGlc, respectively . Transcription of the pts operon is stimulated when glucose is present in the culture medium . One of the two major promoters, P0, is responsible for this glucose induction . However, no regulatory protein responsible for the glucose induction of the pts operon has been identified yet and molecular mechanism by which glucose stimulates the pts transcription is not known . RESULTS: We found by Northern blotting that the pts mRNA levels in cells lacking Mlc, a new global repressor of carbohydrate metabolism, were increased without external glucose and that the addition of glucose had no effect on the pts mRNA levels in the mutant cells . Western blotting revealed that the enzyme I level in the mlc- cells was also elevated without glucose and no further increase in the enzyme I level was observed in the presence of glucose . S1 analysis revealed that transcription of the glucose-sensitive promoter, P0, occurs constitutively in the mlc- cells independently from the external glucose . In vitro transcription studies indicated that Mlc strongly inhibited P0 transcription . DNase I footprinting experiment revealed that Mlc bound to P0 promoter region to prevent RNA polymerase binding at P0 . CONCLUSION: We conclude that Mlc is a repressor for the pts transcription acting as a major regulatory protein involved in the glucose induction of pts operon . We propose that glucose induces the pts transcription by modulating the Mlc activity . The mechanism by which glucose modulates the Mlc action remains to be studied.

Eur J Biochem, 1999 Aug, 263(3), 834 - 9
p-Coumaroyltriacetic acid synthase, a new homologue of chalcone synthase, from Hydrangea macrophylla var . thunbergii; Akiyama T et al.; Chalcone synthase and stilbene synthase are plant-specific polyketide synthases . They catalyze three common consecutive decarboxylative condensations and specific cyclization reactions . They are highly homologous to each other, and are likely to fall into a family of polyketide synthases along with acridone synthase and bibenzyl synthase . Two cDNA clones (named HmC and HmS), both of which show high homology to the known chalcone synthases, were obtained from leaves of Hydrangea macrophylla var . thunbergii . They were expressed in Escherichia coli in order to determine their enzyme functions . Detection of chalcone formation clearly indicated that HmC encoded chalcone synthase, while HmS protein catalyzed the formation of neither chalcone nor stilbene . However, a novel pyrone, a lactonization product of a linear tetraketide was detected in reaction products of HmS protein . This proves that HmS encodes a novel polyketide synthase that catalyzes only chain elongation without cyclization.

Eur J Biochem, 1999 Aug, 263(3), 806 - 16
p55-hGRF, a short natural form of the Ras-GDP exchange factor high yield production and characterization; Meyer P et al.; p55-hGRF, a natural short form of the guanine-nucleotide-releasing factor for p21-Ras from human brain, was expressed at high level in Escherichia coli as well as an engineered truncated form, p39-hGRF . A T7 polymerase expression system was used, resulting in the formation of insoluble cytoplasmic protein aggregates . The recombinant products were resolubilized, renatured and purified to homogeneity . The exchange activity of the refolded hGRF samples on H-Ras was comparable with that published for the soluble catalytic domain of the mouse counterpart, CDC25 Mm . Both p55-hGRF and p39-hGRF form dimers . We established a procedure to prepare and purify the complex with Ras . The results of the characterization study are consistent with a stoichiometry of 1:1 and an equilibrium between dimeric and monomeric forms of the complex.

Eur J Biochem, 1999 Aug, 263(3), 789 - 96
Methylcobalamin:homocysteine methyltransferase from Methanobacterium thermoautotrophicum . Identification as the metE gene product; Schroder I et al.; Methanobacterium thermoautotrophicum is a methane-forming archaeon that grows on H2 and CO2 as sole carbon and energy source . Cell extracts of the methanogen were found to contain methylcobalamin: homocysteine methyltransferase activity which was purified 3000-fold to a specific activity of approximately 500 U.mg-1 protein . SDS/PAGE revealed the presence of a polypeptide with an apparent molecular mass of 34 kDa . Via its N-terminal amino acid sequence, the 34-kDa polypeptide was identified as the metE gene product . The metE gene was heterologously expressed in Escherichia coli . The overproduced protein was recovered in the inclusion body fraction and was found to be inactive . The protein could be partially solubilized by unfolding in 8 M urea and then refolding . The solubilized protein had a specific activity of 450 U.mg-1 . It exhibited first-order kinetics with respect to methylcobalamin concentration and Michaelis-Menten kinetics with respect to L-homocysteine concentration (apparent Km 0.1 mM) . The enzyme was specific for L-homocysteine as methyl acceptor . Methylcobalamin could be substituted with methylcobinamide as methyl donor.

Eur J Biochem, 1999 Aug, 263(3), 782 - 8
Introduction of a C-terminal aromatic sequence from snake venom phospholipases A2 into the porcine pancreatic isozyme dramatically changes the interfacial kinetics; Janssen MJ et al.; Porcine pancreatic phospholipase A2 (PLA2) was modified by single and multiple site-directed mutations at sites thought to be involved in interfacial binding . Charged and polar residues in the C-terminal region were replaced by aromatic residues on the basis of an analogy with snake venom PLA2s, which display high affinity for a zwitterionic interface . The PLA2 variants constructed were N117W, N117W/D119Y and K116Y/N117W/D119Y . Titration with micelles of a zwitterionic substrate suggests that the variants N117W and K116Y/N117W/D119Y possess improved ability to bind to the micellar substrate interface, relative to the wild-type enzyme . Improved interfacial binding was confirmed by direct binding studies with micelles of a zwitterionic substrate analogue, indicating up to five times higher affinity for both variants . Interfacial binding is not improved for the variant N117W/D119Y . Maximal enzyme velocities (Vapp./max) with the zwitterionic substrate were between 25 and 75% of that of the wild-type enzyme . However, competitive inhibition and direct binding studies with a strong inhibitor revealed that the affinity for substrate present at the interface (Km*) is perturbed by the mutations made . For the variant N117W, the slight decrease observed in Vapp./max is most likely made up of a 24-fold reduction in catalytic turnover (kcat) and 18-fold improved substrate binding (Km*).

Eur J Biochem, 1999 Aug, 263(3), 656 - 61
Structure determination of the O-antigenic polysaccharide from the enteroinvasive Escherichia coli O136; Staaf M et al.; The structure of the O-antigen polysaccharide of the lipopolysaccharide from the enteroinvasive Escherichia coli O136 has been elucidated . The composition of the repeating unit was established by sugar and methylation analysis together with 1H and 13C NMR spectroscopy . Two-dimensional nuclear Overhauser effect spectroscopy (NOESY) and heteronuclear multiple-bond correlation experiments were used to deduce the sequence . The absolute configuration for the nonulosonic acid (NonA) could be determined using spin-spin coupling constants, 13C chemical shifts and NOESY . The anomeric configuration of the NonA was determined via vicinal and geminal 13C,1H coupling constants . The structure of the repeating unit of the polysaccharide from E . coli O136 is as follows, in which beta-NonpA is 5,7-diacetamido-3,5,7, 9-tetradeoxy-Lglycero-beta-Lmanno-nonulosonic acid: -->4)-beta-NonpA-(2-->4)-beta-D-Galp-(1-->4)-beta-D-GlcpNAc-(1-->

Proc Natl Acad Sci U S A, 1999 Aug 31, 96(18), 10045 - 50
New routes for lignin biosynthesis defined by biochemical characterization of recombinant ferulate 5-hydroxylase, a multifunctional cytochrome P450-dependent monooxygenase; Humphreys JM et al.; The enzymes and genes of the lignin biosynthetic pathway have been studied for several decades, but the gene encoding ferulate 5-hydroxylase (F5H) was cloned only 3 years ago by T-DNA tagging in Arabidopsis . To characterize the enzyme in detail, we have expressed F5H in yeast . According to current models of the phenylpropanoid pathway, F5H catalyzes the hydroxylation of ferulate to 5-hydroxyferulate; however, our studies indicate that the enzyme also uses coniferaldehyde and coniferyl alcohol as substrates . Unexpectedly, the K(m) values measured for the latter two substrates are three orders of magnitude lower than that measured for ferulic acid, suggesting that in lignifying tissues, syringyl monomers may be derived from their guaiacyl counterparts by hydroxylation and subsequent methylation . Thus, F5H may function later in the lignin biosynthetic pathway than was originally proposed . To further test this model, recombinant F5H was incubated together with ferulic acid, coniferaldehyde, or coniferyl alcohol in the presence of native or recombinant Arabidopsis caffeic acid/5-hydroxyferulic acid O-methyltransferase and {(14)C}S-adenosylmethionine . In all cases, the corresponding radiolabeled sinapyl derivatives were synthesized, indicating that the necessary enzymes required for this pathway are present in Arabidopsis . Taken together, these data suggest that the previously accepted pathway for lignin biosynthesis is likely to be incorrect.

Free Radic Biol Med, 1999 Aug, 27(3-4), 388 - 91
Efficacy of the antioxidant ebselen in experimental uveitis; Bosch-Morell F et al.; Inflammation results in the production of free radicals . In a model of experimental uveitis upon subcutaneous injection of endotoxin to Lewis rats, i.e., endotoxin-induced experimental uveitis (EIU), we have evaluated the status of the antioxidant capacity of ocular tissues . EIU results in a decrease of glutathione (GSH) content and glutathione peroxidase (GPx) activity in whole eye homogenates 24-h after endotoxin administration . Furthermore, an increase in malondialdehyde (MDA) content was observed in these same samples, thus confirming the involvement of oxidative stress in the pathophysiology of the process . In view of the ability of the antioxidant ebselen as GPx enzyme mimic, we tested the effect of the oral treatment with two doses of 100 mg/kg body weight of ebselen (first dose administered at the same time of endotoxin, and the second after 12 h) . Ebselen administration normalized the GSH and MDA contents and protected the GPx activity of the EIU rat eyes . The GPx activity in the eye homogenate of the treated rats could be completely acounted for by the ebselen-dependent GPx-like activity, i.e., GPx activity measured in the acidic supernatant of the homogenate after neutralization . Unmodified ebselen was detected in whole eye homogenates, thus it shows for the first time the penetration of ebselen through the blood-aqueous and blood-retina barrier . The results herein may allow the proposal of ebselen as a suitable antiinflammatory agent in ocular tissues.

Free Radic Biol Med, 1999 Aug, 27(3-4), 271 - 7
Endogenous catechol thioethers may be pro-oxidant or antioxidant; Picklo MJ et al.; Increased catechol thioether formation is associated with Parkinson's disease . In this study, we examined whether catechol thioethers, having a lower oxidation potential than their parent catechols, would cause greater oxidative damage than their parent catechols . We synthesized 5'-S-glutathionyl, cysteinyl, and N-acetylcysteinyl derivatives of dopamine and dopac, encompassing the known catechol thioethers of the mercapturate pathway . Cyclic voltametry studies showed that catechol thioethers had higher reduction potentials than their parent catechols . A higher reduction potential did not correlate with an increase in oxidative damage, measured by metal-catalyzed DNA strand breakage . 5'-S-Glutathionyldopamine and the cysteinyl adducts of dopamine and dopac mediated less oxidative damage than their parent catechols . In contrast, both N-acetylcysteinyl analogs were equipotent to dopamine . Oxygen consumption corresponded to DNA damage except for 5'-S-glutathionyldopamine . The glutathionyl and cysteinyl adducts of dopamine inhibited dopamine-mediated DNA damage indicating that these adducts may have antioxidant properties . 5'-S-Glutathionyldopamine potentiated H2O2-mediated damage whereas 5-S-cysteinyldopamine was inhibitory . Our results show that the ability of catechol thioethers to cause oxidative damage in vitro is not based simply upon the reduction potential but rather, reflects a complex relationship among structures of the parent catechol and thiol adduct, metal catalyst, and oxidant.

Shock, 1999 Jul, 12(1), 75 - 80
Vascular permeability increase and plasma volume loss induced by endotoxin was attenuated by hypertonic saline with or without dextran; de Carvalho H et al.; Endotoxin given intravenously is known to cause plasma leakage and subsequent loss of circulating plasma volume . Hypertonic saline resuscitation has been successfully applied in hemorrhagic and traumatic shock, but its application for the treatment or prevention of septic or endotoxin shock is less well studied . Our aim was to investigate the effects of endotoxin on plasma leakage in hamsters when administered in two different ways: applied locally to the hamster cheek pouch microcirculation or systemically by i.v . injection . The cheek pouch was studied by intravital microscopy using FITC-labeled dextran as a tracer of plasma leakage . Escherichia coli lipopolysaccharide (LPS) was continuously added into the superfusion buffer of the cheek pouch preparation during 120 min in two control groups (each n = 6) and two further groups (each n = 6) treated with either hypertonic saline (HS) or hypertonic saline and dextran (HSD) . Treatment was given as an i.v . injection 0.35 mL NaCl 7.5%/100 g b.w . during 4 min starting 15 min prior to the start of endotoxin application . Endotoxin caused a reversible increase in the number of postcapillary venular leaks with a maximal response at 70 min after start of endotoxin application . The maximal responses were reduced to 36% in the HS-treated and to 37% in the HSD-treated group in comparison to what was seen in the control groups . In the second part of the study endotoxin was given i.v . 0.3 mg/kg to anesthetized hamsters (n = 41) and arterial blood samples were collected at 0, 60, 120, and 180 min after endotoxin injection for measurement of hematocrit and plasma FITC-dextran concentration . Hamsters were divided into seven groups: untreated control group (n = 6); HSC control group given only an i.v . injection of hypertonic saline (n = 6); LPS group given endotoxin 0.3 mg/kg during 1 min (n = 9); HSp group given hypertonic saline (NaCl 7.5%) 10 min prior to i.v . endotoxin (n = 6); HSa group given hypertonic saline 10 min after i.v . endotoxin (n = 6); HSD group given hypertonic saline with dextran 40, 10 min prior to i.v . endotoxin (n = 6); HSD control group given only i.v . hypertonic saline + dextran and no endotoxin (n = 2) . Injection of endotoxin caused a significant increase in hematocrit, which was counteracted by hypertonic saline treatment, with or without dextran, probably due to reduced extravasation of plasma in postcapillary venules.

Int J Biochem Cell Biol, 1999 Jul, 31(7), 769 - 76
Modeling the Leigh syndrome nt8993 T-->C mutation in Escherichia coli F1F0 ATP synthase; Hartzog PE et al.; The mutations in human mitochondrial DNA at nt8993 are associated with a range of neuromuscular disorders . One mutation encodes a proline in place of a leucine conserved in all animal mitochondrial ATPase-6 subunits and bacterial a subunits of F1F0 ATP synthases . This conserved site is leu-156 and leu-207 in humans and Escherichia coli, respectively . An aleu-207-->pro substitution mutation has been constructed in the E . coli F1F0 ATP synthase in order to model the biochemical basis of the human disease mutation . The phenotype of the aleu-207-->pro substitution has been compared to that of the previously studied aleu-207-->arg substitution (Hartzog and Cain, 1993, Journal of Biological Chemistry 268, 12250-12252) . The leu-207-->pro mutation resulted in approximately a 35% decrease in the number of intact enzyme complexes as determined by N, N'-dicyclohexylcarbodiimide-sensitive membrane associated ATP hydrolysis activity and western analysis using an anti-a subunit antibody . A 75% reduction in the efficiency of proton translocation through F1F0 ATP synthase was observed in ATP-driven proton pumping assays . Interestingly, the loss in F1F0 ATP synthase activity resulting from the leu-207-->pro substitution was markedly less dramatic than had been observed for the leu-207-->arg mutation studied earlier . By analogy, the human enzyme may also be affected by the leu-156-->pro substitution to a lesser extent than the leu-156-->arg substitution, and this would account for the milder clinical manifestations of the human leu-156-->pro disease mutations.

Structure Fold Des, 1999 Aug 15, 7(8), 919 - 30
Identification of the Archaeoglobus fulgidus endonuclease III DNA interaction surface using heteronuclear NMR methods; Shekhtman A et al.; BACKGROUND: Endonuclease III is the prototype for a family of DNA-repair enzymes that recognize and remove damaged and mismatched bases from DNA via cleavage of the N-glycosidic bond . Crystal structures for endonuclease III, which removes damaged pyrimidines, and MutY, which removes mismatched adenines, show a highly conserved structure . Although there are several models for DNA binding by this family of enzymes, no experimental structures with bound DNA exist for any member of the family . RESULTS: Nuclear magnetic resonance (NMR) spectroscopy chemical-shift perturbation of backbone nuclei (1H, 15N, 13CO) has been used to map the DNA-binding site on Archaeoglobus fulgidus endonuclease III . The experimentally determined interaction surface includes five structural elements: the helix-hairpin-helix (HhH) motif, the iron-sulfur cluster loop (FCL) motif, the pseudo helix-hairpin-helix motif, the helix B-helix C loop, and helix H . The elements form a continuous surface that spans the active site of the enzyme . CONCLUSIONS: The enzyme-DNA interaction surface for endonuclease III contains five elements of the protein structure and suggests that DNA damage recognition may require several specific interactions between the enzyme and the DNA substrate . Because the target DNA used in this study contained a generic apurinic/apyrimidinic (AP) site, the binding interactions we observed for A . fulgidus endonuclease III should apply to all members of the endonuclease III family and several interactions could apply to the endonuclease III/AlkA (3-methyladenine DNA glycosylase) superfamily.

Chem Biol, 1999 Sep, 6(9), 607 - 15
Heterologous expression, purification, reconstitution and kinetic analysis of an extended type II polyketide synthase; Zawada RJ et al.; BACKGROUND: Polyketide synthases (PKSs) are bacterial multienzyme systems that synthesize a broad range of natural products . The 'minimal' PKS consists of a ketosynthase, a chain length factor, an acyl carrier protein and a malonyl transferase . Auxiliary components (ketoreductases, aromatases and cyclases are involved in controlling the oxidation level and cyclization of the nascent polyketide chain . We describe the heterologous expression and reconstitution of several auxiliary PKS components including the actinorhodin ketoreductase (act KR), the griseusin aromatase/cyclase (gris ARO/CYC), and the tetracenomycin aromatase/cyclase (tcm ARO/CYC) . RESULTS: The polyketide products of reconstituted act and tcm PKSs were identical to those identified in previous in vivo studies . Although stable protein-protein interactions were not detected between minimal and auxiliary PKS components, kinetic analysis revealed that the extended PKS comprised of the act minimal PKS, the act KR and the gris ARO/CYC had a higher turnover number than the act minimal PKS plus the act KR or the act minimal PKS alone . Adding the tcm ARO/CYC to the tcm minimal PKS also increased the overall rate . CONCLUSIONS: Until recently the principal strategy for functional analysis of PKS subunits was through heterologous expression of recombinant PKSs in Streptomyces . Our results corroborate the implicit assumption that the product isolated from whole-cell systems is the dominant product of the PKS . They also suggest that an intermediate is channeled between the various subunits, and pave the way for more detailed structural and mechanistic analysis of these multienzyme systems.

Biotechnol Appl Biochem, 1999 Aug, 30 ( Pt 1), 41 - 5
Detection by HPLC of a trypanothione synthetase activity in vitro from Entamoeba histolytica; Ondarza RN et al.; We have previously demonstrated the presence of glutathione-spermidine (Gsp) and trypanothione {T(SH)(2)} from Entamoeba histolytica trophozoites, on the basis of results obtained with acid extracts purified by Florisil and DEAE-cellulose, derivatized with the fluorescent reagent monobromobimane and separated by HPLC . Gsp was originally found in Escherichia coli and later in trypanosomatids such as Trypanosoma cruzi, T . brucei, T . congolense and the insect trypanosomatid Crithidia fasciculata, along with the novel compound T(SH)(2), N(1), N(8)-bis(glutathionyl)-spermidine . Here we demonstrate the presence of a T(SH)(2) synthetase activity in partly purified extracts from Entamoeba histolytica HK9, incubated at two pH values (6.5 and 7.5) with reduced glutathione (GSH), spermidine and ATP, in the presence of Mg(2+) at different time intervals . The thiol products were detected by HPLC in picomole amounts and compared with commercial Gsp and T(SH)(2) standards . We have used also an extract of Crithidia luciliae as a reference, to compare our results with C . fasciculata, in which the presence of this enzyme has previously been demonstrated and was later purified and separated into two synthetase activities from the same source: one for Gsp and the other for T(SH)(2) . The presence of a T(SH)(2) synthetase activity in Entamoeba histolytica means that this protozoan has a similar metabolism to that of the trypanosomatids and opens the possibility of establishing a rational drug design against this human parasite.

Biotechnol Appl Biochem, 1999 Aug, 30 ( Pt 1), 19 - 25
Purification and properties of Thermus filiformis DNA polymerase expressed in Escherichia coli; Choi JJ et al.; The gene encoding Thermus filiformis (Tfi) DNA polymerase was expressed under the control of the tac promoter on a high-copy plasmid, pJR, in Escherichia coli . The Tfi DNA polymerase was purified by using heat treatment and DEAE-Sephacel column chromatography . The purified enzyme had a molecular mass of 92 kDa, as estimated by SDS/PAGE . The optimum pH and temperature of the enzyme were 8.4-9.0 and 70-72.5 degrees C respectively . The half-life of the enzyme at 94 degrees C was approx . 40 min . The enzyme was activated by the bivalent cations, Mg(2+) and Mn(2+), and was inhibited by EDTA . The optimal Mg(2+) concentration of the enzyme was 4 mM . The optimal conditions for the PCR reaction were slightly different from those for the enzyme activity except for the optimal Mg(2+) concentration . Low concentrations of KCl had no effect on either the enzymic activity or the PCR amplification . The result of the PCR experiment with the enzyme indicates that Tfi DNA polymerase might be useful in DNA amplification.

Nature, 1999 Aug 19, 400(6746), 787 - 92
Four-helical-bundle structure of the cytoplasmic domain of a serine chemotaxis receptor; Kim KK et al.; The bacterial chemotaxis receptors are transmembrane receptors with a simple signalling pathway which has elements relevant to the general understanding of signal recognition and transduction across membranes, how signals are relayed between molecules in a pathway, and how adaptation to a persistent signal is achieved . In contrast to many mammalian receptors which signal by oligomerizing upon ligand binding, the chemotaxis receptors are dimeric even in the absence of their ligands, and their signalling does not depend on a monomer-dimer equilibrium . Bacterial chemotaxis receptors are composed of a ligand-binding domain, a transmembrane domain consisting of two helices TM1 and TM2, and a cytoplasmic domain . All known bacterial chemotaxis receptors have a highly conserved cytoplasmic domain, which unites signals from different ligand domains into a single signalling pathway to flagella motors . Here we report the crystal structure of the cytoplasmic domain of a serine chemotaxis receptor of Escherichia coli, which reveals a 200 A-long coiled-coil of two antiparallel helices connected by a 'U-turn' . Two of these domains form a long, supercoiled, four-helical bundle in the cytoplasmic portion of the receptor.

Nature, 1999 Aug 19, 400(6746), 784 - 7
Nucleosome mobilization catalysed by the yeast SWI/SNF complex; Whitehouse I et al.; The generation of a local chromatin topology conducive to transcription is a key step in gene regulation . The yeast SWI/SNF complex is the founding member of a family of ATP-dependent remodelling activities capable of altering chromatin structure both in vitro and in vivo . Despite its importance, the pathway by which the SWI/SNF complex disrupts chromatin structure is unknown . Here we use a model system to demonstrate that the yeast SWI/SNF complex can reposition nucleosomes in an ATP-dependent reaction that favours attachment of the histone octamer to an acceptor site on the same molecule of DNA (in cis) . We show that SWI/SNF-mediated displacement of the histone octamer is effectively blocked by a barrier introduced into the DNA, suggesting that this redistribution involves sliding or tracking of nucleosomes along DNA, and that it is achieved by a catalytic mechanism . We conclude that SWI/SNF catalyses the redistribution of nucleosomes along DNA in cis, which may represent a general mechanism by which ATP-dependent chromatin remodelling occurs.

Nature, 1999 Aug 19, 400(6746), 781 - 4
Binding of phytochrome B to its nuclear signalling partner PIF3 is reversibly induced by light; Ni M et al.; The phytochrome photoreceptor family directs plant gene expression by switching between biologically inactive and active conformers in response to the sequential absorption of red and farred photons . Several intermediates that act late in the phytochrome signalling pathway have been identified, but fewer have been identified that act early in the pathway . We have cloned a nuclear basic helix-loop-helix protein, PIF3, which can bind to non-photoactive carboxy-terminal fragments of phytochromes A and B and functions in phytochrome signalling in vivo . Here we show that full-length photoactive phytochrome B binds PIF3 in vitro only upon light-induced conversion to its active form, and that photoconversion back to its inactive form causes dissociation from PIF3 . We conclude that photosensory signalling by phytochrome B involves light-induced, conformer-specific recognition of the putative transcriptional regulator PIF3, providing a potential mechanism for direct photoregulation of gene expression.

Lancet, 1999 Aug 21, 354(9179), 635 - 9
Non-pathogenic Escherichia coli versus mesalazine for the treatment of ulcerative colitis: a randomised trial; Rembacken BJ et al.; BACKGROUND: Ulcerative colitis has been suggested to be caused by infection and there is circumstantial evidence linking Escherichia coli with the condition . Our aim was to find out whether the administration of a non-pathogenic strain of E . coli (Nissle 1917) was as effective as mesalazine in preventing relapse of ulcerative colitis . We also examined whether the addition of E . coli to standard medical therapy increased the chance of remission of active ulcerative colitis . METHODS: This was a single-centre, randomised, double-dummy study in which 120 patients with active ulcerative colitis were invited to take part . 116 patients accepted; 59 were randomised to mesalazine and 57 to E . coli . All patients also received standard medical therapy together with a 1-week course of oral gentamicin . After remission, patients were maintained on either mesalazine or E . coli and followed up for a maximum of 12 months . A two-stage, conditional, intention-to-treat analysis was done . FINDINGS: 44 (75%) patients in the mesalazine group attained remission compared with 39 (68%) in the E . coli group . Mean time to remission was 44 days (median 42) in the mesalazine group and 42 days (median 37) for those treated with E . coli . In the mesalazine group, 32 (73%) patients relapsed compared with 26 (67%) in the E . coli group . Mean duration of remission was 206 days in the mesalazine group (median 175) and 221 days (median 185) in the E . coli group . INTERPRETATION: Our results suggest that treatment with a non-pathogenic E . coli has an equivalent effect to mesalazine in maintaining remission of ulcerative colitis . The beneficial effect of live E . coli may provide clues to the cause of ulcerative colitis.

Dev Growth Differ, 1999 Aug, 41(4), 401 - 6
Newt RAD51: cloning of cDNA and analysis of gene expression during spermatogenesis; Yamamoto T et al.; A cDNA encoding a newt homolog of Escherichia coli RecA and yeast RAD51 from a testis cDNA library was isolated . The newt RAD51 (nRAD51) cDNA predicted a 337 amino acid protein with a 95-96% amino acid identity to Xenopus and mammalian RAD51 . Northern blot analysis showed that nRAD51 mRNA, 1.7 kb in length, was expressed strongly in the testis and ovary, but weakly in the liver, kidney and brain . In situ hybridization revealed that expression of nRAD51 mRNA was barely observed in primary spermatogonia (one cell in a cyst) and early secondary spermatogonia (two to four cells in a cyst), but increased in late secondary spermatogonia (> or =eight cells in a cyst), reaching a maximum level in leptotene-zygotene spermatocytes, and thereafter declined . These results suggest that nRAD51 is involved in mitotic recombination in spermatogonia as well as in meiotic recombination in spermatocytes.

J Gen Virol, 1999 Aug, 80 ( Pt 8), 2193 - 203
Epstein-Barr virus lacking latent membrane protein 2 immortalizes B cells with efficiency indistinguishable from that of wild-type virus; Speck P et al.; Epstein-Barr virus (EBV) is a human herpesvirus that efficiently transforms and immortalizes human primary B lymphocytes . In this study, the role of latent membrane protein 2 (LMP2) in EBV growth transformation was investigated . LMP2 is a virally encoded membrane protein expressed in EBV-immortalized B cells previously shown to be nonessential for EBV transformation . However, a recent study reported that LMP2 may be an important determinant for efficient B cell transformation (Brielmeier et al., Journal of General Virology 77, 2807-2818, 1996) . In this study a deletion mutation was introduced into the LMP2 gene using an E . coli mini-EBV construct containing sufficient EBV DNA to result in growth transformation of primary B cells . In an alternative approach, the introduction of the gene encoding the enhanced green fluorescent protein (EGFP) by homologous recombination into the LMP2 gene of EBV strain B95-8, generating the same LMP2 deletion mutation is reported . Careful quantification of B cell transformation using the EGFP+ LMP2- recombinant virus determined that in liquid culture medium or in culture medium containing soft agarose there was no difference in the ability of LMP2- virus to immortalize primary human B cells when compared to that of wild-type virus.

J Gen Virol, 1999 Aug, 80 ( Pt 8), 2157 - 64
Nucleolar localization of the UL3 protein of herpes simplex virus type 2; Yamada H et al.; A rabbit polyclonal antiserum was raised against a recombinant 6 x His-UL3 fusion protein expressed in Escherichia coli and used to examine the intracellular localization of the UL3 protein of herpes simplex virus type 2 (HSV-2) . The antiserum reacted specifically with 31 and 34 kDa proteins in HSV-2 186-infected Vero cells and with 31 and 35 kDa proteins in UL3-expressing COS-7 cells . The UL3 protein localized both in the cytoplasm and in five to ten bright fluorescent granules in the nucleus close to the nuclear membrane at 4 h post-infection (p.i.) . These structures became bigger at 5 h p.i . and showed doughnut-like forms at 6 h p.i . In transfected Vero cells, the UL3 protein localized exclusively in the nucleoplasm and specifically in the nucleolus . Five deletion mutants of the UL3 protein were constructed for transfection assays and the results showed that the region containing amino acids 100-164 was important for nucleolar localization . Moreover, green fluorescent protein (GFP)-targetting experiments showed that the region containing amino acids 100-164 was able to transport non-nucleolar GFP to the nucleolus as a fusion protein.

Lancet, 1999 Aug 21, 354(9179), 649 - 50
Antibodies to lipopolysaccharides of Escherichia coli serogroups O5 and O165 in healthy adults; Jenkins C et al.; The observation that over 50% of healthy blood donors have serum antibodies to the lipopolysaccharide of Escherichia coli O5 and O165 influences the serodiagnosis of infection with verocytotoxin-producing E . coli.

Vet Microbiol, 1999 Jul 1, 67(4), 307 - 10
Genotypic prevalence of F4 variants (ab, ac, and ad) in Escherichia coli isolated from diarrheic piglets in Korea; Choi C et al.; A total of 812 Escherichia coli strains isolated from diarrheic piglets were tested for the presence of the F4 (K88) variant (ab, ac and ad) gene by the polymerase chain reaction . Forty four (5.4%) of the 812 E . coli strains carried genes for F4 . Among the 44 isolates known to carry genes for F4, 42 (96%) isolates contained genes for F4ac and 2 (4%) isolates contained genes for F4ab . None of the E . coli strains carried genes for F4ad . Our data show that F4ac is the predominant F4 variant associated with diarrhea in piglets in Korea.

Biophys J, 1999 Sep, 77(3), 1619 - 26
Cooperative folding units of escherichia coli tryptophan repressor; Wallqvist A et al.; A previously published computational procedure was used to identify cooperative folding units within tryptophan repressor . The theoretical results predict the existence of distinct stable substructures in the protein chain for the monomer and the dimer . The predictions were compared with experimental data on structure and folding of the repressor and its proteolytic fragments and show excellent agreement for the dimeric form of the protein . The results suggest that the monomer, the structure of which is currently unknown, is likely to have a structure different from the one it has within the context of the highly intertwined dimer . Application of this method to the repressor monomer represents an extension of the computations into the realm of evaluating hypothetical structures such as those produced by threading.

J Biol Chem, 1999 Sep 3, 274(36), 25849 - 54
The anion-stimulated ATPase ArsA shows unisite and multisite catalytic activity; Kaur P; ArsA, an anion-stimulated ATPase, consists of two nucleotide binding domains, A1 in the N terminus and A2 in the C terminus of the protein, connected by a linker . The A1 domain contains a high affinity ATP binding site, whereas the A2 domain has low affinity and it requires the allosteric ligand antimonite for binding ATP . ArsA is known to form a UV-activated adduct with {alpha-(32)P}ATP in the linker region . This study shows that on addition of antimonite, much more adduct is formed . Characterization of the nature of the adduct suggests that it is between ArsA and ADP, instead of ATP, indicating that the adduct formation reflects hydrolysis of ATP . The present study also demonstrates that the A1 domain is capable of carrying out unisite catalysis in the absence of antimonite . On addition of antimonite, multisite catalysis involving both A1 and A2 sites occurs, resulting in a 40-fold increase in ATPase activity . Studies with mutant proteins suggest that the A2 site may be second in the sequence of events, so that its role in catalysis is dependent on a functional A1 site . It is also proposed that ArsA goes through an ATP-bound and an ADP-bound conformation, and the linker region, where ADP binds under both unisite and multisite catalytic conditions, may play an important role in the energy transduction process.

Bioorg Med Chem Lett, 1999 Aug 2, 9(15), 2237 - 42
A 2-nitroimidazole carbamate prodrug of 5-amimo-1-(chloromethyl)-3-{(5,6,7-trimethoxyindol-2-yl)carbony l}-1,2-dihydro-3H--benz{E}indole (amino-seco-CBI-TMI) for use with ADEPT and GDEPT; Hay MP et al.; The synthesis of a 2-nitroimidazol-5-ylmethyl carbamate prodrug 10 of the potent minor groove alkylating agent amino-seco-CBI-TMI 3 is described . Chemical, radiolytic, and enzymic reductions of a model 2-nitroimidazol-5-yl carbamate 8 show release of the amine effector upon reduction . Prodrug 10 gives a ten fold increase in cytotoxicity against human ovarian carcinoma SKOV3 cells in the presence of E . coli B nitroreductase (NTR) and a 21-fold increase in cytotoxicity against a SKOV3 cell line (SC3.2) transfected with the gene for NTR . The cytotoxicity of 10 increased 15- to 40-fold under hypoxia . Prodrug 10 has significant potential as a prodrug for use in ADEPT and GDEPT applications, and as a hypoxia-selective cytotoxin.

J Food Prot, 1997 Jan, 60(1), 84 - 7
Growth of an Aspergillus flavus transformant expressing Escherichia coli beta-glucuronidase in maize kernels resistant to aflatoxin production; Brown RL et al.; Kernels of a maize inbred that demonstrated resistance to aflatoxin production in previous studies were inoculated with an Aspergillus flavus strain containing the Escherichia coli beta-D-glucuronidase reporter gene linked to a beta-tubulin gene promoter and assessed for both fungal growth and aflatoxin accumulation . Prior to inoculation, kernels were pin-wounded through the pericarp to the endosperm, pin-wounded in the embryo region, or left unwounded . After 7 days incubation with the fungus, beta-glucuronidase activity (fungal growth) in the kernels was quantified using a fluorogenic assay and aflatoxin B content of the same kernels was analyzed . Kernels of a susceptible inbred, similarly treated, served as controls . Results indicate a positive relationship between aflatoxin levels and the amount of fungal growth . However, resistant kernels wounded through the pericarp to the endosperm before inoculation supported an increase in aflatoxin B over levels observed in nonwounded kernels, without an increase in fungal growth . Wounding kernels of the resistant inbred through the embryo resulted in both the greatest fungal growth and the highest levels of aflatoxin B1 for this genotype . Maintenance of resistance to aflatoxin B1 in endosperm-wounded kernels may be due to the action of a mechanism which limits fungal access to the kernel embryo.

Mol Biotechnol, 1999 Apr, 11(2), 117 - 28
Purification and characterization of two recombinant human granulocyte colony-stimulating factor glycoforms . Pharmacokinetic and activity studies of single-dose administration in mice; Rotondaro L et al.; Two recombinant human granulocyte colony-stimulating factor (rhG-CSF) isoforms were isolated from the medium conditioned by an engineered Chinese hamster ovary (CHO) cell line . The two rhG-CSFs were characterized and were found to differ in the carbohydrate structure attached to Thr-133 . The glycoform, referred to as Peak 1, contains the O-linked glycan Neu5Ac(alpha 2-3)Gal(beta 1-3)GalNAc; the Peak 2 glycoform contains the O-linked glycan Neu5Ac(alpha 2-3)Gal(beta 1-3){Neu5Ac(alpha 2-6)}GalNAc . The two glycoforms displayed a similar biological activity in cultures of a mouse 32D C13 cell line and human bone-marrow myelo-monocytic progenitor cells (CFU-GM) . In the latter test both glycoforms displayed a higher activity than nonglycosylated rMet-hG-CSF from Escherichia coli . The pharmacokinetic profile and activity of the two rhG-CSF glycoforms and of a mixture of them (Pool) were investigated in mice treated with a single injection of rhG-CSF at the doses of 125 micrograms and 250 micrograms/kg, given via the intravenous (i.v.) and the subcutaneous (s.c.) route, respectively . The plasma concentration profiles obtained were similar for all three substances and did not show any relevant differences in absorption or elimination . The pharmacokinetic parameters indicate that the three substances have similar area under the curve (AUCs), volumes of distribution, and terminal half-life . Furthermore, our data indicate a high bioavailability of the two different glycoforms of rhG-CSF when given to mice via the s.c . route either singularly or as a mixture . Detectable levels of rhG-CSF persisted for more than 8 h in the i.v . and more than 24 h in the s.c . route of administration . All three substances induced early neutrophilia in mice . All rhG-CSF-treated mice developed a two-four-fold rise in neutrophil counts as early as 4 h after the intravenous and 2 h after the subcutaneous injection . Relatively high levels of neutrophils were maintained for at least 8 and 24 h after i.v . and s.c . administration, respectively.

J Biol Chem, 1999 Sep 3, 274(36), 25814 - 20
The mouse gene PDCR encodes a peroxisomal delta(2), delta(4)-dienoyl-CoA reductase; Geisbrecht BV et al.; Here we describe the identification and characterization of a novel mouse gene, PDCR, that encodes a peroxisomal Delta(2), Delta(4)-dienoyl-CoA reductase . The mouse PDCR cDNA contains an 892-base pair open reading frame and is predicted to encode a 292-amino acid protein with a deduced molecular mass of 31,298 Da that terminates in a consensus type-1 peroxisomal targeting signal . Purified recombinant PDCR protein was generated from Escherichia coli and catalyzed the NADPH-dependent reduction of Delta(2)-trans, Delta(4)-trans-decadienoyl-CoA with a specific activity of 20 units/mg . Enzymatic characterization followed by high pressure liquid chromatography analysis of the products revealed that PDCR converted Delta(2)-trans,Delta(4)-trans-decadienoyl-CoA to a Delta(3)-enoyl-CoA but not to a Delta(2)-enoyl-CoA . Kinetic analyses demonstrated that PDCR is active on a broad range of Delta(2), Delta(4)-dienoyl-CoAs . Although the observed substrate preference was to Delta(2)-trans,Delta(4)-trans-decadienoyl-CoA, PDCR was also active on a C(22) substrate with multiple unsaturations, a result consistent with the role of peroxisomes in the oxidation of complex, very long chain, polyunsaturated fatty acids . The presence of a type-1 peroxisomal targeting signal Ala-Lys-Leu-COOH at the C terminus of PDCR suggested that this protein may be peroxisomal . We observed that tagged PDCR was efficiently transported to the peroxisome lumen in normal human fibroblasts but not in cells derived from a Zellweger syndrome patient with a specific defect in peroxisomal matrix protein import . We conclude that this protein resides within the peroxisome matrix and therefore represents the first mammalian peroxisomal Delta(2),Delta(4)-dienoyl-CoA reductase to be characterized at the molecular level.

J Biol Chem, 1999 Sep 3, 274(36), 25792 - 800
DNA methylation at mammalian replication origins; Rein T et al.; In Escherichia coli, DNA methylation regulates both origin usage and the time required to reassemble prereplication complexes at replication origins . In mammals, at least three replication origins are associated with a high density cluster of methylated CpG dinucleotides, and others whose methylation status has not yet been characterized have the potential to exhibit a similar DNA methylation pattern . One of these origins is found within the approximately 2-kilobase pair region upstream of the human c-myc gene that contains 86 CpGs . Application of the bisulfite method for detecting 5-methylcytosines at specific DNA sequences revealed that this region was not methylated in either total genomic DNA or newly synthesized DNA . Therefore, DNA methylation is not a universal component of mammalian replication origins . To determine whether or not DNA methylation plays a role in regulating the activity of origins that are methylated, the rate of remethylation and the effect of hypomethylation were determined at origin beta (ori-beta), downstream of the hamster DHFR gene . Remethylation at ori-beta did not begin until approximately 500 base pairs of DNA was synthesized, but it was then completed by the time that 4 kilobase pairs of DNA was synthesized (<3 min after release into S phase) . Thus, DNA methylation cannot play a significant role in regulating reassembly of prereplication complexes in mammalian cells, as it does in E . coli . To determine whether or not DNA methylation plays any role in origin activity, hypomethylated hamster cells were examined for ori-beta activity . Cells that were >50% reduced in methylation at ori-beta no longer selectively activated ori-beta . Therefore, at some loci, DNA methylation either directly or indirectly determines where replication begins.

J Biol Chem, 1999 Sep 3, 274(36), 25599 - 607
Mode analysis of a fatty acid molecule binding to the N-terminal 8-kDa domain of DNA polymerase beta . A 1:1 complex and binding surface; Mizushina Y et al.; We reported previously that long-chain fatty acids are potent inhibitors of mammalian DNA polymerase beta . At present, based on information available from the NMR structure of the N-terminal 8-kDa domain, we examined the structural interaction with the 8-kDa domain using two species, C(18)-linoleic acid (LA) or C(24)-nervonic acid (NA) . In the 8-kDa domain with LA or NA, the structure that forms the interaction interface included helix-1, helix-2, helix-4, the three turns (residues 1-13, 48-51, and 79-87) and residues adjacent to an Omega-type loop connecting helix-1 and helix-2 of the same face . No significant shifts were observed for any of the residues on the opposite side of the 8-kDa domain . The NA interaction interface on the amino acid residues of the 8-kDa domain fragment was mostly the same as that of LA, except that the shifted cross-peaks of Leu-11 and Thr-79 were significantly changed between LA and NA . The 8-kDa domain bound to LA or NA as a 1:1 complex with a dissociation constant (K(D)) of 1.02 or 2.64 mM, respectively.

J Biol Chem, 1999 Sep 3, 274(36), 25583 - 7
Dimerization of guanylyl cyclase-activating protein and a mechanism of photoreceptor guanylyl cyclase activation; Olshevskaya EV et al.; Ca(2+)-binding guanylyl cyclase-activating proteins (GCAPs) stimulate photoreceptor membrane guanylyl cyclase (retGC) in the light when the free Ca(2+) concentrations in photoreceptors decrease from 600 to 50 nM . RetGC activated by GCAPs exhibits tight dimerization revealed by chemical cross-linking (Yu, H., Olshevskaya, E., Duda, T., Seno, K., Hayashi, F., Sharma, R . K., Dizhoor, A . M., and Yamazaki, A . (1999) J . Biol . Chem . 274, 15547-15555) . We have found that the Ca(2+)-loaded GCAP-2 monomer undergoes reversible dimerization upon dissociation of Ca(2+) . The ability of GCAP-2 and its several mutants to activate retGC in vitro correlates with their ability to dimerize at low free Ca(2+) concentrations . A constitutively active GCAP-2 mutant E80Q/E116Q/D158N that stimulates retGC regardless of the free Ca(2+) concentrations forms dimers both in the absence and in the presence of Ca(2+) . Several GCAP-2/neurocalcin chimera proteins that cannot efficiently activate retGC in low Ca(2+) concentrations are also unable to dimerize in the absence of Ca(2+) . Additional mutation that restores normal activity of the GCAP-2 chimera mutant also restores its ability to dimerize in the absence of Ca(2+) . These results suggest that dimerization of GCAP-2 can be a part of the mechanism by which GCAP-2 regulates the photoreceptor guanylyl cyclase . The Ca(2+)-free GCAP-1 is also capable of dimerization in the absence of Ca(2+), but unlike GCAP-2, dimerization of GCAP-1 is resistant to the presence of Ca(2+).

J Biol Chem, 1999 Sep 3, 274(36), 25550 - 4
Additive effects of beta chain mutations in low oxygen affinity hemoglobin betaF41Y,K66T; Baudin-Creuza V et al.; In order to decrease significantly the oxygen affinity of human hemoglobin, we have associated the mutation betaF41Y with another point mutation also known to decrease the oxygen affinity of Hb . We have synthesized a recombinant Hb (rHb) with two mutations in the beta chains: rHb betaF41Y,K66T . In the absence of 2, 3-diphosphoglycerate, additive effects of the mutations are evident, since the doubly mutated Hb exhibits a larger decrease in oxygen affinity than for the individual single mutations . In the presence of 2,3-diphosphoglycerate, the second mutation did not significantly increase the P(50) value relative to the single mutations . However, the kinetics of CO binding still indicate combined effects on the allosteric equilibrium, as evidenced by more of the slow bimolecular phase characteristic of binding to the deoxy conformation . Dimer-tetramer equilibrium studies indicate an increase in stability of the mutants relative to rHb A; the double mutant rHb betaF41Y, K66T at pH 7.5 showed a K(4,2) value of 0.26 microM . Despite the lower oxygen affinity, the single mutant betaF41Y and double mutant betaF41Y,K66T show only a moderate increase of 20% in the autoxidation rate . These mutations are thus of interest in developing a Hb-based blood substitute.

J Biol Chem, 1999 Sep 3, 274(36), 25481 - 9
alpha-synuclein binds to Tau and stimulates the protein kinase A-catalyzed tau phosphorylation of serine residues 262 and 356; Jensen PH et al.; alpha-Synuclein has been implicated in the pathogenesis of several neurodegenerative disorders based on the direct linking of missense mutations in alpha-synuclein to autosomal dominant Parkinson's disease and its presence in Lewy-like lesions . To gain insight into alpha-synuclein functions, we have investigated whether it binds neuronal proteins and modulates their functional state . The microtubule-associated protein tau was identified as a ligand by alpha-synuclein affinity chromatography of human brain cytosol . Direct binding assays using (125)I-labeled human tau40 demonstrated a reversible binding with a IC(50) about 50 pM . The interacting domains were localized to the C terminus of alpha-synuclein and the microtubule binding region of tau as determined by protein fragmentation and the use of recombinant peptides . High concentrations of tubulin inhibited the binding between tau and alpha-synuclein . Functionally, alpha-synuclein stimulated the protein kinase A-catalyzed phosphorylation of tau serine residues 262 and 356 as determined using a phospho-epitope-specific antibody . We propose that alpha-synuclein modulates the phosphorylation of soluble axonal tau and thereby indirectly affects the stability of axonal microtubules.

J Biol Chem, 1999 Sep 3, 274(36), 25335 - 42
Role of walker motif A of RuvB protein in promoting branch migration of holliday junctions . Walker motif a mutations affect Atp binding, Atp hydrolyzing, and DNA binding activities of Ruvb; Hishida T et al.; Escherichia coli RuvB protein, an ATP-dependent hexameric DNA helicase, acts together with RuvA protein to promote branch migration of Holliday junctions during homologous recombination and recombinational repair . To elucidate the role of the Walker motif A of RuvB (GXGKT; X indicates a nonconserved residue) in ATP hydrolysis and branch migration activities, we constructed four ruvB mutant genes by site-directed mutagenesis, altering the highly conserved Lys(68) and Thr(69) . K68R, K68A, and T69A mutants except T69S failed to complement UV-sensitive phenotype of the ruvB strain . These three mutant proteins, when overexpressed, made the wild-type strain UV-sensitive to varying degrees . K68R, K68A, and T69A were defective in ATP hydrolysis and branch migration activities in vitro . In the presence of Mg(2+), K68R showed markedly reduced affinity for ATP, while K68A and T69A showed only mild reduction . K68A and T69A could form hexamers in the presence of Mg(2+) and ATP, while K68R failed to form hexamers and existed instead as a higher oligomer, probably a dodecamer . In contrast to wild-type RuvB, K68R, K68A, and T69A by themselves were defective in DNA binding . However, RuvA could facilitate binding of K68A and T69A to DNA, whereas it could not promote binding of K68R to DNA . All of the three mutant RuvBs could physically interact with RuvA . These results indicate the direct involvement in ATP binding and ATP hydrolysis of the invariant Lys(68) and Thr(69) residues of Walker motif A of RuvB and suggest that these residues play key roles in interrelating these activities with the conformational change of RuvB, which is required for the branch migration activity.

J Biol Chem, 1999 Sep 3, 274(36), 25285 - 90
Functions of the sigma(54) region I in trans and implications for transcription activation; Gallegos MT et al.; Control of transcription frequently involves the direct interaction of activators with RNA polymerase . In bacteria, the formation of stable open promoter complexes by the sigma(54) RNA polymerase is critically dependent on sigma(54) amino Region I sequences . Their presence correlates with activator dependence, and removal allows the holoenzyme to engage productively with melted DNA independently of the activator . Using purified Region I sequences and holoenzymes containing full-length or Region I-deleted sigma(54), we have explored the involvement of Region I in transcription activation . Results show that Region I in trans inhibits a reversible conformational change in the holoenzyme believed to be polymerase isomerization . Evidence is presented indicating that the holoenzyme (and not the promoter DNA per se) is one interacting target used by Region I in preventing polymerase isomerization . Activator overcomes this inhibition in a reaction requiring nucleotide hydrolysis . Region I in trans is able to inhibit activated transcription by the holoenzyme containing full-length sigma(54) . Inhibition appeared to be noncompetitive with respect to the activator, suggesting that a direct activator interaction occurs with parts of the holoenzyme outside Region I . Stabilization of isomerized holoenzyme bound to melted DNA by Region I in trans occurs largely independently of the initiating nucleotide, suggesting a role for Region I in maintaining the open complex.

J Biol Chem, 1999 Sep 3, 274(36), 25254 - 9
Importance of redox potential for the in vivo function of the cytoplasmic disulfide reductant thioredoxin from Escherichia coli; Mossner E et al.; The thioredoxin superfamily consists of enzymes that catalyze the reduction, formation, and isomerization of disulfide bonds and exert their activity through a redox active disulfide in a Cys-Xaa(1)-Xaa(2)-Cys motif . The individual members of the family differ strongly in their intrinsic redox potentials . However, the role of the different redox potentials for the in vivo function of these enzymes is essentially unknown . To address the question of in vivo importance of redox potential for the most reducing member of the enzyme family, thioredoxin, we have employed a set of active site variants of thioredoxin with increased redox potentials (-270 to -195 mV) for functional studies in the cytoplasm of Escherichia coli . The variants proved to be efficient substrates of thioredoxin reductase, providing a basis for an in vivo characterization of NADPH-dependent reductive processes catalyzed by the thioredoxin variants . The reduction of sulfate and methionine sulfoxide, as well as the isomerization of periplasmic disulfide bonds by DsbC, which all depend on thioredoxin as catalyst in the E . coli cytoplasm, proved to correlate well with the intrinsic redox potentials of the variants in complementation assays . The same correlation could be established in vitro by using the thioredoxin-catalyzed reduction of lipoic acid by NADPH as a model reaction . We propose that the rate of direct reduction of substrates by thioredoxin, which largely depends on the redox potential of thioredoxin, is the most important parameter for the in vivo function of thioredoxin, as recycling of reduced thioredoxin through NADPH and thioredoxin reductase is not rate-limiting for its catalytic cycle.

J Bacteriol, 1999 Sep, 181(17), 5534 - 8
Identification of a conserved N-terminal sequence involved in transmembrane signal transduction in EnvZ; Waukau J et al.; To determine whether N-terminal sequences are involved in the transmembrane signaling mechanism of EnvZ, the nucleotide sequences of envZ genes from several enteric bacteria were determined . Comparative analysis revealed that the amino acid sequence between Pro41 and Glu53 was highly conserved . To further analyze the role of the conserved sequence, envZ of Escherichia coli was subjected to random PCR mutagenesis and mutant alleles that produced a high-osmolarity phenotype, in which ompF was repressed, were isolated . The mutations identified clustered within, as well as adjacent to, the Pro41-to-Glu53 sequence . These findings suggest that the conserved Pro41-to-Glu53 sequence is involved in the signal transduction mechanism of EnvZ.

J Bacteriol, 1999 Sep, 181(17), 5527 - 9
Clustering of the chemoreceptor complex in Escherichia coli is independent of the methyltransferase CheR and the methylesterase CheB; Lybarger SR et al.; The Escherichia coli chemoreceptors and their associated cytoplasmic proteins, CheA and CheW, cluster predominantly at the cell poles . The nature of the clustering remains a mystery . Recent studies suggest that CheR binding to and/or methylation of the chemoreceptors may play a role in chemoreceptor complex aggregation . In this study, we examined the intracellular distribution of the chemoreceptors by immunoelectron microscopy in strains lacking either the methyltransferase CheR or the methylesterase CheB . The localization data revealed that, in vivo, aggregation of the chemoreceptor complex was independent of either CheR or CheB.

J Bacteriol, 1999 Sep, 181(17), 5516 - 20
Molecular characterization of the PhoP-PhoQ two-component system in Escherichia coli K-12: identification of extracellular Mg2+-responsive promoters; Kato A et al.; We identified Mg2+-responsive promoters of the phoPQ, mgtA, and mgrB genes of Escherichia coli K-12 by S1 nuclease analysis . Expression of these genes was induced by magnesium limitation and depended on PhoP and PhoQ . The transcription start sites were also determined, which allowed us to find a (T/G)GTTTA direct repeat in their corresponding promoter regions.

J Bacteriol, 1999 Sep, 181(17), 5509 - 11
The Escherichia coli NadR regulator is endowed with nicotinamide mononucleotide adenylyltransferase activity; Raffaelli N et al.; The first identification and characterization of a catalytic activity associated with NadR protein is reported . A computer-aided search for sequence similarity revealed the presence in NadR of a 29-residue region highly conserved among known nicotinamide mononucleotide adenylyltransferases . The Escherichia coli nadR gene was cloned into a T7-based vector and overexpressed . In addition to functionally specific DNA binding properties, the homogeneous recombinant protein catalyzes NAD synthesis from nicotinamide mononucleotide and ATP.

J Bacteriol, 1999 Sep, 181(17), 5461 - 6
Molecular analysis of the gene encoding a novel transglycosylative enzyme from Alteromonas sp . strain O-7 and its physiological role in the chitinolytic system; Tsujibo H et al.; We purified from the culture supernatant of Alteromonas sp . strain O-7 and characterized a transglycosylating enzyme which synthesized beta-(1-->6)-(GlcNAc)2, 2-acetamido-6-O-(2-acetamido-2-deoxy-beta-D-glucopyranosyl)-2- deoxyglucopyranose from beta-(1-->4)-(GlcNAc)2 . The gene encoding a novel transglycosylating enzyme was cloned into Escherichia coli, and its nucleotide sequence was determined . The molecular mass of the deduced amino acid sequence of the mature protein was determined to be 99,560 Da which corresponds very closely with the molecular mass of the cloned enzyme determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis . The molecular mass of the cloned enzyme was much larger than that of enzyme (70 kDa) purified from the supernatant of this strain . These results suggest that the native enzyme was the result of partial proteolysis occurring in the N-terminal region . The enzyme showed significant sequence homology with several bacterial beta-N-acetylhexosaminidases which belong to family 20 glycosyl hydrolases . However, this novel enzyme differs from all reported beta-N-acetylhexosaminidases in its substrate specificity . To clarify the role of the enzyme in the chitinolytic system of the strain, the effect of beta-(1-->6)-(GlcNAc)2 on the induction of chitinase was investigated . beta-(1-->6)-(GlcNAc)2 induced a level of production of chitinase similar to that induced by the medium containing chitin . On the other hand, GlcNAc, (GlcNAc)2, and (GlcNAc)3 conversely repressed the production of chitinase to below the basal level of chitinase activity produced constitutively in medium without a carbon source.

J Bacteriol, 1999 Sep, 181(17), 5303 - 8
The napF and narG nitrate reductase operons in Escherichia coli are differentially expressed in response to submicromolar concentrations of nitrate but not nitrite; Wang H et al.; Escherichia coli synthesizes two biochemically distinct nitrate reductase enzymes, a membrane-bound enzyme encoded by the narGHJI operon and a periplasmic cytochrome c-linked nitrate reductase encoded by the napFDAGHBC operon . To address why the cell makes these two enzymes, continuous cell culture techniques were used to examine napF and narG gene expression in response to different concentrations of nitrate and/or nitrite . Expression of the napF-lacZ and narG-lacZ reporter fusions in strains grown at different steady-state levels of nitrate revealed that the two nitrate reductase operons are differentially expressed in a complementary pattern . The napF operon apparently encodes a "low-substrate-induced" reductase that is maximally expressed only at low levels of nitrate . Expression is suppressed under high-nitrate conditions . In contrast, the narGHJI operon is only weakly expressed at low nitrate levels but is maximally expressed when nitrate is elevated . The narGHJI operon is therefore a "high-substrate-induced" operon that somehow provides a second and distinct role in nitrate metabolism by the cell . Interestingly, nitrite, the end product of each enzyme, had only a minor effect on the expression of either operon . Finally, nitrate, but not nitrite, was essential for repression of napF gene expression . These studies reveal that nitrate rather than nitrite is the primary signal that controls the expression of these two nitrate reductase operons in a differential and complementary fashion . In light of these findings, prior models for the roles of nitrate and nitrite in control of narG and napF expression must be reconsidered.

J Bacteriol, 1999 Sep, 181(17), 5263 - 72
The Cpx envelope stress response is controlled by amplification and feedback inhibition; Raivio TL et al.; In Escherichia coli, the Cpx two-component regulatory system activates expression of protein folding and degrading factors in response to misfolded proteins in the bacterial envelope (inner membrane, periplasm, and outer membrane) . It is comprised of the histidine kinase CpxA and the response regulator CpxR . This response plays a role in protection from stresses, such as elevated pH, as well as in the biogenesis of virulence factors . Here, we show that the Cpx periplasmic stress response is subject to amplification and repression through positive and negative autofeedback mechanisms . Western blot and operon fusion analyses demonstrated that the cpxRA operon is autoactivated . Conditions that lead to elevated levels of phosphorylated CpxR cause a concomitant increase in transcription of cpxRA . Conversely, overproduction of CpxP, a small, Cpx-regulated protein of previously unknown function, represses the regulon and can block activation of the pathway . This repression is dependent on an intact CpxA sensing domain . The ability to autoactivate and then subsequently repress allows for a temporary amplification of the Cpx response that may be important in rescuing cells from transitory stresses and cueing the appropriately timed elaboration of virulence factors.

J Bacteriol, 1999 Sep, 181(17), 5257 - 62
Suppression of nonsense mutations induced by expression of an RNA complementary to a conserved segment of 23S rRNA; Chernyaeva NS et al.; We identified a short RNA fragment, complementary to the Escherichia coli 23S rRNA segment comprising nucleotides 735 to 766 (in domain II), which when expressed in vivo results in the suppression of UGA nonsense mutations in two reporter genes . Neither UAA nor UAG mutations, examined at the same codon positions, were suppressed by the expression of this antisense rRNA fragment . Our results suggest that a stable phylogenetically conserved hairpin at nucleotides 736 to 760 in 23S rRNA, which is situated close to the peptidyl transferase center, may participate in one or more specific interactions during peptide chain termination.

J Bacteriol, 1999 Sep, 181(17), 5210 - 8
Effect of ionic strength on initial interactions of Escherichia coli with surfaces, studied on-line by a novel quartz crystal microbalance technique; Otto K et al.; A novel quartz crystal microbalance (QCM) technique was used to study the adhesion of nonfimbriated and fimbriated Escherichia coli mutant strains to hydrophilic and hydrophobic surfaces at different ionic strengths . This technique enabled us to measure both frequency shifts (Deltaf), i.e., the increase in mass on the surface, and dissipation shifts (DeltaD), i.e., the viscoelastic energy losses on the surface . Changes in the parameters measured by the extended QCM technique reflect the dynamic character of the adhesion process . We were able to show clear differences in the viscoelastic behavior of fimbriated and nonfimbriated cells attached to surfaces . The interactions between bacterial cells and quartz crystal surfaces at various ionic strengths followed different trends, depending on the cell surface structures in direct contact with the surface . While Deltaf and DeltaD per attached cell increased for nonfimbriated cells with increasing ionic strengths (particularly on hydrophobic surfaces), the adhesion of the fimbriated strain caused only low-level frequency and dissipation shifts on both kinds of surfaces at all ionic strengths tested . We propose that nonfimbriated cells may get better contact with increasing ionic strengths due to an increased area of contact between the cell and the surface, whereas fimbriated cells seem to have a flexible contact with the surface at all ionic strengths tested . The area of contact between fimbriated cells and the surface does not increase with increasing ionic strengths, but on hydrophobic surfaces each contact point seems to contribute relatively more to the total energy loss . Independent of ionic strength, attached cells undergo time-dependent interactions with the surface leading to increased contact area and viscoelastic losses per cell, which may be due to the establishment of a more intimate contact between the cell and the surface . Hence, the extended QCM technique provides new qualitative information about the direct contact of bacterial cells to surfaces and the adhesion mechanisms involved.

J Bacteriol, 1999 Sep, 181(17), 5185 - 92
Amino acid-DNA contacts by RhaS: an AraC family transcription activator; Bhende PM et al.; RhaS, an AraC family protein, activates rhaBAD transcription by binding to rhaI, a site consisting of two 17-bp inverted repeat half-sites . In this work, amino acids in RhaS that make base-specific contacts with rhaI were identified . Sequence similarity with AraC suggested that the first contacting motif of RhaS was a helix-turn-helix . Assays of rhaB-lacZ activation by alanine mutants within this potential motif indicated that residues 201, 202, 205, and 206 might contact rhaI . The second motif was identified based on the hypothesis that a region of especially high amino acid similarity between RhaS and RhaR (another AraC family member) might contact the nearly identical DNA sequences in one major groove of their half-sites . We first made targeted, random mutations and then made alanine substitutions within this region of RhaS . Our analysis identified residues 247, 248, 250, 252, 253, and 254 as potentially important for DNA binding . A genetic loss-of-contact approach was used to identify whether any of the RhaS amino acids in the first or second contacting motif make base-specific DNA contacts . In motif 1, we found that Arg202 and Arg206 both make specific contacts with bp -65 and -67 in rhaI1, and that Arg202 contacts -46 and Arg206 contacts -48 in rhaI2 . In motif 2, we found that Asp250 and Asn252 both contact the bp -79 in rhaI1 . Alignment with the recently crystallized MarA protein suggest that both RhaS motifs are likely helix-turn-helix DNA-binding motifs.

J Bacteriol, 1999 Sep, 181(17), 5167 - 75
Timing of FtsZ assembly in Escherichia coli; Den Blaauwen T et al.; The timing of the appearance of the FtsZ ring at the future site of division in Escherichia coli was determined by in situ immunofluorescence microscopy for two strains grown under steady-state conditions . The strains, B/rA and K-12 MC4100, differ largely in the duration of the D period, the time between termination of DNA replication and cell division . In both strains and under various growth conditions, the assembly of the FtsZ ring was initiated approximately simultaneously with the start of the D period . This is well before nucleoid separation or initiation of constriction as determined by fluorescence and phase-contrast microscopy . The durations of the Z-ring period, the D period, and the period with a visible constriction seem to be correlated under all investigated growth conditions in these strains . These results suggest that (near) termination of DNA replication could provide a signal that initiates the process of cell division.

J Bacteriol, 1999 Sep, 181(17), 5140 - 8
Evaluation of acyl coenzyme A oxidase (Aox) isozyme function in the n-alkane-assimilating yeast Yarrowia lipolytica; Wang HJ et al.; We have identified five acyl coenzyme A (CoA) oxidase isozymes (Aox1 through Aox5) in the n-alkane-assimilating yeast Yarrowia lipolytica, encoded by the POX1 through POX5 genes . The physiological function of these oxidases has been investigated by gene disruption . Single, double, triple, and quadruple disruptants were constructed . Global Aox activity was determined as a function of time after induction and of substrate chain length . Single null mutations did not affect growth but affected the chain length preference of acyl-CoA oxidase activity, as evidenced by a chain length specificity for Aox2 and Aox3 . Aox2 was shown to be a long-chain acyl-CoA oxidase and Aox3 was found to be active against short-chain fatty acids, whereas Aox5 was active against molecules of all chain lengths . Mutations in Aox4 and Aox5 resulted in an increase in total Aox activity . The growth of mutant strains was analyzed . In the presence of POX1 only, strains did not grow on fatty acids, whereas POX4 alone elicited partial growth, and the growth of the double POX2-POX3-deleted mutant was normal excepted on plates containing oleic acid as the carbon source . The amounts of Aox protein detected by Western blotting paralleled the Aox activity levels, demonstrating the regulation of Aox in cells according to the POX genotype.

Microbiology, 1999 Aug, 145 ( Pt 8), 2087 - 94
Mycoplasma synoviae surface protein MSPB as a recombinant antigen in an indirect ELISA; Noormohammadi AH et al.; Mycoplasma synoviae is a poultry pathogen causing respiratory disease and synovitis . A number of serological assays have been developed for diagnosis of M . synoviae infection; however, they lack sensitivity and/or are prone to false-positive reactions . Using a combination of PCR and expression cloning, four overlapping regions (regions 1-4) of the surface antigen MSPB of M . synoviae WVU-1853 were expressed in a bacterial expression system . Immunostaining of the resultant polypeptides with chicken sera raised against different M . synoviae strains, or Mycoplasma gallisepticum S6, suggested that region 4 contained a highly antigenic and species-specific domain (amino acids 212-317) {corrected} of MSPB . A fusion protein of region 4 was expressed in the pMAL expression system and purified from cold-osmotic-shock fluids of Escherichia coli cells for use in an indirect ELISA . The potential of the purified antigen for detection of M . synoviae antibodies was assessed with sera obtained from chickens experimentally infected with different strains of M . synoviae or M . gallisepticum, or from uninoculated chickens . All the sera from M . synoviae-inoculated chickens provided higher absorbance values than those from M . gallisepticum-inoculated or uninoculated chickens . Chickens inoculated with M . synoviae 86079/7NS had detectable increases of serum anti-MSPB immunoglobulins at day 7 after inoculation . These studies have identified the most antigenic region of one of the major species-specific surface proteins of M . synoviae, and shown the potential of this protein as a serodiagnostic reagent.

FASEB J, 1999 Sep, 13(12), 1637 - 46
Endogenous peroxynitrite mediates mitochondrial dysfunction in rat diaphragm during endotoxemia; Boczkowski J et al.; It has been shown that nitric oxide (NO), synthesized by the inducible NO synthase (iNOS) expressed in the diaphragm during endotoxemia, participates in the development of muscular contractile failure . The aim of the present study was to investigate whether this deleterious action of NO was related to its effects on cellular oxidative pathways . Rats were inoculated with E . coli lipopolysaccharide (LPS) or sterile saline solution (controls) and studied at 3 and 6 h after inoculation . iNOS protein and activity could be detected in the rat diaphragm as early as 3 h after LPS, with a sustained steady-state concentration of 0.5 microM NO in the muscle associated with increased detection of hydrogen peroxide (H(2)O(2)) . In vitro, the same NO concentration produced a marked increase in H(2)O(2) production by isolated control diaphragm mitochondria, thus reflecting a higher intramitochondrial concentration of nondiffusible superoxide anion (O(2)(-.)) . In a similar way, whole diaphragmatic muscle and diaphragm mitochondria from endotoxemic rats showed a progressive increase in H(2)O(2) production associated with uncoupling and decreased phosphorylating capacity . Simultaneous with the maximal impairment in respiration (6 h after LPS), nitration of mitochondrial proteins (a peroxynitrite footprint) was detected and diaphragmatic force was reduced . Functional mitochondrial abnormalities, nitration of mitochondrial proteins, and the decrease in force were significantly attenuated by administration of the NOS inhibitor L-NMMA . These results show that increased and sustained NO levels lead to a consecutive formation of O(2)(-.) that reacts with NO to form peroxynitrite, which in turn impairs mitochondrial function, which probably contributes to the impairment of muscle contractility . during endotoxemia.

Dev Biol Stand, 1999, 97, 39 - 47
Chromatography of recombinant proteins; O'Connor JV; Variants of intact polypeptides/proteins ranging in mass from 6,500 to 70,000 Da were easily separated using reversed-phaseHPLC (rpHPLC) or affinity chromatography . A variant of rhlGF-I, where the racemization of a serine residue was detected in the intact molecule, was resolved from rhlGF-I within 25 minutes by rpHPLC . Other variants of rhlGF-I separated by this method include methionine sulphoxide at position 59, des Gly1, des Gly1Pro2, Glu for Asp substitution at position 20 and incorrectly folded IGF-I . For rhDNase (approximately 40 kDa), affinity chromatography was able to clearly resolve three different amino acids (Asn, Asp and iso-Asp) at position 74 of the intact glycoprotein . The presence or absence of O-linked sugars on Thr -37 of recombinant human thrombopoietin was rapidly demonstrated by rpHPLC . While the separation of these types of variants is essential, the demonstration of biological activity is critical for designing specifications that allow the administration of these proteins into humans . Once a correlation exists between the variant and its biological activity, control of the manufacturing process can be better achieved with analytical methodology.

Microbiology, 1999 Aug, 145 ( Pt 8), 2153 - 62
Genetic analysis of the GcvA binding site in the gcvA control region; Jourdan AD et al.; The GcvA protein both activates and represses the gcv operon and negatively regulates its own transcription . GcvA binds to three sites in the gcv control region and to one site in the gcvA control region; each of these binding sites contains the conserved 5 bp DNA sequence 5'-CTAAT-3' . This report describes the role this DNA sequence plays in autoregulation and expression of gcvA . Through single base-pair mutations, the importance of three of these five basepairs in the autoregulation of gcvA expression is shown . Two of the gcvA control region mutations described cause a gcvA::lacZ fusion to be overexpressed at 9-24 times the wild-type level . The increase in expression is due in part to a complete loss of autoregulation and in part to a GcvA-independent mechanism . One of the mutants was shown by Western blot analysis to increase the intracellular concentration of GcvA . This high level of gcvA expression subsequently causes the loss of purine-mediated repression of a gcvT::lacZ fusion . However, overexpression of gcvR re-established purine-mediated repression of the gcvT::lacZ fusion, supporting the model for gcv regulation that suggests the need for a relatively constant GcvA to GcvR ratio for appropriate regulation of gcv expression in response to glycine and purine availability.

Microbiology, 1999 Aug, 145 ( Pt 8), 2135 - 44
Analysis of the CoIE1 stability determinant Rcd; Sharpe ME et al.; Multimer formation is an important cause of instability for many multicopy plasmids . Plasmid CoIE1 is maintained stably because multimers are converted to monomers by Xer-mediated site-specific recombination at the cer site . However, multimer resolution is not the whole story; inactivation of a promoter (Pcer) within cer causes plasmid instability even though recombination is unaffected . The promoter directs the synthesis of a short transcript (Rcd) which is proposed to delay the division of multimer-containing cells . Mapping of the 5' terminus of Rcd confirms that transcription initiates from Pcer . The 3' terminus shows considerable heterogeneity, consistent with a primary transcript of 95 nt being degraded via intermediates of 79 and 70 nt . Secondary structure predictions for Rcd are presented . Of four mutations which abolish Rcd-mediated growth inhibition, one reduces the activity of Pcer while the other three map to the rcd coding sequence and reduce the steady-state level of the transcript . RNA folding analysis suggests that these three mutant transcripts adopt a common secondary structure in which the major stem-loop differs from that of wild-type Rcd . A survey of 24 cer-like multimer resolution sites revealed six which contain Pcer-like sequences . The putative transcripts from these sites have similar predicted secondary structures to Rcd and contain a highly conserved 15 base sequence . To test the hypothesis that Rcd acts as an anti-sense RNA, interacting with its target gene(s) through the 15 nt sequence, we used DNA hybridization and sequence analysis to find matches to this sequence in the Escherichia coli chromosome . Our failure to find plausible anti-sense targets has led to the suggestion that Rcd may interact directly with a protein target.

Microbiology, 1999 Aug, 145 ( Pt 8), 2129 - 33
On-line monitoring of gene expression; Biran I et al.; Gene expression in cultures of Escherichia coli has been determined in situ and on-line by the use of an electrochemical sensor . Intact bacteria were used to monitor the induction of the lacZ gene; the onset of stationary phase was also monitored, using a reporter gene fused to the RpoS-dependent promoter of the osmY gene . The technique described can in principle be used to determine the activity of any promoter, with a variety of reporter genes . This technology is non-intrusive, allows real-time monitoring of gene expression, and will be useful in the study of growth regulation and development.

Microbiology, 1999 Aug, 145 ( Pt 8), 2077 - 85
Use of primate model system to identify Chlamydia trachomatis protein antigens recognized uniquely in the context of infection; Bannantine JP et al.; A primate model system was used to identify Chlamydia trachomatis antigens uniquely recognized in the context of infection . Serum antibody titres were measured in cynomolgus monkeys challenged urethrally with C . trachomatis serovar L2 elementary bodies (EBs) . High-titre sera from these primates were used, in parallel with antisera against killed C . trachomatis EBs, to differentially screen an expression library of C . trachomatis serovar L2 DNA . Four clones were recognized only by antisera from infected monkeys . Sequence analysis revealed that three of these immunoreactive clones overlap a common ORF, designated ORF D242 (encoding p242), in the C . trachomatis genome database . The fourth clone contains two complete ORFs, each encoding 32 kDa proteins that share identity with Treponema pallidum TroA and TroB (ORFs D067 and D068 in the C . trachomatis database, respectively) . Immunoblot analysis of Escherichia coli lysates expressing C . trachomatis TroA, TroB and p242 fusion proteins showed that p242 and TroA, but not TroB, were detected by the sera collected from infected primates . Antibodies directed at TroA and p242 were also detected in sera from several C . trachomatis-infected patients, demonstrating that these proteins are also recognized by humans following infection . Immunoblot analysis with antibody against TroA and p242 also demonstrated that both antigens are present in higher abundance in infected ChoK1 cells relative to purified C . trachomatis EBs . Immunofluorescence microscopy shows that TroA and p242 are both localized to intracellular developmental forms at the margins of growing inclusions . Collectively, these studies identify two C . trachomatis proteins that are under-represented in EBs and are recognized uniquely in the context of infection.

Microbiology, 1999 Aug, 145 ( Pt 8), 2011 - 21
Involvement of the N- and C-terminal domains of Mycobacterium tuberculosis KatG in the protection of mutant Escherichia coli against DNA-damaging agents; Mulder MA et al.; The Mycobacterium tuberculosis KatG enzyme, like most hydroperoxidase I (HPI)-type catalases, consists of two related domains, each with strong similarity to the yeast cytochrome c peroxidase . The catalase-peroxidase activity is associated with the amino-terminal domain but currently no definite function has been assigned to the carboxy-terminal domain, although it may play a role in substrate binding . This paper reports another possible function of the KatG protein involving protection of the host cell against DNA-damaging agents . The M . tuberculosis katG gene, the 5' domain and the 3' domain were cloned separately, in-frame with the maltose-binding protein, into the vector pMAL-c2 . These constructs were introduced into four DNA-repair mutants of Escherichia coli, DK1 (recA), AB1884 (uvrC), AB1885 (uvrB) and AB1886 (uvrA), which were then tested for their ability to survive treatment with UV light (254 nm), hydrogen peroxide (1.6 mg ml-1) and mitomycin C (6 micrograms ml-1) . All three constructs conferred resistance to UV upon the recA E . coli cells, whereas resistance to mitomycin C was found in all repair mutants tested . Protection against hydrogen peroxide damage was less pronounced and predominantly found in the recA host . These results indicated that the M . tuberculosis katG gene can enhance DNA repair in E . coli, and that the 5' and 3' domains can function separately . UV sensitivity tests on Mycobacterium intracellulare and M . tuberculosis strains mutant in katG revealed that the katG gene product does not play an additive role in the survival of mycobacterial cells after exposure to short-wavelength UV irradiation, in repair-competent cells.

Microbiology, 1999 Aug, 145 ( Pt 8), 1937 - 43
Isolation of the gene encoding an immunodominant membrane protein of the apple proliferation phytoplasma, and expression and characterization of the gene product; Berg M et al.; An immunodominant membrane protein (IMP) of the apple proliferation (AP) phytoplasma was detected in preparations from AP-diseased periwinkle plants using monoclonal and polyclonal antibodies to the AP agent . Following isolation from Western blots and partial sequencing, degenerate oligonucleotides derived from the IMP sequence were used as probes to identify a DNA fragment containing the ORF encoding the IMP . Complete sequencing and subsequent analysis of the cloned DNA fragment revealed the presence of two ORFs, predicted to encode proteins with molecular masses of 25 kDa (P-318A) and 19 kDa (P-318B) . Whilst database searches failed to assign a possible function to P-318A, analysis of P-318B predicted an amphiphilic membrane protein with a positively charged N-terminal portion, followed by a hydrophobic segment forming an alpha-helix, and a hydrophilic C-terminal part located outside of the cell . The amphiphilic nature of P-318B was confirmed by its solubility in Triton X-114 . The gene encoding P-318B was expressed in Escherichia coli and the resulting protein was used to immunize rabbits . The antiserum obtained reacted specifically with P-318B . The same protein was also detected by an antiserum raised against antigen preparations from AP-diseased plants . The P-318B antiserum did not react with antigen preparations from plants infected with the closely related pear decline phytoplasma . However, in Southern hybridization studies, the gene encoding the IMP hybridized to genomic fragments of the pear decline and European stone fruit yellows phytoplasmas . It also showed significant sequence similarity to a gene encoding an antigenic membrane protein of the sweet potato witches' broom phytoplasma, but not to a gene encoding a major immunogenic membrane protein of an aster yellows group phytoplasma . Since it appears that most phytoplasmas possess a major immunogenic membrane protein which may have a function in pathogenesis, this work may be a basis for further studies on fundamental aspects of host-pathogen interactions . It also describes a new approach to obtain suitable immunogens to produce specific antibodies for detection and characterization of the non-culturable phytoplasmas.

Adv Biophys, 1999, 36, 1 - 25
Biophysical studies on ATP synthase; Kagawa Y; The isolation of ATP synthase (F0F1) (82) and F0 (83) 34 years ago finally revealed that F0F1 is a motor composed of F0 (ion-motor, abc subunits) and F1 (ATP-motor, alpha 3 beta 3 gamma delta epsilon subunits) (Fig . 1) . The single molecule videotape (4, 5, 65, 66) revealed that gamma epsilon axis of F1 rotates counterclockwise, proceeds by each 2 pi/3 step, and is driven by torque of 42 pN.nm (12) with nearly 100% efficiency (5) (Fig . 4) . The motor is composed of a rotor (gamma epsilon-F0-c) and a stator (alpha 3 beta 3 delta-F0-ab), and the rotor is connected to a shaft (gamma epsilon) . Since F0F1 is driven by delta microH+ (9, 10, 84), biophysical studies on stable TF0F1 (1, 7) are essential to elucidate the mechanism . These include nanomechanics (4, 5) (Fig . 4), crystallography (2, 3) (Figs . 2 and 3), NMR (51, 52), ESR (56), synchrotron analysis (3, 28), and electrophysiology (10, 25) . The KmATP value of rotation is 0.8 microM, with the Vmax of 3.9 rps (5) . This corresponds to the bi-site catalysis in proton transport by F0F1 (10, 70, 84) . X-ray crystallography of MF1 (2) and the alpha 3 beta 3 oligomer of TF1 (3) (Fig . 2) together with mutation analyses revealed the role of residues in the rotation . The idea of elastic energy store is proposed in alpha 3 beta 3 gamma during the stepping time (up to a few sec) after the ATP binding . Biological studies have partially clarified the genetic and kinetic regulation of the rotation in MF1 . Both theories (6, 7, 62, 64, 85) and the biological significance (17) of the intramolecular rotation of F0F1 await further studies, especially those of F0 and minor subunits.

Commun Dis Public Health, 1999 Jan, 2(1), 27 - 31
Outbreak of gastroenteritis associated with contamination of a private borehole water supply; Reacher M et al.; An outbreak of gastroenteritis affected 58 of 700 people served by a private water supply at a biological research institute located in a village . No cases were detected in 250 residents of the same village served by a public water supply over the same period . Consumer complaints of tainting and laboratory evidence of bacterial and chemical contamination were obtained for the private water supply, but not for the public water supply . The outbreak was probably caused by contamination from a nearby sewer of a borehole used for the private supply . The outbreak showed how a large, private water supply posed a substantial risk to public health . The regulatory framework for such water supplies should be modified to ensure their safer design and operation.

Environ Mol Mutagen, 1999, 34(1), 1 - 8
Spectra of gpt mutations in ethylnitrosourea-treated and untreated transgenic mice; Masumura K et al.; We have established a new transgenic mouse mutagenicity assay for the efficient detection of point mutations and deletions in vivo (Nohmi et al . {1996} Env . Mol . Mutagen . 28:465-470) . In this assay, the gpt gene of Escherichia coli is used as a reporter for the detection of point mutations . Treatment of mice with ethylnitrosourea (ENU, 150 mg/kg) enhances by several-fold the mutant frequency of gpt in bone marrow . Here, we report the mutation spectra of the gpt gene recovered from bone marrow of ENU-treated and untreated transgenic mice . In the gpt mutants rescued from ENU-treated mice, more than 90% of the mutations were base change mutations; the predominant types were A:T to T:A transversions and G:C to A:T transitions . On the contrary, in the mutants rescued from untreated mice, 54% were base substitutions and the remainders were short deletions and insertions . Among untreated mice, the most frequently observed base substitution was G:C to A:T transitions (7/14 mutants) . Three of these occurred at 5'-CpG-3' sites . Interestingly, the mutation spectra of the gpt gene were different from those of the gpt gene in ENU-treated and untreated E.coli, whereas they were similar to those of the lacZ and lacI genes in ENU-treated and untreated other transgenic mice or cultured mammalian cells . We also report the establishment of homozygous transgenic mice that have transgene lambdaEG10 DNA in both chromosome 17 of C57BL/6J mouse .

J Cell Sci, 1999 Sep, 112 Pt 18, 3061 - 70
Characterization of tGLP-1, a Golgi and lysosome-associated, transmembrane glycoprotein of African trypanosomes; Lingnau A et al.; Purification of endosomal/lysosomal vesicles of Trypanosoma brucei brucei bloodstream forms and generation of monoclonal antibodies led to the isolation of antibodies directed against an 85 kDa, Golgi and endocytic traffic-associated protein termed tGLP-1, Trypanosoma Golgi/lysosome protein-1 . Preliminary immunoelectron microscopical analysis revealed that the protein is present in, but not restricted to, the limiting membrane of multivesicular lysosomes and is more abundant in bloodstream forms compared to the procyclic stage . The corresponding gene was cloned and is present as a single copy . Blast searches did not reveal any homologies to other proteins and genes published . The nucleotide sequence of the gene (1848 base pairs) predicted a type 1 membrane topology with an N-terminal signal sequence (20 aa), a luminal domain with 2 N-glycosylation sites (524 aa), a transmembrane domain (23 aa), and a long cytosolic tail domain (49 aa) . Polyclonal antibodies raised against the cytosolic tail confirmed the localization of the gene product to multivesicular lysosomes but revealed that the majority of the protein was in the Golgi apparatus . Colabelling with an antibody against p67, a lysosomal glycoprotein of trypanosomes, revealed extensive overlap between the proteins with opposing relative abundance . Expression of the tGLP-1 open reading frame in Leishmania resulted in Golgi localization, and in Toxoplasma, in localization to both the Golgi and endoplasmic reticulum . These data indicate conservation in the functionality of the Golgi-targeting sequence of tGLP-1.

Arch Biochem Biophys, 1999 Sep 1, 369(1), 30 - 41
Prediction of the active-site structure and NAD(+) binding in SQD1, a protein essential for sulfolipid biosynthesis in Arabidopsis; Essigmann B et al.; Sulfolipids of photosynthetic bacteria and plants are characterized by their unique sulfoquinovose headgroup, a derivative of glucose in which the 6-hydroxyl group is replaced by a sulfonate group . These sulfolipids have been discussed as promising anti-tumor and anti-HIV therapeutics based on their inhibition of DNA polymerase and reverse transcriptase . To study sulfolipid biosynthesis, in particular the formation of UDP-sulfoquinovose, we have combined computational modeling with biochemical methods . A database search was performed employing the derived amino acid sequence from SQD1, a gene involved in sulfolipid biosynthesis of Arabidopsis thaliana . This sequence shows high similarity to other sulfolipid biosynthetic proteins of different organisms and also to sugar nucleotide modifying enzymes, including UDP-glucose epimerase and dTDP-glucose dehydratase . Additional biochemical data on the purified SQD1 protein suggest that it is involved in the formation of UDP-sulfoquinovose, the first step of sulfolipid biosynthesis . To understand which aspects of epimerase catalysis may be shared by SQD1, we built a three-dimensional model of SQD1 using the 1.8 A crystallographic structure of UDP-glucose 4-epimerase as a template . This model predicted an NAD(+) binding site, and the binding of NAD(+) was subsequently confirmed by enzymatic assay and mass spectrometry . The active-site interactions together with biochemical data provide the basis for proposing a reaction mechanism for UDP-sulfoquinovose formation .

Vaccine, 1999 Aug 6, 17(23-24), 3072 - 82
Immunization of rhesus monkeys with a mucosal prime, parenteral boost strategy protects against infection with Helicobacter pylori; Lee CK et al.; Rhesus monkeys were immunized with recombinant Helicobacter pylori urease vaccine given solely by the parenteral route or preceded by a priming dose given by the oral route . Two groups of monkeys received parenteral urease with either a synthetic glycolipid adjuvant (Bay) or aluminum hydroxide (alum) as adjuvants . A third group of monkeys received a priming dose of oral urease given with the mucosal adjuvant LT (Escherichia coli heat labile enterotoxin), followed by parenterally administered booster doses of urease adsorbed to alum . Monkeys receiving placebo served as controls . The monkeys received a total of 4 doses of vaccine with the first 3 doses given every 3 weeks and the last booster dose administered 14 weeks later . The monkeys were challenged orally with H . pylori one week after the last vaccine dose and euthanized 10 weeks after challenge, at which time, their stomachs were collected for determination of bacterial colonization and histopathology . Monkeys primed with the oral vaccine and boosted with the parenteral vaccine showed a statistically significant reduction in bacterial colonization when compared to sham-immunized control animals (P = 0.05; Wilcoxon rank sums test) . Monkeys receiving parenteral only regimes of urease plus Bay or alum showed no difference in bacterial colonization compared with sham-immunized controls (P = 1.00 and P = 0.33, respectively) . The mucosal prime-parenteral boost regime did not cause gastropathy . There was no difference in any of the 3 treatment groups with respect to gastric epithelial changes compared to control animals . There was also no difference in the type and extent of gastric inflammatory cell infiltrates between animals vaccinated by the mucosal prime-parenteral boost strategy and sham immunized controls . However, monkeys receiving the two parenteral-only regimens had slightly elevated gastritis scores.

Vaccine, 1999 Aug 6, 17(23-24), 3039 - 49
Differentiation of convalescent animals from those vaccinated against foot-and-mouth disease by a peptide ELISA; Shen F et al.; We have identified continuous antigenic determinants within the amino acid sequences of the conserved nonstructural region containing proteins 2C and 3ABC of foot-and-mouth disease virus which can distinguish between the sera from vaccinated and infected animals . An ELISA based on a 3B peptide gave a positive reaction with sera from cattle, pigs, sheep and guinea pigs infected with all seven serotypes of the virus, but not with sera from vaccinated animals . In experiments with cattle and pigs to determine the duration of the antibody response, positive reactions were obtained as late as one year after infection . The advantages of using peptides from the nonstructural viral proteins instead of recombinant proteins for differentiating vaccinees from infected animals include their exquisite specificity, nonreactivity with antibodies against host cell-derived proteins (e.g . E . coli and insect cell proteins), and their ease of preparation.

Pharmacol Biochem Behav, 1999 Aug, 63(4), 629 - 37
Endotoxin- and interleukin-1-induced hypophagia are not affected by adrenergic, dopaminergic, histaminergic, or muscarinic antagonists; Swiergiel AH et al.; Endotoxin (lipopolysaccharide, LPS) and interleukin-1 (IL-1) administration induce hypophagia in rodents . Both IL-1 and LPS are known to activate cerebral norepinephrine and serotonin metabolism, and IL-1 affects that of acetylcholine and histamine . Each of these neurotransmitters has been implicated in feeding behavior . Therefore, the ability of specific antagonists of the above neurotransmitter systems to counteract feeding responses to peripherally injected mIL-1beta and LPS was studied . Feeding was assessed in nondeprived mice by measuring the intake of sweetened milk in a 30-min period, as well as daily food pellet intake . LPS and mIL-1beta reliably reduced milk intake, and often reduced food pellet intake and body weight . Treatment of the mice with peripherally administered alpha-adrenergic (phentolamine or prazosin) or 3-adrenergic antagonists (propranolol), either alone or in combination, did not significantly alter the hypophagic responses to mIL-1beta or LPS . Mice in which cerebral norepinephrine was depleted with DSP-4 or 6-hydroxydopamine also displayed the usual hypophagia in response to mIL-1beta and LPS . The hypophagic responses to mIL-1beta and LPS were not affected by the histaminergic antagonists, pyrilamine (H1), cimetidine (H2), thioperamide (H3), or the histamine-depleting agent, alpha-fluoromethylhistidine, nor by the muscarinic cholinergic antagonist, scopolamine . The responses to mIL-l1 were also unaffected by the dopamine receptor antagonist, haloperidol, the opioid receptor antagonist, naloxone, and the NO synthase inhibitor, L-NAME . These results suggest that adrenergic, dopaminergic, histaminergic, cholinergic, opioid or nitric oxide systems are not essential for the hypophagia induced by IL-1, and that multiple redundant pathways may be involved in illness-related hypophagia.

FEBS Lett, 1999 Aug 13, 456(3), 370 - 4
Structural and enzymatic characterization of human recombinant GDP-D-mannose-4,6-dehydratase; Bisso A et al.; GDP-D-mannose-4,6-dehydratase (GMD) is the key enzyme in the 'de novo' pathway of GDP-L-fucose biosynthesis . The reported cDNA sequences for human GMD predict three forms of different length, whose 'in vivo' occurrence and molecular properties are completely undefined . Here, we report the expression in Escherichia coli and the properties of each native recombinant GMD form . Only the 42 kDa long GMD (L-GMD) and the 40.2 kDa (M-GMD) forms were recovered as soluble functional proteins, while the 38.7 kDa form, short GMD (S-GMD), lacking an N-terminal domain critical for dinucleotide binding, was inactive and formed a precipitate . Both L-GMD and M-GMD are homodimers and contain 1 mol of tightly bound NADP+ . Their kinetic properties (Km, Kcat) are apparently identical and both forms are non-competitively feedback-inhibited by GDP-L-fucose to a similar extent . M-GMD is the predominant enzyme form expressed in several human cell lines . These data seem to suggest that modulation of the 'de novo' pathway of GDP-L-fucose biosynthesis involves mechanisms other than differential 'in vivo' expression of GMD forms.

J Anim Sci, 1999 Aug, 77(8), 1985 - 93
Effect of level of chronic immune system activation on the lactational performance of sows; Sauber TE et al.; The effect of the level of chronic immune system (IS) activation on sow lactational performance was determined in 11 pairs of littermate, primiparous sows . Sows with a low level of IS activation were created by rearing the animals via early weaning, isolated rearing schemes . During lactation, two levels of IS activation were achieved in each littermate sow pair by subcutaneous administration of either 0 (saline) or 5 microg/kg of sow BW of Escherichia coli lipopolysaccharide (LPS) in a mineral oil adjuvant emulsion on d 2 and 10 of lactation . Litters were standardized to 13 pigs by 8 h postpartum . Sows were offered daily 6.0 kg of a corn-soy diet formulated to contain a minimum of 250% of the dietary nutrient concentrations estimated to be needed by lactating sows . Based on antibody titers to LPS and serum concentrations of alpha-1 acid glycoprotein (AGP), high IS sows mounted an immune response to the LPS during lactation, and low IS sows maintained a low level of IS activation . Over an 18-d lactation, a high level of chronic activation of the sows' immune systems depressed daily sow feed intakes by .56 kg, litter weight gains by .32 kg, and daily milk by 1.4 kg, milk energy by 1.7 Mcal, and milk protein yields by 71 g, but did not alter sow body weight loss . The reductions in yields of milk and milk nutrients likely were because of proinflammatory cytokine-induced inhibition of the lactogenic hormones resulting from high chronic IS activation . Based on these data, the level of chronic IS activation alters the lactational performance of sows.

J Neurochem, 1999 Sep, 73(3), 1164 - 74
Oxidative damage to the c-fos gene and reduction of its transcription after focal cerebral ischemia; Cui J et al.; We investigated oxidative damage to the c-fos gene and to its transcription in the brain of Long-Evans rats using a transient focal cerebral ischemia and reperfusion (FCIR) model . We observed a significant (p < 0.001) increase in the immunoreactivity to 8-hydroxy-2'-guanine (oh8G) and its deoxy form (oh8dG) in the ischemic cortex at 0-30 min of reperfusion in all 27 animals treated with 15-90 min of ischemia . Treatment with a neuronal nitric oxide synthase (nNOS) inhibitor, 3-bromo-7-nitroindazole (60 mg/kg, i.p.), abolished the majority but not all of the oh8G/oh8dG immunoreactivity . Treatment with RNase A reduced the oh8G immunoreactivity, suggesting that RNA may be targeted . This observation was further supported by decreased levels of mRNA transcripts of the c-fos and actin genes in the ischemic core within 30 min of reperfusion using in situ hybridization . The reduction in mRNA transcription occurred at a time when nuclear gene damage, detected as sensitive sites to Escherichia coli Fpg protein in the transcribed strand of the c-fos gene, was increased 13-fold (p < 0.01) . Our results suggest that inhibiting nNOS partially attenuates FCIR-induced oxidative damage and that nNOS or other mechanisms induce nuclear gene damage that interferes with gene transcription in the brain.

Rheumatology (Oxford), 1999 Jul, 38(7), 631 - 5
Depressed proliferative responses by peripheral blood mononuclear cells from early arthritis patients to mycobacterial heat shock protein 60; Ramage JM et al.; OBJECTIVES: T-cell responses to mycobacterial heat shock protein 60 (M.hsp60) have been implicated in the pathogenesis of adjuvant arthritis, but whether they play a role in rheumatoid arthritis (RA) is undefined . We therefore examined T-cell responses to M.hsp60 and to other recall antigens in a cohort of patients with early RA and in healthy controls . METHODS: In vitro peripheral blood mononuclear cells' (PBMC) proliferative responses to antigen were measured by {3H}thymidine incorporation, and results correlated with clinical and laboratory features of disease . RESULTS: Whereas responses to the recall antigens tetanus toxin and purified protein derivative (PPD) were equivalent in the two groups, responses to both M.hsp60 and the Escherichia coli hsp60 were lower in the RA patients . These results could not be explained by either the higher prevalence of HLA-DR4 in the RA group, or the disease severity of the patients . CONCLUSION: In the light of results from the adjuvant arthritis model which suggest that arthritis may be ameliorated by the actions of an hsp60-reactive T-cell population, the lack of response to M.hsp60 in RA could contribute to disease persistence.

Photochem Photobiol, 1999 Aug, 70(2), 254 - 60
Cloning and molecular characterization of the cDNA for the Brazilian larval click-beetle Pyrearinus termitilluminans luciferase; Viviani VR et al.; The larval click-beetle Pyrearinus termitilluminans elicits the phenomenon of luminous termite mounds in the central-west region of Brazil . The bioluminescence (BL) spectrum of this larva (lambda max = 534 nm) is one of the most blue-shifted reported among known luminescent Coleoptera . We have isolated mRNA from larval thoracic lanterns and constructed a cDNA library into a lambda ZAP II vector . An expression library was obtained after excision of the pBluescript plasmid . This library was screened by photodetection and one clone that emitted green BL (lambda max = 538 nm) was isolated . The 2.2 kb cDNA insert includes a 543 residue open reading frame showing 82% homology with the luciferase isoenzymes of Pyrophorus plagiophthalamus (Coleoptera: Elateridae) . As expected, the region between residues 223 and 247 that contains the putative active site for BL color determination showed a higher degree of homology among click-beetle luciferases that elicit closer BL colors . The in vitro BL spectrum of recombinant P . termitilluminans luciferase also peaks at 538 nm and, as in the case of native enzyme, does not show any bathochromic shift upon decreasing the pH.

J Biol Inorg Chem, 1999 Aug, 4(4), 431 - 40
Natural and synthetic double-stranded DNA binding studies of macrocyclic tetraamine zinc(II) complexes appended with polyaromatic groups; Kikuta E et al.; The characteristic binding mode of zinc(II) complexes of macrocyclic tetraamines (1,4,7,10-tetraazacyclododecane, cyclen) appended with one or two arylmethyl group(s) {(4-quinolyl)methyl-, 1,7-bis(4-quinolyl)methyl-, (1-naphthyl)methyl-, 1,7-bis(1-naphthyl)methyl-, and (9-acridinyl)methyl-cyclen} to double-stranded calf thymus DNA and synthetic DNAs {poly(dA)-poly(dT), poly(dA-dT)2, poly(dI).poly(dC), poly(dI-dC)2, poly(dG) poly(dC), and poly(dG-dC)2} has been examined by spectrophotometric methods, Tm measurement, and inhibition of these DNA-directed transcriptions in vitro . Various hypochromic and bathochromic effects on the pendant aromatic absorption spectra of the complexes were observed in titration with the native and synthetic DNA . The binding constants Kapp (={bound cyclen derivatives}/{unbound cyclen derivatives}{DNA phosphates} M(-1)), at 25 degrees C in 10 mM EPPS (pH 8.0) containing 0.1 M Na+, were determined and compared with those of the corresponding Zn2+ -free ligands . The results showed that the Zn2+ -cyclen complexes interact with the DNA more strongly than the corresponding diprotonated ligands, leading to a stronger stacking of the pendant aromatic rings . The binding of Zn2+ -(9-acridinyl)methyl-cyclen to calf thymus DNA was competed by an AT-selective, minor groove binder, distamycin, but not by a major groove binder, methyl green . In an unusual interaction of excess Zn2+ -(9-acridinyl)methyl-cyclen with poly(dA).poly(dT), the Zn2+ -cyclen moiety went into the minor groove to make coordination bonds with the deprotonated imides of the thymines, resulting in disruption of the poly(dA).poly(dT) duplex . Thymine-containing DNA-directed transcription with Escherichia coli RNA polymerase in vitro was inhibited by the Zn2+ -(9-acridinyl)methyl-cyclen . The 50% inhibition concentrations of the transcription (IC50) were 22-45 microM with poly(dA).poly(dT) or poly(dA-dT)2 as templates, while with poly(dG-dC)2 as a template the IC50 value was 110 microM.

Eur Biophys J, 1999, 28(6), 447 - 56
Incoherent neutron scattering of copper azurin: a comparison with molecular dynamics simulation results; Paciaroni A et al.; The low-frequency dynamics of copper azurin has been studied at different temperatures for a dry and deuterium hydrated sample by incoherent neutron scattering and the experimental results have been compared with molecular dynamics (MD) simulations carried out in the same temperature range . Experimental Debye-Waller factors are consistent with a dynamical transition at approximately 200 K which appears partially suppressed in the dry sample . Inelastic and quasielastic scattering indicate that hydration water modulates both vibrational and diffusive motions . The low-temperature experimental dynamical structure factor of the hydrated protein shows an excess of inelastic scattering peaking at about 3 meV and whose position is slightly shifted downwards in the dry sample . Such an excess is reminiscent of the "boson peak" observed in glass-like materials . This vibrational peak is quite well reproduced by MD simulations, although at a lower energy . The experimental quasielastic scattering of the two samples at 300 K shows a two-step relaxation behaviour with similar characteristic times, while the corresponding intensities differ only by a scale factor . Also, MD simulations confirm the two-step diffusive trend, but the slow process seems to be characterized by a decay faster than the experimental one . Comparison with incoherent neutron scattering studies carried out on proteins having different structure indicates that globular proteins display common elastic, quasielastic and inelastic features, with an almost similar hydration dependence, irrespective of their secondary and tertiary structure.

Biochemistry, 1999 Aug 24, 38(34), 11147 - 55
Domain interactions in protein tyrosine kinase Csk; Sondhi D et al.; Csk (C-terminal Src kinase) is a protein tyrosine kinase that phosphorylates Src family member C-terminal tails, resulting in downregulation of Src family members . It is composed of three principal domains: an SH3 (Src homology 3) domain, an SH2 (Src homology 2) domain, and a catalytic domain . The impact of the noncatalytic domains on kinase catalysis was investigated . The Csk catalytic domain was expressed in Escherichia coli as a recombinant glutathione S-transferase-fusion protein and demonstrated to have 100-fold reduced catalytic efficiency . Production of the catalytic domain by proteolysis of full-length Csk afforded a similar rate reduction . This suggested that the reduction in catalytic efficiency of the recombinant catalytic domain was intrinsic to the sequence and not an artifact related to faulty expression . This rate reduction was similar for peptide and protein substrates and was due almost entirely to a reduced k(cat) rather than to effects on substrate K(m)s . Viscosity experiments on the catalytic fragment kinase reaction demonstrated that the chemical (phosphoryl transfer) step had a reduced rate . While the Csk SH2 domain had no intermolecular effect on the kinase activity of the Csk catalytic domain, the SH3 domain and SH3-SH2 fragment led to a partial rescue (4-5-fold) of the lost kinase activity . This rescue was not achieved with two other SH3 domains (lymphoid cell kinase, Abelson kinase) . The extrapolated K(d) of interaction for the Csk catalytic domain with the Csk SH3 domain was 2.2 microM and that of the Csk catalytic domain with the Csk SH3-SH2 fragment was 8.8 microM . Taken together, these findings suggest that there is likely an intramolecular interaction between the catalytic and SH3 domains in full-length Csk that is important for efficient catalysis . By employing a Csk SH3 specific type II polyproline helix peptide and carrying out site-directed mutagenesis, it was established that the SH3 surface that interacts with the catalytic domain was distinct from the surface that binds type II polyproline helix peptides . This finding suggests a novel mode of protein-protein interaction for an SH3 domain . The implications for Csk substrate selectivity, regulation, and function are discussed.

Biochemistry, 1999 Aug 24, 38(34), 11122 - 9
Engineering cytochrome c peroxidase into cytochrome P450: a proximal effect on heme-thiolate ligation; Sigman JA et al.; In an effort to investigate factors required to stabilize heme-thiolate ligation, key structural components necessary to convert cytochrome c peroxidase (CcP) into a thiolate-ligated cytochrome P450-like enzyme have been evaluated and the H175C/D235L CcP double mutant has been engineered . The UV-visible absorption, magnetic circular dichroism (MCD) and electron paramagnetic resonance (EPR) spectra for the double mutant at pH 8.0 are reported herein . The close similarity between the spectra of ferric substrate-bound cytochrome P450cam and those of the exogenous ligand-free ferric state of the double mutant with all three techniques support the conclusion that the latter has a pentacoordinate, high-spin heme with thiolate ligation . Previous efforts to prepare a thiolate-ligated mutant of CcP with the H175C single mutant led to Cys oxidation to cysteic acid {Choudhury et al . (1994) J . Biol . Chem . 267, 25656-25659} . Therefore it is concluded that changing the proximal Asp235 residue to Leu is critical in forming a stable heme-thiolate ligation in the resting state of the enzyme . To further probe the versatility of the CcP double mutant as a ferric P450 model, hexacoordinate low-spin complexes have also been prepared . Addition of the neutral ligand imidazole or of the anionic ligand cyanide results in formation of hexacoordinate adducts that retain thiolate ligation as determined by spectral comparison to the analogous derivatives of ferric P450cam . The stability of these complexes and their similarity to the analogous forms of P450cam illustrates the potential of the H175C/D235L CcP double mutant as a model for ferric P450 enzymes . This study marks the first time a stable cyanoferric complex of a model P450 has been made and demonstrates the importance of the environment around the primary coordination ligands in stabilizing metal-ligand ligation.

Biochemistry, 1999 Aug 24, 38(34), 11079 - 85
The methionyl aminopeptidase from Escherichia coli can function as an iron(II) enzyme; D'souza VM et al.; The identity of the physiologically relevant metal ions for the methionyl aminopeptidase (MetAP) from Escherichia coli was investigated and is suggested to be Fe(II) . The metal content of whole cells in the absence and presence of expression of the type I MetAP from E . coli was determined by inductively coupled plasma (ICP) emission analysis . The observed change in whole cell concentrations of cobalt, cadmium, copper, nickel, strontium, titanium, and vanadium upon expression of MetAP was negligible . On the other hand, significant increases in the cellular metal ion concentrations of chromium, zinc, manganese, and iron were observed with the increase in iron concentration being 4.4 and 6.2 times greater than that of manganese and zinc, respectively . Activity assays of freshly lysed BL21(DE3) cells containing the pMetAAP plasmid revealed detectable levels (>2 units/mg) of MetAP activity . Control experiments with BL21(DE3) without the MetAP plasmid showed no detectable enzymatic activity . Since MetAP is active upon expression, these data strongly suggest that cobalt is not the in vivo metal ion for the MetAP from E . coli . The MetAP from E . coli as purified was found to be catalytically inactive (</=2 units/mg) . ICP emission analysis of the as-purified enzyme revealed no catalytically relevant metal ions . Both the Co(II)- and Fe(II)-MetAP enzymes are susceptible to autoxidation, so strict care must be taken to remove all dissolved oxygen . Enzymatic assays performed under anaerobic conditions indicated that of the di- and trivalent metal cations tested to date, only Co(II) (37.3 units/mg), Fe(II) (29.7 units/mg), Mn(II) (7.0 units/mg), and Zn(II) (3.3 units/mg) provided detectable levels of enzymatic activity . In each case, excess metal ions were found to be inhibitory . The observed specific activity of Co(II)-MetAP is more than 3 times greater than that previously reported for the MetAP from E . coli {Ben-Bassat, A., et al . (1987) J . Bacteriol . 169, 751-757} . This increase in activity is likely due to the strict exclusion of air from reaction samples . Oxidation of either the Fe(II) or Co(II) form of the enzyme resulted in the complete loss of catalytic activity . The substrate binding constants (K(m)) for Met-Gly-Met-Met binding to the Co(II)- or Fe(II)-substituted MetAP enzymes, under anaerobic conditions, were found to be 3.16 and 1.95 mM, respectively . The combination of these data suggests that the in vivo metal ions for the MetAP enzyme from E . coli are likely Fe(II) ions.

Biochemistry, 1999 Aug 24, 38(34), 11012 - 20
Identification of linker regions and domain borders of the transcription activator protein NtrC from Escherichia coli by limited proteolysis, in-gel digestion, and mass spectrometry; Bantscheff M et al.; We have developed a mass spectrometry based method for the identification of linker regions and domain borders in multidomain proteins . This approach combines limited proteolysis and in-gel proteolytic digestions and was applied to the determination of linkers in the transcription factor NtrC from Escherichia coli . Limited proteolysis of NtrC with thermolysin and papain revealed that initial digestion yielded two major bands in SDS-PAGE that were identified by mass spectrometry as the R-domain and the still covalently linked OC-domains . Subsequent steps in limited proteolysis afforded further cleavage of the OC-fragment into the O- and the C-domain at accessible amino acid residues . Mass spectrometric identification of the tryptic/thermolytic peptides obtained after in-gel total proteolysis of the SDS-PAGE-separated domains determined the domain borders and showed that the protease accessible linker between R- and O-domain comprised amino acids Val-131 and Gln-132 within the "Q-linker" in agreement with papain and subtilisin digestion . The region between amino acid residues Thr-389 and Gln-396 marked the hitherto unknown linker sequence that connects the O- with the C-domain . High abundances of proline-, alanine-, serine-, and glutamic acid residues were found in this linker structure (PASE-linker) of related NtrC response regulator proteins . While R- and C-domains remained stable under the applied limited proteolysis conditions, the O-domain was further truncated yielding a core fragment that comprised the sequence from Ile-140 to Arg-320 . ATPase activity was lost after separation of the R-domain from the OC-fragment . However, binding of OC- and C- fragments to specific DNA was observed by characteristic band-shifts in migration retardation assays, indicating intact tertiary structures of the C-domain . The outlined strategy proved to be highly efficient and afforded lead information of tertiary structural features necessary for protein design and engineering and for structure-function studies.

Biochemistry, 1999 Aug 24, 38(34), 11006 - 11
Effect of cysteine residues on the activity of arginyl-tRNA synthetase from Escherichia coli; Liu M et al.; Arginyl-tRNA synthetase (ArgRS) from Escherichia coli (E . coli) contains four cysteine residues . In this study, the role of cysteine residues in the enzyme has been investigated by chemical modification and site-directed mutagenesis . Titration of sulfhydryl groups in ArgRS by 5, 5'-dithiobis(2-nitro benzoic acid) (DTNB) suggested that a disulfide bond was not formed in the enzyme and that, in the native condition, two DTNB-sensitive cysteine residues were located on the surface of ArgRS, while the other two were buried inside . Chemical modification of the native enzyme by iodoacetamide (IAA) affected only one DTNB-sensitive cysteine residue and resulted in 50% loss of enzyme activity, while modification by N-ethylmeimide (NEM) affected two DTNB-sensitive residues and caused a complete loss of activity . These results, when combined with substrate protection experiments, suggested that at least the two cysteine residues located on the surface of the molecule were directly involved in substrates binding and catalysis . However, changing Cys to Ala only resulted in slight loss of enzymatic activity and substrate binding, suggesting that these four cysteine residues in E . coli ArgRS were not essential to the enzymatic activity . Moreover, modifications of the mutant enzymes indicated that the two DTNB- and NEM-sensitive residues were Cys(320) and Cys(537) and the IAA-sensitive was Cys(320) . Our study suggested that inactivation of E . coli ArgRS by sulfhydryl reagents is a result of steric hindrance in the enzyme.

Biochemistry, 1999 Aug 24, 38(34), 10989 - 96
Site-specific tamoxifen-DNA adduct formation: lack of correlation with mutational ability in Escherichia coli; Lowes DA et al.; We have mapped sites of tamoxifen adduct formation, in the lacI gene using the polymerase STOP assay, following reaction in vitro with alpha-acetoxytamoxifen and horseradish peroxidase (HRP)/H(2)O(2) activated 4-hydroxytamoxifen . For both compounds, most adduct formation occurred on guanines . However, one adenine, within a run of guanines, generated a strong polymerase STOP site with activated 4-hydroxytamoxifen, and a weaker STOP site with alpha-acetoxytamoxifen at the same location . In Escherichia coli the lac I gene reacted with 4-hydroxytamoxifen was more likely to be mutated (2 orders of magnitude) than when reacted with alpha-acetoxytamoxifen, despite the greater DNA adduct formation by alpha-acetoxytamoxifen . This correlates with the greater predicted ability of activated 4-hydroxytamoxifen adducts to disrupt DNA structure than alpha-acetoxytamoxifen adducts . For lac I reacted with activated 4-hydroxytamoxifen, a hot spot of base mutation was located in the region of the only adenosine adduct . No mutational hot spots were observed with alpha-acetoxytamoxifen . Our data clearly shows a lack of correlation between gross adduct number, as assayed by (32)P-postlabeling and mutagenic potential . These data indicate the importance of minor adduct formation in mutagenic potential and further that conclusions regarding the mutagenicity of a chemical may not be reliably derived from the gross determination of adduct formation.

Biochemistry, 1999 Aug 24, 38(34), 10929 - 39
Mechanism of DNA binding by the DnaB helicase of Escherichia coli: analysis of the roles of domain gamma in DNA binding; Biswas EE et al.; We have analyzed the mechanism of single-stranded DNA (ssDNA) binding mediated by the C-terminal domain gamma of the DnaB helicase of Escherichia coli . Sequence analysis of this domain indicated a specific basic region, "RSRARR", and a leucine zipper motif that are likely involved in ssDNA binding . We have carried out deletion as well as in vitro mutagenesis of specific amino acid residues in this region in order to determine their function(s) in DNA binding . The functions of the RSRARR domain in DNA binding were analyzed by site-directed mutagenesis . DnaBMut1, with mutations R(328)A and R(329)A, had a significant decrease in the DNA dependence of ATPase activity and lost its DNA helicase activity completely, indicating the important roles of these residues in DNA binding and helicase activities . DnaBMut2, with mutations R(324)A and R(326)A, had significantly attenuated DNA binding as well as DNA-dependent ATPase and DNA helicase activities, indicating that these residues also play a role in DNA binding and helicase activities . The role(s) of the leucine zipper dimerization motif was (were) determined by deletion analysis . The DnaB Delta 1 mutant with a 55 amino acid C-terminal deletion, which left the leucine zipper and basic DNA binding regions intact, retained DNA binding as well as DNA helicase activities . However, the DnaB Delta 2 mutant with a 113 amino acid C-terminal deletion that included the leucine zipper dimerization motif, but not the RSRARR sequence, lost DNA binding, DNA helicase activities, and hexamer formation . The major findings of this study are (i) the leucine zipper dimerization domain, I(361)-L(389), is absolutely required for (a) dimerization and (b) ssDNA binding; (ii) the base-rich RSRARR sequence is required for DNA binding; (iii) three regions of domain gamma (gamma I, gamma II, and gamma III) differentially regulate the ATPase activity; (iv) there are likely three ssDNA binding sites per hexamer; and (v) a working model of DNA unwinding by the DnaB hexamer is proposed.

Biochemistry, 1999 Aug 24, 38(34), 10919 - 28
Mechanism of DnaB helicase of Escherichia coli: structural domains involved in ATP hydrolysis, DNA binding, and oligomerization; Biswas EE et al.; We describe the delineation of three distinct structural domains of the DnaB helicase of Escherichia coli: domain alpha, amino acid residues (aa) 1-156; domain beta, aa 157-302; and domain gamma, aa 303-471 . Using mutants with deletion in these domains, we have examined their role(s) in hexamer formation, DNA-dependent ATPase, and DNA helicase activities . The mutant DnaBbetagamma protein, in which domain alpha was deleted, formed a hexamer; whereas the mutant DnaBalphabeta, in which domain gamma was deleted, could form only dimers . The dimerization of DnaBalphabeta was Mg(2+) dependent . These data suggest that the oligomerization of DnaB helicase involves at least two distinct protein-protein interaction sites; one of these sites is located primarily within domain beta (site 1), while the other interaction site is located within domain gamma (site 2) . The mutant DnaBbeta, a polypeptide of 147 aa, where both domains alpha and gamma were deleted, displayed a completely functional ATPase activity . This domain, thus, constitutes the "central catalytic domain" for ATPase activity . The ATPase activity of DnaBalphabeta was kinetically comparable to that of DnaBbeta, indicating that domain alpha had little or no influence on the ATPase activity . In both cases, the ATPase activities were DNA independent . DnaBbetagamma had a DNA-dependent ATPase activity that was kinetically comparable to the ATPase activity of wild-type DnaB protein (wtDnaB), indicating a specific role for C-terminal domain gamma in enhancement of the ATPase activity of domain beta as well as in DNA binding . Mutant DnaBbetagamma, which lacked domain alpha, was devoid of any helicase activity pointing to a significant role for domain alpha . The major findings of this study are (i) domain beta contained a functional ATPase active site; (ii) domain gamma appeared to be the DNA binding domain and a positive regulator of the ATPase activity of domain beta; (iii) although domain alpha did not have any significant effect on the ATPase, DNA binding activities, or hexamer formation, it definitely plays a pivotal role in transducing the energy of ATP hydrolysis to DNA unwinding by the hexamer; and (iv) all three domains are required for helicase activity.

Biochemistry, 1999 Aug 24, 38(34), 10909 - 14
Phenylalanine residues in the active site of tyrosine hydroxylase: mutagenesis of Phe300 and Phe309 to alanine and metal ion-catalyzed hydroxylation of Phe300; Ellis HR et al.; Residues Phe300 and Phe309 of tyrosine hydroxylase are located in the active site in the recently described three-dimensional structure of the enzyme, where they have been proposed to play roles in substrate binding . Also based on the structure, Phe300 has been reported to be hydroxylated due to a naturally occurring posttranslational modification {Goodwill, K . E., Sabatier, C., and Stevens, R . C . (1998) Biochemistry 37, 13437-13445} . Mutants of tyrosine hydroxylase with alanine substituted for Phe300 or Phe309 have now been purified and characterized . The F309A protein possesses 40% less activity than wild-type tyrosine hydroxylase in the production of DOPA, but full activity in the production of dihydropterin . The F300A protein shows a 2.5-fold decrease in activity in the production of both DOPA and dihydropterin . The K(6-MPH4) value for F300A tyrosine hydroxylase is twice the wild-type value . These results are consistent with Phe309 having a role in maintaining the integrity of the active site, while Phe300 contributes less than 1 kcal/mol to binding tetrahydropterin . Characterization of Phe300 by MALDI-TOF mass spectrometry and amino acid sequencing showed that hydroxylation only occurs in the isolated catalytic domain after incubation with a large excess of 7, 8-dihydropterin, DTT, and Fe(2+) . The modification is not observed in the untreated catalytic domain or in the full-length protein, even in the presence of excess iron . These results establish that hydroxylation of Phe300 is an artifact of the crystallography conditions and is not relevant to catalysis.

FEMS Immunol Med Microbiol, 1999 Aug 15, 25(3), 275 - 82
Electrotransformation of the human pathogenic fungus Scedosporium prolificans mediated by repetitive rDNA sequences; Ruiz-Diez B et al.; The regions encoding the 5.8S rRNA and the flanking internal transcribed spacers (ITSI and ITSII) from two isolates of the human pathogenic fungus Scedosporium prolificans and one isolate of the taxonomically related species Pseudallescheria boydii (S . apiospermum) were sequenced . The sequences of the two S . prolificans isolates were identical . However, there were minor differences between both species . Phylogenetic analysis of known fungal sequences confirmed a close relationship between S . prolificans and P . boydii . An attempt was made to transform S . prolificans by electroporation using a plasmid vector, pMLF2, bearing the Escherichia coli hygromycin B phosphotransferase gene (hph) under the control of Aspergillus nidulans promoter and terminator sequences . To increase transformation efficiency, the sequenced ribosomal cluster of S . prolificans was used to construct a new vector for homologous recombination.

FEMS Immunol Med Microbiol, 1999 Aug 15, 25(3), 265 - 74
F165(1) fimbriae of the P fimbrial family inhibit the oxidative response of porcine neutrophils; Ngeleka M et al.; The F165(1) fimbrial system has been associated with the resistance of Escherichia coli O115:K"V165" to phagocytic killing by porcine polymorphonuclear leukocytes (PMNLs) . One mechanism of this resistance seemed to be inhibition of the oxidative response as observed following induction of PMNLs by phorbol myristate acetate (PMA) and treatment with bacteria possessing the F165(1) fimbriae . In order to confirm whether or not the F165(1) fimbriae are involved in this inhibition, we evaluated the effect of F165(1)-positive strains (a pathogenic wild-type strain 5131, and a recombinant strain HB101(pCJ7)) or an F165(1)-negative strain HB101 (used as negative control) on the oxidative response of porcine neutrophils (pNs) stimulated with PMA . Incubation of pNs with pathogenic E . coli strain 5131 resulted in significant inhibition of the oxidative response as compared to that observed for pNs incubated without bacteria, as assessed by hydrogen peroxide (H2O2) and superoxide anion (O2-) release from the phagocytes, and by the chemiluminescence assay . Similarly, incubation of pNs with the F165(1)-producing cloned strain HB101(pCJ7) resulted in significant inhibition of the pN oxidative response as compared to that observed for pNs incubated without bacteria or with strain HB101 . In contrast, addition of purified F165(1) fimbriae to the pNs had no effect on the oxidative response.

J Med Virol, 1999 Oct, 59(2), 198 - 203
Prevalence of TT virus DNA in eastern Taiwan aborigines; Lo SY et al.; We studied the prevalence of TT virus (TTV) DNA in the general population of the eastern Taiwan aborigine villages, about 11% (34 of 317) . There is no association between the presence of HBsAg and TTV DNA or between the presence of HCV RNA and TTV DNA . Therefore, the infection of HBV or HCV and the presence of TTV DNA appear to be independent from each other . The association between the presence of TTV DNA and the elevated alanine aminotransferase (and/or aspartate aminotransferase) activity was also investigated . The presence of TTV DNA was not found to be correlated with abnormal liver function (P = 0.574) when age, gender, and the presence of HBsAg, HCV RNA, and HGV RNA were all considered in the assay . The sequence homology of TTV DNA fragments between different isolates from Taiwan and N22 (the clone obtained from the original prototype strain) from Japan ranged from 84 to 97% . The recombinant protein encoded by the TTV DNA fragment corresponding to the open reading frame of N22 was expressed in E . coli successfully . However, no serum response against the recombinant protein was detected .

Nature, 1999 Aug 12, 400(6745), 693 - 6
Trigger factor and DnaK cooperate in folding of newly synthesized proteins; Deuerling E et al.; The role of molecular chaperones in assisting the folding of newly synthesized proteins in the cytosol is poorly understood . In Escherichia coli, GroEL assists folding of only a minority of proteins and the Hsp70 homologue DnaK is not essential for protein folding or cell viability at intermediate growth temperatures . The major protein associated with nascent polypeptides is ribosome-bound trigger factor, which displays chaperone and prolyl isomerase activities in vitro . Here we show that delta tig::kan mutants lacking trigger factor have no defects in growth or protein folding . However, combined delta tig::kan and delta dnaK mutations cause synthetic lethality . Depletion of DnaK in the delta tig::kan mutant results in massive aggregation of cytosolic proteins . In delta tig::kan cells, an increased amount of newly synthesized proteins associated transiently with DnaK . These findings show in vivo activity for a ribosome-associated chaperone, trigger factor, in general protein folding, and functional cooperation of this protein with a cytosolic Hsp70 . Trigger factor and DnaK cooperate to promote proper folding of a variety of E . coli proteins, but neither is essential for folding and viability at intermediate growth temperatures.

Infect Immun, 1999 Sep, 67(9), 4908 - 11
Adhesion of Escherichia coli to HeLa cells mediated by Trypanosoma cruzi surface glycoprotein-derived peptides inserted in the outer membrane protein LamB; Pereira CM et al.; Peptides derived from the surface glycoprotein gp82 of Trypanosoma cruzi, previously implicated in the parasite's invasion of host cells, were expressed as fusions to the protein LamB of Escherichia coli in a region known to be exposed on the cell surface . Bacteria expressing these proteins adhered to HeLa cells in a manner that mimics the pattern of parasite invasion of mammalian cells . Purified LamB fusion proteins were shown to bind to HeLa cells and to inhibit infection by T . cruzi, supporting the notion that these gp82-derived peptides can mediate interaction of the parasite with its host.

Gene Ther, 1999 Jun, 6(6), 956 - 65
Targeting endogenous platelet-derived growth factor B-chain by adenovirus-mediated gene transfer potently inhibits in vivo smooth muscle proliferation after arterial injury; Deguchi J et al.; Platelet-derived growth factor (PDGF), especially its B chain, has been implicated in the pathogenesis of vascular proliferative disorders such as atherosclerosis and restenosis after angioplasty . We constructed a replication-deficient recombinant adenovirus containing the gene encoding the extracellular region of PDGF beta-receptor (PDGFXR) that binds PDGF-B chain and acts as its antagonist . The administration into balloon-injured rat carotid arteries of an adenovirus containing the Escherichia coli lacZ gene as a marker gene at 5 days after injury markedly facilitated efficacy of gene transfer, as compared with its administration immediately after injury . Adenovirus-mediated gene transfer of PDGFXR into injured arteries performed at 5 days resulted in a more than 50% reduction in the neointimal area of injured arteries at 14 days . In contrast, the administration of control adenoviruses containing lacZ gene or containing no foreign gene was without suppressive effects on neointima formation . The inhibition of neointima formation by the expression of PDGFXR was accompanied by a reduction in bromodeoxyuridine-labeled cells and nearly complete inhibition of tyrosine phosphorylation of both alpha- and beta-receptors for PDGF, but not of epidermal growth factor receptor, in injured arteries . This is the first report to indicate the usefulness of targeting a growth factor by expressing an extracellular binding region of a receptor using an adenovirus for the treatment of vascular proliferative disorders, and provide direct evidence that PDGF-B chain plays an essential role in neointimal formation.

Biotechnol Bioeng, 1999 Oct 20, 65(2), 233 - 9
Construction of a multi RE module: exploitation of mechanochemistry of restriction endonucleases; Agarwal PK et al.; We describe the construction of a multi-immobilized restriction endonuclease module (Multi RE module) . We demonstrate that the applied mechanical stress enables modulation of enzyme activity and modulation of recognition site selectivity (in oligonucleotides of approximately 200 bp) of immobilized restriction endonucleases . The central module which is consisted of different strips of immobilized restriction endonucleases allows limited digestion of a large DNA sample in a controlled manner as a function of applied mechanical stress on strips . The stress-activity relationship and the effect of repeated cycles of stress and relaxation on the immobilized strips are presented here . &copy 1999 John Wiley & Sons, Inc.

Biotechnol Bioeng, 1999 Oct 20, 65(2), 151 - 9
Monitoring of genes that respond to process-related stress in large-scale bioprocesses; Schweder T et al.; In large-scale aerobic fed-batch processes, cells are exposed to local zones of high glucose concentrations that can also cause local oxygen limitations at high cell densities . The mRNA levels of four stress genes (clpB, dnaK, uspA, and proU) and three genes responding to oxygen limitation or glucose excess (pfl, frd, and ackA) were investigated in an industrial 20-m(3) Escherichia coli process and in a scale-down reactor with defined high-glucose and low-oxygen zones . The mRNA levels of ackA and proU were high during the batch growth phase, but declined drastically when glucose became limited, whereas the mRNA levels of the other stress genes were relatively constant throughout the process . In the industrial-scale reactor, the stress gene mRNA levels were, in most cases, highest in the middle part and at the top of the reactor, where the substrate was fed . Cells passing through the high glucose zone of the scale-down reactor had elevated mRNA levels for the oxygen limitation genes and had also elevated heat-shock gene mRNA levels . Both responses to stress occurred within seconds . The approach presented in this study offers a tool for monitoring process-related changes in the transcriptional regulation of genes .

Chem Res Toxicol, 1999 Aug, 12(8), 690 - 9
2,2',3,3',6,6'-hexachlorobiphenyl hydroxylation by active site mutants of cytochrome P450 2B1 and 2B11; Waller SC et al.; The structural basis of species differences in cytochrome P450 2B-mediated hydroxylation of 2,2',3,3',6,6'-hexachlorobiphenyl (236HCB) was evaluated by using 14 site-directed mutants of cytochrome P450 2B1 and three point mutants of 2B11 expressed in Escherichia coli . To facilitate metabolite identification, seven possible products, including three hydroxylated and four dihydroxylated hexachlorobiphenyls, were synthesized by direct functionalization of precursors and Ullmann and crossed Ullmann reactions . HPLC and GC/MS analysis and comparison with authentic standards revealed that 2B1, 2B11, and all their mutants produced 4, 5-dihydroxy-236HCB and 5-hydroxy-236HCB, while 2B11 L363V and 2B1 I114V mutants also catalyzed hydroxylation at the 4-position . The amount of products formed by 2B1 mutants I114V, F206L, L209A, T302S, V363A, V363L, V367A, I477A, I477L, G478S, I480A, and I480L was smaller than that of the wild type . I477V exhibited unaltered 236HCB metabolism, and I480V produced twice as much dihydroxy product as the wild type . For 2B11, substitution of Val-114 or Asp-290 with Ile decreased the product yields . Replacement of Leu-363 with Val dramatically altered the profile of 236HCB metabolites . In addition to an increase in the overall level of hydroxylation, the mutant mainly catalyzed hydroxylation at the 4-position . Incubation of P450 2B1 with 5-hydroxy-236HCB produced 4,5-dihydroxy-236HCB, which indicates that 4,5-dihydroxy-236HCB may be formed by a direct hydroxylation of 5-hydroxy-236HCB . The findings from this study demonstrate the importance of residues 114, 206, 209, 302, 363, 367, 477, 478, and 480 in 2B1 and 114, 290, and 363 in 2B11 for 236HCB metabolism.

Cell, 1999 Aug 6, 98(3), 397 - 408
Structure of the DNA repair enzyme endonuclease IV and its DNA complex: double-nucleotide flipping at abasic sites and three-metal-ion catalysis; Hosfield DJ et al.; Endonuclease IV is the archetype for a conserved apurinic/apyrimidinic (AP) endonuclease family that primes DNA repair synthesis by cleaving the DNA backbone 5' of AP sites . The crystal structures of Endonuclease IV and its AP-DNA complex at 1.02 and 1.55 A resolution reveal how an alpha8beta8 TIM barrel fold can bind dsDNA . Enzyme loops intercalate side chains at the abasic site, compress the DNA backbone, bend the DNA approximately 90 degrees, and promote double-nucleotide flipping to sequester the extrahelical AP site in an enzyme pocket that excludes undamaged nucleotides . These structures suggest three Zn2+ ions directly participate in phosphodiester bond cleavage and prompt hypotheses that double-nucleotide flipping and sharp bending by AP endonucleases provide exquisite damage specificity while aiding subsequent base excision repair pathway progression.

Biotechniques, 1999 Aug, 27(2), 356 - 61
Flow microsphere immunoassay-based method of virus quantitation; Samoylova TI et al.; A sensitive assay for adenovirus quantitation in vitro was developed using the flow microsphere immunoassay (FMIA) approach . Polystyrene microspheres were covalently coated with purified anti-adenoviral antibodies and incubated with virus-containing samples . After incubation, the samples were stained with DNA-specific fluorescent dyes, and microsphere-associated fluorescence was quantitated with a flow cytometer . The adsorption of virus to microspheres was examined under different experimental conditions . The flow cytometric assay was determined to be as accurate in detecting adenovirus as titering on 293 cells . The proposed method can be used to quantify virus in viral stocks and in biological samples.

Biotechniques, 1999 Aug, 27(2), 350 - 5
Direct observation of nucleocytoplasmic transport by microinjection of GFP-tagged proteins in living cells; Rosorius O et al.; We established a straightforward experimental system to investigate directly the requirements for nucleocytoplasmic transport in live cells . For this purpose, substrates were created containing nuclear localization signals (NLS) or nuclear export signals (NES) linked to a chimeric protein composed of the glutathione S-transferase (GST) fused to the green fluorescent protein (GFP) . The combination of GST/GFP-tagging allowed us to control protein expression in bacteria and to monitor protein purification during chromatography . Following microinjection into somatic cells, nuclear export/import of the highly fluorescent substrates could be observed directly by fluorescence microscopy . This system sets the stage to quantitate, in real time, the kinetics of nuclear import/export in living cells and to evaluate qualitative differences in various NLS/NES signals and pathways.

Biotechniques, 1999 Aug, 27(2), 328 - 30, 332-4
Simplified gene-fragment phage display system for epitope mapping; Gupta S et al.; We describe a simple and efficient system for epitope mapping by cloning random gene fragments into a specially designed gIIIp-based phage display vector . DNA encoding the antigen of interest is PCR-amplified and partially digested with DNaseI to generate 50-150-bp-long fragments, which are polished with T4 DNA polymerase and dephosphorylated . These fragments are cloned at the 5' end of the gIII after linearizing the vector with SmaI/SrfI, and the ligation is carried out in the presence of restriction enzyme SrfI . The restriction enzyme in the ligation reaction recuts the self-ligated vector but not the recombinants, since ligation with foreign fragments destroys the enzyme recognition site . Dephosphorylation of inserts prevents their chimerization and ensures ligation of single insert per vector molecule . Thus, using the above strategy, which prevents self-ligation of both the insert and the vector, the overall cloning efficiency and, thereby the library size, is improved more than 10-fold compared to the standard blunt-end, ligation-based methods for making similar libraries . The library is further enriched by a single-step infection of E . coli by phages obtained from primary transformants . This step eliminates all the phages that carry insert that are not in-frame with gIIIp and therefore do not display gIIIp . We have shown the utility of the above system in constructing a glutathione-S-transferase (GST) gene-fragment library in phages and identifying the epitope recognized by a monoclonal antibody against GST.

J Chromatogr A, 1999 Jul 23, 849(2), 403 - 12
Comparative studies on chemically and enzymatically coupled DNA-Sepharose columns for purification of a lac repressor chimeric fusion protein; Robinson FD et al.; The length of a DNA sequence attached to an affinity chromatography column affects column retention of transcription factors . Even when unrelated sequences such as a poly(A):poly(T) tail are included in a DNA sequence, transcription factors such as the lac repressor are bound more tightly by the column . The position of the additional sequences is also important . To compare coupling procedures, an identical DNA sequence was covalently attached to Sepharose by chemical coupling or produced enzymatically by template driven enzymatic primer extension . These two types of supports, containing the O1 operator sequence bound by lac repressor, were packed into identical columns and compared by purification of a lac repressor-beta-galactosidase fusion protein . We found that the purity and yield of proteins eluted from the two columns were similar . Overall, the results suggest that there is no significant advantage to either type of support for the purification of some proteins . The study revealed a potentially important effect of the length of DNA sequences on column selectivity.

J Gastrointest Surg, 1998 Nov-Dec, 2(6), 537 - 46
Mucosal production of complement C3 and serum amyloid A is differentially regulated in different parts of the gastrointestinal tract during endotoxemia in mice; Wang Q et al.; The effect of endotoxemia and sepsis on mucosal production of the acute-phase proteins complement component C3 and serum amyloid A (SAA) was studied in mice . In addition, the role of the proinflammatory cytokines tumor necrosis factor-alpha, interleukin (IL)(-1)beta, and IL-6 on mucosal C3 and SAA production was examined . Endotoxemia was induced by the subcutaneous injection of 250 microg/mouse of lipopolysaccharide . Control mice were injected with corresponding volumes of sterile saline solution . Sepsis was induced by cecal ligation and puncture, and sham-operated mice served as controls . Endotoxemia resulted in increased mucosal C3 levels in all parts of the gastrointestinal tract examined, from the stomach to the colon, with the most pronounced effects noticed in the proximal gastrointestinal tract . The influence of endotoxemia on mucosal SAA production was more differentiated with increased levels noted in the jejunum and ileum, and no changes seen in gastric and colonic mucosa . Sepsis resulted in similar changes in mucosal C3 and SAA levels as seen in endotoxemic mice, except that SAA levels were increased in colonic mucosa of septic mice . Among the cytokines, IL(-1)beta resulted in the most pronounced changes in mucosal acute-phase proteins . The increase in C3 and SAA levels in the mucosa of the small intestine during endotoxemia was partially blocked by IL(-1) receptor antagonist . The results suggest that endotoxemia is associated with increased mucosal C3 production in different parts of the gastrointestinal tract and increased SAA production in the mucosa of the small intestine . Mucosal acute-phase protein synthesis may, at least in part, be regulated by IL(-1)beta.

J Bone Miner Res, 1999 Aug, 14(8), 1290 - 301
Adenovirus-mediated gene therapy of osteoblasts in vitro and in vivo; Mehrara BJ et al.; Modulation of biological pathways governing osteogenesis may accelerate osseous regeneration and reduce the incidence of complications associated with fracture healing . Transforming growth factor beta1 (TGF-beta1) is a potent growth factor implicated in the regulation of osteogenesis and fracture repair . The use of recombinant proteins, however, has significant disadvantages and has limited the clinical utility of these molecules . Targeted gene therapy using adenovirus vectors is a technique that may circumvent difficulties associated with growth factor delivery . In this study, we investigate the efficacy of replication-deficient adenoviruses containing the human TGF-beta1 and the bacterial lacZ genes in transfecting osteoblasts in vitro and osseous tissues in vivo . We demonstrate that adenovirus-mediated gene therapy efficiently transfects osteoblasts in vitro with the TGF-beta1 virus causing a marked up-regulation in TGF-beta1 mRNA expression even 7 days after transfection . Increased TGF-beta1 mRNA expression was efficiently translated into protein production and resulted in approximately a 46-fold increase in TGF-beta1 synthesis as compared with control cells (vehicle- or B-galactosidase-transfected) . Moreover, virally produced TGF-beta1 was functionally active and regulated the expression of collagen IalphaI (5-fold increase) and the vascular endothelial growth factor (2.5-fold increase) . Using an adenovirus vector encoding the Escherichia coli LacZ gene, we demonstrated that adenovirus-mediated gene transfer efficiently transfects osteoblasts and osteocytes in vivo and that transfection can be performed by a simple percutaneous injection . Finally, we show that delivery of the hTGF-beta1 gene to osseous tissues in vivo results in significant changes in the epiphyseal plate primarily as a result of increased thickness of the provisional calcification zone.

Immunology, 1999 Aug, 97(4), 706 - 13
Mucosal immunogenicity and adjuvant activity of the recombinant A subunit of the Escherichia coli heat-labile enterotoxin; De Haan L et al.; The Escherichia coli heat-labile enterotoxin (LT) is an exceptionally effective mucosal immunogen and mucosal immunoadjuvant towards coadministered antigens . Although, in general, the molecular basis of these properties is poorly understood, both the toxic ADP-ribosylation activity of the LTA subunit and the cellular toxin receptor, ganglioside, GM1-binding properties of the LTB-pentamer have been suggested to be involved . In recent studies we found that GM1-binding is not essential for the adjuvanticity of LT, suggesting an important role for the LTA subunit in immune stimulation . We now describe the immunomodulatory properties of recombinant LTA molecules with or without ADP-ribosylation activity, LTA(His)10 and LTA-E112K(His)10, respectively . These molecules were expressed as fusion proteins with an N-terminal His-tag to allow simple purification on nickel-chelate columns . Their immunogenic and immunoadjuvant properties were assessed upon intranasal administration to mice, and antigen-specific serum immunoglobulin-isotype and -subtype responses and mucosal secretory immunoglobulin A (IgA) responses were monitored using enzyme-linked immunosorbent assay . With respect to immunogenicity, both LTA(His)10 and LTA-E112K(His)10 failed to induce antibody responses . On the other hand, immunization with both LT and the non-toxic LT-E112K mutant not only induced brisk LTB-specific, but also LTA-specific serum and mucosal antibody responses . Therefore, we conclude that linkage of LTA to the LTB pentamer is essential for the induction of LTA-specific responses . With respect to adjuvanticity, both LTA(His)10 and LTA-E112K(His)10 were found to stimulate serum and mucosal antibody responses towards coadministered influenza subunit antigen . Remarkably, responses obtained with LTA(His)10 were comparable in both magnitude and serum immunoglobulin isotype and subtype distributions to those observed after coimmunization with LT, LT-E112K, or recombinant LTB . We conclude that LTA, by itself, can act as a potent adjuvant for intranasally administered antigens in a fashion independent of ADP-ribosylation activity and association with the LTB pentamer.

Immunology, 1999 Aug, 97(4), 699 - 705
DNA activates human immune cells through a CpG sequence-dependent manner; Bauer M et al.; While bacterial DNA and cytosine-guanosine-dinucleotide-containing oligonucleotides (CpG ODN) are well described activators of murine immune cells, their effect on human cells is inconclusive . We investigated their properties on human peripheral blood mononuclear cells (PBMC) and subsets thereof, such as purified monocytes, T and B cells . Here we demonstrate that bacterial DNA and CpG ODN induce proliferation of B cells, while other subpopulations, such as monocytes and T cells, did not proliferate . PBMC mixed cell cultures, as well as purified monocytes, produced interleukin-6 (IL-6), IL-12 and tumour necrosis factor-alpha upon stimulation with bacterial DNA; however, only IL-6 and IL-12 secretion became induced upon CpG ODN stimulation . We conclude that monocytes, but not B or T cells, represent the prime source of cytokines . Monocytes up-regulated expression of antigen-presenting, major histocompatibility complex class I and class II molecules in response to CpG DNA . In addition, both monocytes and B cells up-regulate costimulatory CD86 and CD40 molecules . The activation by CpG ODN depended on sequence motifs containing the core dinucleotide CG since destruction of the motif strongly reduced immunostimulatory potential.

Infect Immun, 1999 Sep, 67(9), 4751 - 6
The gene locus yijP contributes to Escherichia coli K1 invasion of brain microvascular endothelial cells; Wang Y et al.; Most cases of Escherichia coli meningitis develop as a result of hematogenous spread, but it is not clear how circulating E . coli crosses the blood-brain barrier . A TnphoA mutant of E . coli K1 RS218 was shown to be significantly less invasive than its parent strain in bovine and human brain microvascular endothelial cells (BMEC), which constitute the blood-brain barrier . More importantly, traversal of the blood-brain barrier was significantly less with this mutant than with the parent strain in newborn rats with experimental hematogenous meningitis . A DNA segment containing the TnphoA insertion site was cloned from RS218, and the cloned DNA complemented the TnphoA mutant in invasion of BMEC . Nucleotide sequence revealed a near identity to that of a hypothetical yijP gene (also called f577) in the E . coli K-12 genome . Sequence analysis indicated that the E . coli K1 yijP gene likely encodes a 66 . 6-kDa membrane protein . Deletion and complementation experiments indicated that the yijP gene was involved in E . coli K1 invasion of BMEC, i.e., the invasive ability of E . coli K1 was significantly reduced after yijP was deleted and was restored by complementation with a plasmid containing the yijP open reading frame . This is the first demonstration that the yijP gene locus plays a role in the pathogenesis of E . coli K1 meningitis.

Infect Immun, 1999 Sep, 67(9), 4713 - 9
Isolation of recombinant protective Helicobacter pylori antigens; Hocking D et al.; A total of seven clones producing both new and previously described Helicobacter pylori proteins were isolated from a library of H . pylori genomic DNA . The screening approach by which these proteins were detected relied on the use of antisera raised in mice vaccinated with Helicobacter felis sonicate plus cholera toxin, a regimen which protects mice from H . pylori challenge . This strategy was designed to maximize the possibility of obtaining antigens which might be capable of conferring protection from H . pylori infection . Two of the clones were shown to encode the urease enzyme and the heat shock protein HspB, which have already been identified as protective antigens . The other five clones were sequenced, protein coding regions were deduced, and these sequences were amplified by PCR for incorporation into Escherichia coli expression vectors . The proteins produced from these expression systems were purified to allow testing for protective efficacy in an H . pylori mouse model . All five proteins were able to facilitate the clearance of a challenge with H . pylori, as judged by an assay of gastric urease activity and light microscopy on stomach sections . These results clearly indicate that the screening strategy has successfully identified candidate vaccine antigens.

Infect Immun, 1999 Sep, 67(9), 4407 - 17
Cloning and molecular characterization of plasmid-encoded antigens of Borrelia burgdorferi; Skare JT et al.; Thirteen independent clones that encode Borrelia burgdorferi antigens utilizing antiserum from infection-immune rabbits were identified . The serum was adsorbed against noninfectious B . burgdorferi B31 to enrich for antibodies directed against either infection-associated antigens of B . burgdorferi B31 or proteins preferentially expressed during mammalian infection . The adsorption efficiency of the immune rabbit serum (IRS) was assessed by Western immunoblot analysis with protein lysates derived from infectious and noninfectious B . burgdorferi B31 . The adsorbed IRS was used to screen a B . burgdorferi expression library to identify immunoreactive phage clones . Clones were then expressed in Escherichia coli and subsequently analyzed by Western blotting to determine the molecular mass of the recombinant B . burgdorferi antigens . Southern blot analysis of the 13 clones indicated that 10 contained sequences unique to infectious B . burgdorferi . Nucleotide sequence analysis indicated that the 13 clones were composed of 9 distinct genetic loci and that all of the genes identified were plasmid encoded . Five of the clones carried B . burgdorferi genes previously identified, including those encoding decorin binding proteins A and B (dbpAB), a rev homologue present on the 9-kb circular plasmid (cp9), a rev homologue from the 32-kb circular plasmid (cp32-6), erpM, and erpX . Additionally, four previously uncharacterized loci with no known homologues were identified . One of these unique clones encoded a 451-amino-acid lipoprotein with 21 consecutive, invariant 9-amino-acid repeats near the amino terminus that we have designated VraA (for "virulent strain-associated repetitive antigen A") . Since all the antigens identified are recognized by serum from infection immune rabbits, these antigens represent potential vaccine candidates and, based on the identification of dbpAB in this screen, may also be involved in pathogenic processes operative in Lyme borreliosis.

Infect Immun, 1999 Sep, 67(9), 4400 - 6
Genetically detoxified mutants of heat-labile toxin from Escherichia coli are able to act as oral adjuvants; Douce G et al.; Detoxified mutants of the Escherichia coli heat-labile toxin (LT) act as mucosal adjuvants to intranasally presented coadministered antigens . Here, we compare the adjuvant activity of a panel of detoxified derivatives of LT, using both intranasal (i.n.) and oral (p.o.) routes of administration . The mutants used as adjuvants varied in sensitivity to proteases and toxicity . With keyhole limpet hemocyanin (KLH) as the bystander antigen, the immune responses to i . n . immunizations were consistently higher than the equivalent p.o . -delivered proteins . LT-G192, a mutant which demonstrates a 10-fold reduction in toxicity in vitro, demonstrated wild-type adjuvant activity both i.n . and p.o., inducing similar titers of KLH specific antibody in the sera and immunoglobulin A in local mucosal secretions as wild-type LT . In line with previous data, the nontoxic holotoxoid LT-K63 induced intermediate immune responses in both the serum and mucosal secretions which were lower than those achieved with wild-type LT but at least 10-fold higher than those measured when the antigen was administered with LT-B . Although significant levels of local and systemic anti-KLH antibodies were induced following p.o . immunization with LT-K63, cellular proliferative responses to KLH was poor or undetectable . In contrast, LT and LT-G192 induced significant T-cell responses to KLH following p.o . immunization . These proliferating cells secreted both gamma interferon and interleukin-5, suggesting that the type of immune response induced following p.o . coimmunization with LT and purified protein is a mixed Th1/Th2 response.

FEBS Lett, 1999 Aug 6, 456(2), 253 - 6
Selection of ganglioside GM1-binding peptides by using a phage library; Matsubara T et al.; Ganglioside Gal beta1 --> 3GalNAc beta1 --> 4(NeuAc alpha2 --> 3) Gal beta1 --> 4Glc beta1 -->1'Cer (GM1)-binding peptides were obtained from a phage-displayed pentadecapeptide library by an affinity selection . The selection processes were in situ-monitored by a quartz-crystal microbalance method, on which a ganglioside GM1 monolayer was transferred . After five rounds of biopanning, the DNA sequencing of 18 selected phages showed that only three individual clones were selected . The peptide sequences of the random region were found to be DFRRLPGAFWQLRQP, GWWYKGRARPVSAVA and VWRLLAPPFSNRLLP . Binding constants of these phage clones to the GM1 monolayer were 10(10) M(-1) . Three synthetic pentadecapeptides inhibited the binding of cholera toxin B subunit to the GM1 monolayer with an IC50 of 24, 13 and 1.0 microM, respectively . These peptides will be useful for searching functional roles of ganglioside GMI.

J Ind Microbiol Biotechnol, 1999 Jul, 23(1), 668 - 76
A single-use luciferase-based mercury biosensor using Escherichia coli HB101 immobilized in a latex copolymer film; Lyngberg OK et al.; A single-use Hg(II) patch biosensor has been developed consisting of 1.25-cm diameter patches of two acrylic vinyl acetate copolymer layers coated on polyester . The top layer copolymer was 47 microm thick whereas the bottom layer of copolymer plus E . coli cells was 30 microm thick . The immobilized E . coli HB101 cells harbored a mer-lux plasmid construct and produced a detectable light signal when exposed to Hg(II) . The immobilized-cell Hg(II) biosensor had a sensitivity similar to that of suspended cells but a significantly larger detection range . The levels of mercury detected by the patches ranged from 0.1 nM to 10 000 nM HgCl2 in pyruvate buffer, and luciferase induction as a function of Hg(II) concentration was sigmoidal . Luciferase activity was detected in immobilized cells for more than 78 h after exposure of the cells to HgCl2 . Addition of 1 mM D-cysteine to the pyruvate buffer increased luciferase induction more than 100-fold in the immobilized cell patches and 3.5-fold in a comparable suspension culture . The copolymer patches with immobilized cells were stable at -20 degrees C for at least 3 months, and the Hg(II)-induced luciferase activity after storage was similar to that of samples assayed immediately after coating . Patches stored desiccated at room temperature for 2 weeks showed lower mercury-induced luciferase activity when compared to freshly prepared patches, but they still had a considerable detection range of 1 to 10 000 nM HgCl2.

Br J Pharmacol, 1999 Aug, 127(7), 1611 - 8
Influence of CGRP (8-37), but not adrenomedullin (22-52), on the haemodynamic responses to lipopolysaccharide in conscious rats; Gardiner SM et al.; 1 . The functional involvement of the vasodilator peptides, adrenomedullin (ADM) and calcitonin gene-related peptide (CGRP), in the haemodynamic sequelae of continuous infusion of lipopolysaccharide (LPS) was assessed in conscious, male, Long Evans rats, by the use of peptide antagonists . 2 . It was demonstrated that ADM (22-52) at a dose of 500 nmol kg-1 h-1 caused significant inhibition of the effects of ADM (1 nmol kg-1), without affecting responses to CGRP (0.1 or 1 nmol kg-1) . 3 . Even when the regional vasodilator responses to LPS infusion were enhanced (by pre-treatment with dexamethasone and the endothelin antagonist, SB 209670, or by pretreatment with SB 209670 and the AT1-receptor antagonist, losartan), ADM (22-52) had no significant cardiovascular effects . In contrast, the CGRP1-receptor antagonist, CGRP (8-37), caused small, but significant, inhibitions of the hypotensive and renal and mesenteric vasodilator effects of LPS, but only 6 h after onset of infusion in the presence of dexamethasone and SB 209670 . 4 . The results indicate that, in this model of endotoxaemia, the marked regional vasodilatations seen in the presence of dexamethasone and SB 209670 do not involve ADM, but do involve CGRP, albeit only to a small extent.

J Biol Chem, 1999 Aug 27, 274(35), 25144 - 50
Enzymatic repair of 5-formyluracil . II . Mismatch formation between 5-formyluracil and guanine during dna replication and its recognition by two proteins involved in base excision repair (AlkA) and mismatch repair (MutS); Terato H et al.; 5-Formyluracil (fU), a major methyl oxidation product of thymine, forms correct (fU:A) and incorrect (fU:G) base pairs during DNA replication . In the accompanying paper (Masaoka, A., Terato, H., Kobayashi, M., Honsho, A., Ohyama, Y., and Ide, H . (1999) J . Biol . Chem . 274, 25136-25143), it has been shown that fU correctly paired with A is recognized by AlkA protein (Escherichia coli 3-methyladenine DNA glycosylase II) . In the present work, mispairing frequency of fU with G and cellular repair protein that specifically recognized fU:G mispairs were studied using defined oligonucleotide substrates . Mispairing frequency of fU was determined by incorporation of 2'-deoxyribonucleoside 5'-triphosphate of fU opposite template G using DNA polymerase I Klenow fragment deficient in 3'-5' exonuclease . Mispairing frequency of fU was dependent on the nearest neighbor base pair in the primer terminus and 2-12 times higher than that of thymine at pH 7.8 and 2.6-6.7 times higher at pH 9.0 with an exception of the nearest neighbor T(template):A(primer) . AlkA catalyzed the excision of fU placed opposite G, as well as A, and the excision efficiencies of fU for fU:G and fU:A pairs were comparable . In addition, MutS protein involved in methyl-directed mismatch repair also recognized fU:G mispairs and bound them with an efficiency comparable to T:G mispairs, but it did not recognize fU:A pairs . Prior complex formation between MutS and a heteroduplex containing an fU:G mispair inhibited the activity of AlkA to fU . These results suggest that fU present in DNA can be restored by two independent repair pathways, i.e . the base excision repair pathway initiated by AlkA and the methyl-directed mismatch repair pathway initiated by MutS . Biological relevance of the present results is discussed in light of DNA replication and repair in cells.

J Biol Chem, 1999 Aug 27, 274(35), 25136 - 43
Enzymatic repair of 5-formyluracil . I . Excision of 5-formyluracil site-specifically incorporated into oligonucleotide substrates by alka protein (Escherichia coli 3-methyladenine DNA glycosylase II); Masaoka A et al.; 5-Formyluracil (fU) is a major thymine lesion produced by reactive oxygen radicals and photosensitized oxidation . We have previously shown that fU is a potentially mutagenic lesion due to its elevated frequency to mispair with guanine . Therefore, fU can exist in DNA as a correctly paired fU:A form or an incorrectly paired fU:G form . In this work, fU was site-specifically incorporated opposite A in oligonucleotide substrates to delineate the cellular repair mechanism of fU paired with A . The repair activity for fU was induced in Escherichia coli upon exposure to N-methyl-N'-nitro-N-nitrosoguanidine, and the induction was dependent on the alkA gene, suggesting that AlkA (3-methyladenine DNA glycosylase II) was responsible for the observed activity . Activity assay and determination of kinetic parameters using purified AlkA and defined oligonucleotide substrates containing fU, 5-hydroxymethyluracil (hU), or 7-methylguanine (7mG) revealed that fU was recognized by AlkA with an efficiency comparable to that of 7mG, a good substrate for AlkA, whereas hU, another major thymine methyl oxidation products, was not a substrate . (1)H and (13)C NMR chemical shifts of 5-formyl-2'-deoxyuridine indicated that the 5-formyl group caused base C-6 and sugar C-1' to be electron deficient, which was shown to result in destabilization of the N-glycosidic bond . These features are common in other good substrates for AlkA and are suggested to play key roles in the differential recognition of fU, hU, and intact thymine . Three mammalian repair enzymes for alkylated and oxidized bases cloned so far (MPG, Nth1, and OGG1) did not recognize fU, implying that the mammalian repair activity for fU resided on a yet unidentified protein . In the accompanying paper (Terato, H., Masaoka, A., Kobayashi, M., Fukushima, S., Ohyama, Y., Yoshida, M., and Ide, H., J . Biol . Chem . 274, 25144-25150), possible repair mechanisms for fU mispaired with G are reported.

J Biol Chem, 1999 Aug 27, 274(35), 25108 - 12
Kinetic analysis of the actinorhodin aromatic polyketide synthase; Dreier J et al.; Type II polyketide synthases (PKSs) are bacterial multienzyme systems that catalyze the biosynthesis of a broad range of natural products . A core set of subunits, consisting of a ketosynthase, a chain length factor, an acyl carrier protein (ACP) and possibly a malonyl CoA:ACP transacylase (MAT) forms a "minimal" PKS . They generate a poly-beta-ketone backbone of a specified length from malonyl-CoA derived building blocks . Here we (a) report on the kinetic properties of the actinorhodin minimal PKS, and (b) present further data in support of the requirement of the MAT . Kinetic analysis showed that the apoACP is a competitive inhibitor of minimal PKS activity, demonstrating the importance of protein-protein interactions between the polypeptide moiety of the ACP and the remainder of the minimal PKS . In further support of the requirement of MAT for PKS activity, two new findings are presented . First, we observe hyperbolic dependence of PKS activity on MAT concentration, saturating at very low amounts (half-maximal rate at 19.7 +/- 5.1 nM) . Since MAT can support PKS activity at less than 1/100 the typical concentration of the ACP and ketosynthase/chain length factor components, it is difficult to rule out the presence of trace quantities of MAT in a PKS reaction mixture . Second, an S97A mutant was constructed at the nucleophilic active site of the MAT . Not only can this mutant protein support PKS activity, it is also covalently labeled by {(14)C}malonyl-CoA, demonstrating that the serine nucleophile (which has been the target of PMSF inhibition in earlier studies) is dispensible for MAT activity in a Type II PKS system.

J Biol Chem, 1999 Aug 27, 274(35), 25033 - 41
PriA-directed assembly of a primosome on D loop DNA; Liu J et al.; Escherichia coli strains carrying null mutations in priA are chronically induced for the SOS response and are defective in homologous recombination, repair of UV damaged DNA, double-strand break repair, and both induced and constitutive stable DNA replication . This led to the proposal that PriA directed replication fork assembly at D loops formed by the homologous recombination machinery . The demonstration that PriA specifically recognized and bound D loop DNA supported this hypothesis . Using DNA footprinting as an assay, we show here that PriA also directs the assembly of a varphiX174-type primosome on D loop DNA . The ability to load a complete primosome on D loop DNA is a step necessary for replication fork assembly.

J Biol Chem, 1999 Aug 27, 274(35), 24953 - 8
Site-directed mutagenesis of the calcium-binding site of blood coagulation factor XIIIa; Lai TS et al.; Blood coagulation factor XIIIa is a calcium-dependent enzyme that covalently ligates fibrin molecules during blood coagulation . X-ray crystallography studies identified a major calcium-binding site involving Asp(438), Ala(457), Glu(485), and Glu(490) . We mutated two glutamic acid residues (Glu(485) and Glu(490)) and three aspartic acid residues (Asp(472), Asp(476), and Asp(479)) that are in close proximity . Alanine substitution mutants of these residues were constructed, expressed, and purified from Escherichia coli . The K(act) values for calcium ions increased by 3-, 8-, and 21-fold for E485A, E490A, and E485A,E490A, respectively . In addition, susceptibility to proteolysis was increased by 4-, 9-, and 10-fold for E485A, E490A, and E485A,E490A, respectively . Aspartic acids 472, 476, and 479 are not involved directly in calcium binding since the K(act) values were not changed by mutagenesis . However, Asp(476) and Asp(479) are involved in regulating the conformation for exposure of the secondary thrombin cleavage site . This study provides biochemical evidence that Glu(485) and Glu(490) are Ca(2+)-binding ligands that regulate catalysis . The binding of calcium ion to this site protects the molecule from proteolysis . Furthermore, Asp(476) and Asp(479) play a role in modulating calcium-dependent conformational changes that cause factor XIIIa to switch from a protease-sensitive to a protease-resistant molecule.

J Biol Chem, 1999 Aug 27, 274(35), 24921 - 9
Tetrahydrobiopterin inhibits monomerization and is consumed during catalysis in neuronal NO synthase; Reif A et al.; The biosynthesis of nitric oxide (NO) is catalyzed by homodimeric NO synthases (NOS) . For unknown reasons, all NOS co-purify with substoichiometric amounts of (6R)-5,6,7,8-tetrahydrobiopterin (H(4)Bip) and require additional H(4)Bip for maximal activity . We examined the effects of H(4)Bip and pterin-derived inhibitors (anti-pterins) on purified neuronal NOS-I quaternary structure and H(4)Bip content . During L-arginine turnover, NOS-I dimers time dependently dissociated into inactive monomers, paralleled by a loss of enzyme-associated pterin . Dimer dissociation was inhibited when saturating levels of H(4)Bip were added during catalysis . Similar results were obtained with pterin-free NOS-I expressed in Escherichia coli . This stabilizing effect of H(4)Bip was mimicked by the anti-pterin 2-amino-4,6-dioxo-3,4,5,6,8,8a,9, 10-octahydro-oxazolo{1,2f}-pteridine (PHS-32), which also displaced NOS-associated H(4)Bip in a competitive manner . Surprisingly, H(4)Bip not only dissociated from NOS during catalysis, but was only partially recovered in the solute (50.0 +/- 16.5% of control at 20 min) . NOS-associated H(4)Bip appeared to react with a NOS catalysis product to a derivative distinct from dihydrobiopterin or biopterin . Under identical conditions, reagent H(4)Bip was chemically stable and fully recovered (95.5 +/- 3.4% of control) . A similar loss of both reagent and enzyme-bound H(4)Bip and dimer content was observed by NO generated from spermine NONOate . In conclusion, we propose a role for H(4)Bip as a dimer-stabilizing factor of neuronal NOS during catalysis, possibly by interfering with enzyme destabilizing products.

J Biol Chem, 1999 Aug 27, 274(35), 24888 - 95
The protozoan parasite Toxoplasma gondii expresses two functional plant-like glycolytic enzymes . Implications for evolutionary origin of apicomplexans; Dzierszinski F et al.; The recent discovery of a vestigial, nonphotosynthetic plastid ("apicoplast") in the Apicomplexa has considerably modified our perception of the evolutionary origin of these parasites . Phylogenetic analysis and the presence of four surrounding membranes of the apicoplast provide important support for the hypothesis that apicomplexans have acquired their apicoplast by secondary endosymbiosis, probably from a green alga . This suggests that genes encoding predicted homologs of proteins of green algae or related photosynthetic lineages could have entered the nucleus of apicomplexan parasites by transfer from the ancestor harboring the apicoplast . We describe here complementary DNAs encoding two Toxoplasma gondii glycolytic enzymes, glucose-6-phosphate isomerase (G6-PI) and enolase, which have considerable identities with land plant counterparts . Both cDNAs of T . gondii complement Escherichia coli mutants lacking G6-PI and enolase genes and lead to the expression of active enzymes . In the drug untreatable encysted bradyzoites of T . gondii, G6-PI and enolase genes are overexpressed or exclusively expressed at both transcriptional and protein levels . Moreover, three-dimensional models and protein phylogeny confirmed that G6-PIs and enolases of T . gondii, Plasmodium falciparum, and land plants are closely related . Because these glycolytic enzymes are plant homologs, which differ from those of animals, they will be useful to trace the evolutionary origin of Apicomplexa and might offer novel chemotherapeutic targets in diseases caused by apicomplexan parasites.

J Biol Chem, 1999 Aug 27, 274(35), 24617 - 24
A pH-dependent conformational change of NhaA Na(+)/H(+) antiporter of Escherichia coli involves loop VIII-IX, plays a role in the pH response of the protein, and is maintained by the pure protein in dodecyl maltoside; Gerchman Y et al.; Digestion with trypsin of purified His-tagged NhaA in a solution of dodecyl maltoside yields two fragments at alkaline pH but only one fragment at acidic pH . Determination of the amino acid sequence of the N terminus of the cleavage products show that the pH-sensitive cleavage site of NhaA, both in isolated everted membrane vesicles as well as in the pure protein in detergent, is Lys-249 in loop VIII-IX, which connects transmembrane segment VIII to IX . Interestingly, the two polypeptide products of the split antiporter remain complexed and co-purify on Ni(2+)-NTA column . Loop VIII-IX has also been found to play a role in the pH regulation of NhaA; three mutations introduced into the loop shift the pH profile of the Na(+)/H(+) antiporter activity as measured in everted membrane vesicles . An insertion mutation introducing Ile-Glu-Gly between residues Lys-249 and Arg-250 (K249-IEG-R250) and Cys replacement of either Val-254 (V254C) or Glu-241 (E241C) cause acidic shift of the pH profile of the antiporter by 0.5, 1, and 0.3 pH units, respectively . Interestingly, the double mutant E241C/V254C introduces a basic shift of more than 1 pH unit with respect to the single mutation V254C . Taken together these results imply the involvement of loop VIII-IX in the pH-induced conformational change, which leads to activation of NhaA at alkaline pH.

Biochem J, 1999 Sep 1, 342 ( Pt 2), 465 - 72
Overexpression of the FAD-binding domain of the sulphite reductase flavoprotein component from Escherichia coli and its inhibition by iodonium diphenyl chloride; Coves J et al.; SiR-FP43, the NADPH- and FAD-binding domain of the Escherichia coli sulphite reductase flavoprotein component (SiR-FP), has been overexpressed and characterized . It folds independently, retaining FAD as a cofactor and the catalytic properties associated with the presence of this cofactor . Iodonium diphenyl chloride (IDP) was shown to be a very efficient inhibitor of SiR-FP43 and SiR-FP60, the monomeric form of SiR-FP, containing both FMN and FAD as cofactors (K(i) = 18.5 +/- 5 microM, maximal inactivation rate = 0.053 +/- 0.005 s(-1)) . In both cases, inactivation was shown to result from covalent binding of a phenyl group to FAD exclusively, in marked contrast with previous results obtained with cytochrome P450 reductase (CPR), where FMN and a tryptophan were phenylated, but not FAD . However, our kinetic analyses are in agreement with the inhibition mechanism demonstrated with CPR {Tew (1993) Biochemistry 32, 10209-10215} . Nine different FAD phenylated adducts were isolated and, for the first time, two FAD phenylated adducts were identified directly after extraction from a protein . Taken together, our results have shown that flavoprotein inactivation by IDP is not a reliable indicator for a flavin radical intermediate in catalysis.

Biochem J, 1999 Sep 1, 342 ( Pt 2), 287 - 92
Phosphorylation of Ser-241 is essential for the activity of 3-phosphoinositide-dependent protein kinase-1: identification of five sites of phosphorylation in vivo; Casamayor A et al.; 3-phosphoinositide-dependent protein kinase-1 (PDK1) expressed in unstimulated 293 cells was phosphorylated at Ser-25, Ser-241, Ser-393, Ser-396 and Ser-410 and the level of phosphorylation of each site was unaffected by stimulation with insulin-like growth factor-1 . Mutation of Ser-241 to Ala abolished PDK1 activity, whereas mutation of the other phosphorylation sites individually to Ala did not affect PDK1 activity . Ser-241, unlike the other phosphorylation sites on PDK1, was resistant to dephosphorylation by protein phosphatase 2A(1) . Ser-241 lies in the activation loop of the PDK1 kinase domain between subdomains VII and VIII in the equivalent position to the site that PDK1 phosphorylates on its protein kinase substrates . PDK1 expressed in bacteria was active and phosphorylated at Ser-241, suggesting that PDK1 can phosphorylate itself at this site, leading to its own activation.

Methods, 1999 Jul, 18(3), 401 - 6
Drosophila replication and repair proteins: proliferating cell nuclear antigen (PCNA); Mozzherin DJ et al.; Proliferating cell nuclear antigen (PCNA), a protein intimately involved in both replication and repair, has been identified in eukaryotes at all levels of evolution . Is primary sequence, Drosophila melanogaster PCNA is 73% identical to mammalian PCNA . Moreover, it is able to substitute for mammalian PCNA in at least one intricate cell-free replication assay . Mutations in the gene for Drosophila PCNA, including some that are temperature sensitive, have been reported . Procedures are described for the biochemical purification of wild-type PCNA from a population of 6- to 18-h-old Drosophila embryos . Procedures were also developed for purification of unmodified wild-type Drosophila PCNA after induction of expression in Escherichia coli . An NH(2)-terminally His-tagged but otherwise wild-type Drosophila PCNA, as well as mutant His-tagged PCNA, were also engineered and purified to apparent homogeneity . Finally, an in situ polyacrylamide gel technique allows DNA polymerase assays to be performed on portions of single adults as well as single Drosophila embryos . This assay should tremendously facilitate systematic genetic studies of metazoan replication and repair .

Shock, 1999 Jun, 11(6), 411 - 5
Macrophage TNF mRNA expression induced by LPS is regulated by sphingomyelin metabolites; Lo CJ et al.; Metabolism of macrophage (MO) membrane phospholipids produces key mediators of inflammation and major second messengers that modulate inflammatory responses during sepsis . Sphingomyelin is a major class of phospholipid that releases ceramide and sphingosine . This study was designed to investigate the involvement of sphingomyelin metabolites in MO activation by lipopolysaccharide (LPS) . Rabbit alveolar MO were obtained by bronchoalveolar lavage and exposed to C6-ceramide, a cell-permeable analogue of natural ceramide, or sphingosine in the presence of Escherichia coli LPS (100 ng/mL) . Tumor necrosis factor (TNF) mRNA expression was measured by Northern blot assays . Total nuclear extract was harvested for the measurement of nuclear factor KB (NFkappaB) with electrophoretic mobility shift assays . MO TNF production was measured by L929 bioassays . C-6 ceramide did not have any effects on MO TNF production or TNF mRNA expression with or without LPS stimulation . Inhibition of ceramide metabolism with 1-phenyl-2-decanoylamino-3-morpholino-1-propanol (PDMP), or N-oleoyl-ethanolamine (NOE) also did not induce TNF mRNA or TNF production . In comparison, sphingosine inhibited TNF mRNA expression as well as TNF production of LPS-stimulated MO . LPS-induced MO NFkappaB activity was also reduced by sphingosine . Our data indicate that ceramide alone has no effect on macrophage activity, but its metabolite sphingosine down-regulates MO activation induced by LPS stimulation . Therefore, the sphingomyelin pathway is involved in the regulation of MO activation.

Nucleic Acids Res, 1999 Aug 15, 27(16), 3283 - 90
Facile characterization of translation initiation via nonsense codon suppression; Karginov AV et al.; A new strategy for studying the mechanism of translation initiation in eukaryotes has been developed . The strategy involves the use of an in vitro translation system to incorporate a non-natural fluorescent amino acid into a protein from a suppressor tRNAPheCUA misacylated with that amino acid . It is thereby possible to monitor translation initiation efficiency at an AUG codon in different contexts; this is illustrated for three constructs encoding Escherichia coli dihydrofolate reductase mRNA with different translation initiation regions . Fluorescence measurements after in vitro translation of the mRNAs in rabbit reticulocyte lysate reflected differences in the position and efficiency of translation initiation and, therefore, can be used for characterization of the translation initiation process.

Nucleic Acids Res, 1999 Aug 15, 27(16), 3245 - 52
A chicken embryo protein related to the mammalian DEAD box protein p68 is tightly associated with the highly purified protein-RNA complex of 5-MeC-DNA glycosylase; Jost JP et al.; We have shown previously that DNA demethylation by chick embryo 5-methylcytosine (5-MeC)-DNA glycosylase needs both protein and RNA . Amino acid sequences of nine peptides derived from a highly purified 5-MeC-DNA glycosylase complex were identified by Nanoelectrospray ionisation mass spectrometry to be identical to the mammalian nuclear DEAD box protein p68 RNA helicase . Antibodies directed against human p68 helicase cross-reacted with the purified 5-MeC-DNA glycosylase complex and immunoprecipitated the glycosylase activity . A 2690 bp cDNA coding for the chicken homologue of mammalian p68 was isolated and sequenced . Its derived amino acid sequence is almost identical to the human p68 DEAD box protein up to amino acid position 473 (from a total of 595) . This sequence contains all the essential conserved motifs from the DEAD box proteins which are the ATPase, RNA unwinding and RNA binding motifs . The rest of the 122 amino acids in the C-terminal region rather diverge from the human p68 RNA helicase sequence . The recombinant chicken DEAD box protein expressed in Escherichia coli cross-reacts with the same p68 antibodies as the purified chicken embryo 5-MeC-DNA glycosylase complex . The recombinant protein has an RNA-dependent ATPase and an ATP-dependent helicase activity . However, in the presence or absence of RNA the recombinant protein had no 5-MeC-DNA glycosylase activity . In situ hybridisation of 5 day-old chicken embryos with antisense probes of the chicken DEAD box protein shows a high abundance of its transcripts in differentiating embryonic tissues.

Nucleic Acids Res, 1999 Aug 1, 27(15), 3197 - 204
Substrate recognition by Escherichia coli MutY using substrate analogs; Chepanoske CL et al.; The Escherichia coli adenine glycosylase MutY is involved in the repair of 7,8-dihydro-8-oxo-2"-deoxyguanosine (OG):A and G:A mispairs in DNA . Our approach toward understanding recognition and processing of DNA damage by MutY has been to use substrate analogs that retain the recognition properties of the substrate mispair but are resistant to the glycosylase activity of MutY . This approach provides stable MutY-DNA complexes that are amenable to structural and biochemical characterization . In this work, the interaction of MutY with the 2"-deoxyadenosine analogs 2"-deoxy-2"-fluoroadenosine (FA), 2"-deoxyaristeromycin (R) and 2"-deoxyformycin A (F) was investigated . MutY binds to duplexes containing the FA, R or F analogs opposite G and OG within DNA with high affinity; however, no enzymatic processing of these duplexes is observed . The specific nature of the interaction of MutY with an OG:FA duplex was demonstrated by MPE-Fe(II) hydroxyl radical footprinting experiments which showed a nine base pair region of protection by MutY surrounding the mispair . DMS footprinting experiments with an OG:A duplex revealed that a specific G residue located on the OG-containing strand was protected from DMS in the presence of MutY . In contrast, a G residue flanking the substrate analogs R, F or FA was observed to be hypersensitive to DMS in the presence of MutY . These results suggest a major conformational change in the DNA helix upon binding of MutY that exposes the substrate analog-containing strand . This finding is consistent with a nucleotide flipping mechanism for damage recognition by MutY . This work demonstrates that duplex substrates for MutY containing FA, R or F instead of A are excellent substrate mimics that may be used to provide insight into the recognition by MutY of damaged and mismatched base pairs within DNA.

Nucleic Acids Res, 1999 Aug 1, 27(15), 3153 - 8
Repair of oxidative DNA base lesions induced by fluorescent light is defective in xeroderma pigmentosum group A cells; Lipinski LJ et al.; Fluorescent light (FL) has been shown to generate free radicals within cells, however, the specific chemical nature of DNA damage induced by FL has not previously been determined . Using gas chromatography/isotope dilution mass spectrometry, we have detected induction of the oxidative DNA lesions 5-hydroxycytosine (5-OH-Cyt), 2,6-diamino-4-hydroxy-5-formamidopyrimidine (FapyGua) and 4, 6-diamino-5-formamidopyrimidine (FapyAde) in cultured cells irradiated with FL . We followed the repair of these lesions in normal and xeroderma pigmentosum group A (XP-A) cells . 5-OH-Cyt and FapyGua were repaired efficiently in normal cells within 6 h following FL exposure . XP-A cells were unable to repair these oxidative DNA base lesions . Additionally, to compare the repair of oxidative lesions induced by various sources, in vitro repair studies were performed using plasmid DNA damaged by FL, gamma-irradiation or OsO(4)treatment . Whole cell extracts from normal cells repaired damaged substrates efficiently, whereas there was little repair in XP-A extracts . Our data demon-strate defective repair of oxidative DNA base lesions in XP-A cells in vivo and in vitro.

Nucleic Acids Res, 1999 Aug 1, 27(15), 3138 - 45
Exposition of a family of RNA m(5)C methyltransferases from searching genomic and proteomic sequences; Reid R et al.; The Escherichia coli fmu gene product has recently been determined to be the 16S rRNA m(5)C 967 methyltransferase . As such, Fmu represents the first protein identified as an S -adenosyl-L-methionine (AdoMet)- dependent RNA m(5)C methyltransferase whose amino acid sequence is known . Using the amino acid sequence of Fmu as an initial probe in an iterative search of completed DNA sequence databases, 27 homologous ORF products were identified as probable RNA m(5)C methyltransferases . Further analysis of sequences in undeposited genomic sequencing data and EST databases yielded more than 30 additional homologs . These putative RNA m(5)C methyltransferases are grouped into eight subfamilies, some of which are predicted to consist of direct genetic counterparts, or orthologs . The enzymes proposed to be RNA m(5)C methyltransferases have sequence motifs closely related to signature sequences found in the well-studied DNA m(5)C methyltransferases and other AdoMet-dependent methyltransferases . Structure-function correlates in the known AdoMet methyltransferases support the assignment of this family as RNA m(5)C methyltransferases.

Nucleic Acids Res, 1999 Aug 1, 27(15), 3104 - 10
Direct identification of NH...N hydrogen bonds in non-canonical base pairs of RNA by NMR spectroscopy; Wohnert J et al.; It is shown that the recently developed quantitative J(NN)HNN-COSY experiment can be used for the direct identification of hydrogen bonds in non-canonical base pairs in RNA . Scalar(2h)J(NN)couplings across NH.N hydrogen bonds are observed in imino hydrogen bonded GA base pairs of the hpGA RNA molecule, which contains a tandem GA mismatch, and in the reverse Hoogsteen AU base pairs of the E-loop of Escherichia coli 5S rRNA . These scalar couplings correlate the imino donor(15)N nucleus of guanine or uridine with the acceptor N1 or N7 nucleus of adenine . The values of the corresponding(2h)J(NN)coupling constants are similar in size to those observed in Watson-Crick base pairs . The reverse Hoogsteen base pairs could be directly detected for the E-loop of E.coli 5S rRNA both in the free form and in a complex with the ribosomal protein L25 . This supports the notion that the E-loop is a pre-folded RNA recognition site that is not subject to significant induced conformational changes . Since Watson-Crick GC and AU base pairs are also readily detected the HNN-COSY experiment provides a useful and sensitive tool for the rapid identification of RNA secondary structure elements.

Nucleic Acids Res, 1999 Aug 1, 27(15), 3064 - 70
Evolutionary conserved mechanism of transcriptional repression by even-skipped; McKay LM et al.; Even-skipped (Eve) is a transcriptional repressor involved in segment formation in Drosophila melano-gaster . In order to gain further insights into the mechanism of action of Eve we tested whether it would function as a transcriptional repressor in mammalian cells . We found that Eve was indeed a potent repressor in two different mammalian cell types and at several promoters . In vitro transcription assays confirmed that Eve directly represses transcription initiation when specifically targeted to a promoter . We also found that, unlike the case with transcriptional activators, Eve does not repress transcription synergistically . Analysis of the effect of Eve on preinitiation complex assembly in a crude HeLa cell nuclear extract demonstrated that the Eve repression domain functions by preventing the assembly of TFIID with the promoter . Our data support the hypothesis that Eve contains an active repression domain that functions specifically to prevent preinitiation complex formation.

Nucleic Acids Res, 1999 Aug 1, 27(15), 3049 - 56
RecG helicase activity at three- and four-strand DNA structures; McGlynn P et al.; The RecG helicase of Escherichia coli is necessary for efficient recombination and repair of DNA in vivo and has been shown to catalyse the unwinding of DNA junctions in vitro . Despite these findings, the precise role of RecG remains elusive . However, models have been proposed in which RecG promotes the resolution of linked duplexes by targeting three-strand junctions present at D-loops . One such model postulates that RecG catalyses the formation of four-strand (Holliday) junctions from three-strand junctions . To test this model, the DNA binding and unwinding activities of RecG were analysed using synthetic three- and four-strand junctions . The substrate specificity of RecG was found to depend critically on the concentrations of ATP and MgCl(2)and under certain conditions RecG preferentially unwound three-strand junction DNA . This was at least partly due to the larger inhibitory effect of MgCl(2)on the binding of four-strand as opposed to three-strand junctions by RecG . Thus RecG may be targeted to three-strand junctions in vivo whilst still being able to branch migrate the four-strand junctions formed as a result of the initial helicase reaction . The increase in the dissociation constant of RecG on conversion of a three-strand into a four-strand junction may also facilitate resolution of the four-strand junction by the RuvABC complex.

Nucleic Acids Res, 1999 Aug 1, 27(15), 3042 - 8
Regulation of the ribonucleotide reductase small subunit gene by DNA-damaging agents in Dictyostelium discoideum; Gaudet P et al.; In Escherichia coli, yeast and mammalian cells, the genes encoding ribonucleotide reductase, an essential enzyme for de novo DNA synthesis, are up-regulated in response to DNA damaging agents . We have examined the response of the rnrB gene, encoding the small subunit of ribonucleotide reductase in Dictyostelium discoideum, to DNA damaging agents . We show here that the accumulation of rnrB transcript is increased in response to methyl methane sulfonate, 4-nitroquinoline-1-oxide and irradiation with UV-light, but not to the ribonucleotide reductase inhibitor hydroxyurea . This response is rapid, transient and independent of protein synthesis . Moreover, cells from different developmental stages are able to respond to the drug in a similar fashion, regardless of the basal level of expression of the rnrB gene . We have defined the cis -acting elements of the rnrB promoter required for the response to methyl methane sulfonate and 4-nitroquinoline-1-oxide by deletion analysis . Our results indicate that there is one element, named box C, that can confer response to both drugs . Two other boxes, box A and box D, specifically conferred response to methyl methane sulfonate and 4-nitroquinoline-1-oxide, respectively.

Mol Cell Biol, 1999 Sep, 19(9), 6012 - 9
Base pairing between U3 small nucleolar RNA and the 5' end of 18S rRNA is required for pre-rRNA processing; Sharma K et al.; The loop of a stem structure close to the 5' end of the 18S rRNA is complementary to the box A region of the U3 small nucleolar RNA (snoRNA) . Substitution of the 18S loop nucleotides inhibited pre-rRNA cleavage at site A(1), the 5' end of the 18S rRNA, and at site A(2), located 1.9 kb away in internal transcribed spacer 1 . This inhibition was largely suppressed by a compensatory mutation in U3, demonstrating functional base pairing . The U3-pre-rRNA base pairing is incompatible with the structure that forms in the mature 18S rRNA and may prevent premature folding of the pre-rRNA . In the Escherichia coli pre-rRNA the homologous region of the 16S rRNA is also sequestered, in that case by base pairing to the 5' external transcribed spacer (5' ETS) . Cleavage at site A(0) in the yeast 5' ETS strictly requires base pairing between U3 and a sequence within the 5' ETS . In contrast, the U3-18S interaction is not required for A(0) cleavage . U3 therefore carries out at least two functionally distinct base pair interactions with the pre-rRNA . The nucleotide at the site of A(1) cleavage was shown to be specified by two distinct signals; one of these is the stem-loop structure within the 18S rRNA . However, in contrast to the efficiency of cleavage, the position of A(1) cleavage is not dependent on the U3-loop interaction . We conclude that the 18S stem-loop structure is recognized at least twice during pre-rRNA processing.

J Pharmacol Exp Ther, 1999 Sep, 290(3), 1467 - 74
Retroviral-mediated expression of the P140A, but not P140A/G156A, mutant form of O6-methylguanine DNA methyltransferase protects hematopoietic cells against O6-benzylguanine sensitization to chloroethylnitrosourea treatment; Maze R et al.; O(6)-Benzylguanine (6-BG) inactivates mammalian O(6)-methylguanine DNA methyltransferase (MGMT), an important DNA repair protein that protects cells against chloroethylnitrosourea (CENU) cytotoxicity . 6-BG is being tested as an approach to treat CENU-resistant tumors that overexpress endogenous MGMT . However, in addition to restoring CENU tumor cell sensitivity, 6-BG also increases the cytotoxic effects of CENUs on hematopoietic cells . Several 6-BG-resistant human MGMT mutants have been characterized in Escherichia coli and are predicted to protect mammalian cells against the combination of 6-BG and CENU treatment in vivo . Two mutants, P140A and P140A/G156A, demonstrated 20- and 1200-fold more resistance to 6-BG depletion of MGMT activity compared with wild-type MGMT (WTMGMT) . Here, we analyzed retroviral vectors that express either WTMGMT, the P140A or P140A/G156A mutant forms of MGMT . Retroviral-infected L1210 hematopoietic cells demonstrated similar levels of RNA in all transduced clones . However, the amount of MGMT protein and DNA repair activity was reduced in clones expressing the P140A/G156A mutant compared with those expressing WTMGMT or P140A . Expression of P140A was associated with a 4- to 8-fold increase in resistance to 6-BG depletion of MGMT in transduced L1210 clones and a 1, 3-bis(2-chloroethyl)-1-nitrosourea IC(50) of 50 microM (compared with 27.5 microM for WTMGMT) in primary murine hematopoietic cells . These results demonstrate the utility of screening 6-BG-resistant MGMT proteins in hematopoietic cells and provide evidence that the P140A mutant form of MGMT generates 6-BG- and CENU-resistant hematopoietic cells . Retrovirus vectors expressing this mutant may be useful in future human gene therapy trials.

Learn Mem, 1998 May-Jun, 5(1-2), 157 - 65
Dopamine and mushroom bodies in Drosophila: experience-dependent and -independent aspects of sexual behavior; Neckameyer WS; Depletion of dopamine in Drosophila melanogaster adult males, accomplished through systemic introduction of the tyrosine hydroxylase inhibitor 3-iodo-tyrosine, severely impaired the ability of these flies to modify their courtship responses to immature males . Mature males, when first exposed to immature males, will perform courtship rituals; the intensity and duration of this behavior rapidly diminishes with time . Dopamine is also required for normal female sexual receptivity; dopamine-depleted females show increased latency to copulation . One kilobase of 5' upstream information from the Drosophila tyrosine hydroxylase (DTH) gene, when fused to the Escherichia coli beta-galactosidase reporter and transduced into the genome of Drosophila melanogaster, is capable of directing expression of the reporter gene in the mushroom bodies, which are believed to mediate learning acquisition and memory retention in flies . Ablation of mushroom bodies by treatment of newly hatched larva with hydroxyurea resulted in the inability of treated mature adult males to cease courtship when placed with untreated immature males . However, functional mushroom bodies were not required for the dopaminergic modulation of an innate behavior, female sexual receptivity . These data suggest that dopamine acts as a signaling molecule within the mushroom bodies to mediate a simple form of learning.

J Hepatol, 1999 Aug, 31(2), 315 - 22
Overexpression of Bcl-2 protects human hepatoma cells from Fas-antibody-mediated apoptosis; Takahashi M et al.; BACKGROUND/AIMS: Fas is a cell surface antigen, that triggers apoptosis upon specific ligand or antibody binding . The proto-oncogene bcl-2 prevents apoptosis induced by various treatments . The aim of our study was to evaluate whether Bcl-2 protects hepatoma cells from Fas-mediated apoptosis . METHODS: Two human cell lines, HCC-T and HepG2 were used . Expression of Fas antigen and Bcl-2 was detected by flow cytometry and Western blotting . Cell viability and apoptotic change were examined after anti-Fas- and antisense oligodeoxynucleotide treatments . Apoptotic cells were detected by nick-end labelling and the TUNEL method . To test if Bcl-2 expression can protect HepG2 cells from Fas-mediated apoptosis, the cells were transduced using retroviral vector, LZBC, designed to coexpress E . coli beta-galactosidase and human Bcl-2 . To further confirm the protective effect of Bcl-2 expression against Fas-mediated apoptosis in HepG2, Bcl-2 expressing plasmid vector was produced and a cell line stably expressing Bcl-2 was cloned . RESULTS: Western blot analysis showed constitutive Bcl-2 expression in HCC-T cells, but not in HepG2 cells . HCC-T was resistant to apoptosis after treatment with an agonist anti-Fas antibody (1 microg/ml for 3 days), whereas 33% of the HepG2 cells were killed by this treatment . Inhibition of Bcl-2 expression by transfection of antisense oligodeoxynucleotides caused spontaneous apoptosis in HCC-T, but not in HepG2 cells, suggesting that Bcl-2 is essential for survival of HCC-T cells, whereas other proteins may substitute for it in HepG2 cells . Following LZBC infection, 10% HepG2 cells were beta-galactosidase-positive by X-gal staining and Bcl-2-positive . In cells surviving after anti-Fas treatment, the proportion of beta-galactosidase-positive cells increased to 50% and the beta-galactosidase activity increased 6-fold, indicating that Bcl-2 expression protected the cells from Fas-mediated apoptosis . In the cloned HepG2 cells stably expressing Bcl-2, the extent of Fas-mediated apoptosis was inversely related to the level of Bcl-2 expression . CONCLUSION: Bcl-2 confers protection to human hepatoma cells against Fas-mediated apoptosis, and is essential for survival of some, but not all, hepatoma cells.

Zhongguo Yi Xue Ke Xue Yuan Xue Bao, 1997 Dec, 19(6), 409 - 13
{Study on the effect of continuous administration of IL-1ra in BXSB mice}; Yang G et al.; OBJECTIVE: To explore the consequences of IL-1 blocking in BXSB mice which is an experimental model for human SLE . METHODS: rh IL-1ra was expressed in E . coli and injected in BXSB mice . 13 of 4.5 month-age male BXSB mice were divided into two groups, one group was injected rh IL-1ra 10 times (twice a week) at dose of 400 micrograms per mouse each time, another group was injected PBS at the same time as control . We monitored serum ANA and proteinuria weekly, and detected IgG, C3 deposition and IL-6 expression in kidneys at 40th day . RESULTS: The results showed that the increased level of serum ANA and proteinuria in treatment group were not higher than in control group, the IgG, C3 deposition and IL-6 expression in kidneys and IL-6 activity in serum of the treaed group were lower than the control group, whereas no difference of GPT level in the two groups . CONCLUSION: IL-1 might play a pathogenic role in BXSB mice . Blocking or reducing IL-1 secretion would be of beneficial to the treatment of SLE.

Zhongguo Yi Xue Ke Xue Yuan Xue Bao, 1997 Apr, 19(2), 145 - 9
{Experimental studies on somatic gene therapy for diabetes . I . Structuring of recombinant from human insulin gene and ammalian expression vector PRC/CMV}; Xiao X et al.; To develop a model somatic gene therapy system for diabetes, we constructed a human insulin expression vector in non B cells . As the first step, an insulin cDNA fragment of 260 bp, generated by a complet digestion of PBCA with EcoR I and BamH I, was inserted into the EcoR I/BamH I site of plasmid PBS.SK by ligation of cohevise-ended DNA to construct transition plasmid PBS.INS . Then the plasmid PBS.INS was completly digested by Hind III and Xbal I . The small DNA fragment containing insulin cDNA gene was subcloned to the expression plasmid PRC/CMV to form recombinant PRC/CMV.INS.

Gene, 1999 Aug 20, 236(2), 293 - 301
Cloning and characterization of the gene encoding Aspergillus nidulans DNA topoisomerase II; Kim KH et al.; We have determined the complete nucleotide sequence of a 5544bp genomic DNA fragment from Aspergillus nidulans that encodes DNA topoisomerase II (topo II) . It contains a single open reading frame of 4740bp that codes for 1579 amino acid residues with a molecular weight of 178kDa; when expressed in Escherichia coli and Saccharomyces cerevisiae the molecular weight was 180kDa . The gene (TOP2) is divided into three exons . Two introns, 54bp and 60bp in length, are located at nucleotide positions 187 and 3214 respectively . Comparison of the deduced amino acid sequence with other eukaryotic topo II sequences showed a higher degree of identity with other fungal enzymes than the human topo IIalpha . One of monoclonal antibodies raised against human topo II, 6H8, can cross-react with Aspergillus topo II.

Placenta, 1999 Sep, 20(7), 561 - 6
Expression of thioredoxin and thioredoxin reductase in placentae of pregnant mice exposed to lipopolysaccharide; Ejima K et al.; We have previously shown that thioredoxin and thioredoxin reductase were immunohistochemically localized in cytotrophoblasts, decidua and stromal cells in the stem villi of human placenta and that the addition of exogenous thioredoxin and thioredoxin reductase to mitochondrial fractions from human placenta displayed a protective effect on fumarase activity against oxidative stress . In this study, to investigate further the roles of thioredoxin and thioredoxin reductase in protecting pregnancy against oxidative stress, we examined the effect of lipopolysaccharide (LPS), which induces a variety of cytokines and produces radical oxygen species, on the expression of thioredoxin and thioredoxin reductase in mouse placenta . We focused on the placental protective effect in the second trimester, when the onset of placental dysfunction might occasionally lead to a critical state for the fetus . Thus we analysed placentae from mice on day 13 of pregnancy at various time points after they were injected with LPS (50 microg/kg i.p.) or saline as a control . The expressions of thioredoxin and thioredoxin reductase were evaluated by Western blotting and immunohistochemistry . Western blot analysis revealed that LPS approximately quadrupled the expression of both thioredoxin and thioredoxin reductase in the placentae of pregnant mice . When both proteins were localized immunohistochemically, it was found that the decidua and the diploid trophoblasts in the basal zone were intensively stained . Furthermore, the expression of 4-hydroxy-2-nonenal (HNE)-modified proteins, which are markers of oxidative stress, was enhanced in placenta by LPS . Our study suggests that the induced thioredoxin and thioredoxin reductase might protect the placenta from the stress induced by LPS .

J Mol Biol, 1999 Aug 27, 291(4), 877 - 98
High resolution crystal structures of the Escherichia coli lytic transglycosylase Slt70 and its complex with a peptidoglycan fragment; van Asselt EJ et al.; The 70 kDa soluble lytic transglycosylase (Slt70) from Escherichia coli is an exo-muramidase, that catalyses the cleavage of the glycosidic bonds between N -acetylmuramic acid and N -acetylglucosamine residues in peptidoglycan, the main structural component of the bacterial cell wall . This cleavage is accompanied by the formation of a 1,6-anhydro bond between the C1 and O6 atoms in the N -acetylmuramic acid residue (anhMurNAc) . Crystallographic studies at medium resolution revealed that Slt70 is a multi-domain protein consisting of a large ring-shaped alpha-superhelix with on top a catalytic domain, which resembles the fold of goose-type lysozyme . Here we report the crystal structures of native Slt70 and of its complex with a 1,6-anhydromuropeptide solved at nominal resolutions of 1.65 A and 1.90 A, respectively . The high resolution native structure reveals the details on the hydrogen bonds, electrostatic and hydrophobic interactions that stabilise the catalytic domain and the alpha-superhelix . The building-block of the alpha-superhelix is an "up-down-up-down" four-alpha-helix bundle involving both parallel and antiparallel helix pairs . Stabilisation of the fold is provided through an extensive packing of apolar atoms, mostly from leucine and alanine residues . It lacks, however, an internal consensus sequence that characterises other super-secondary helical folds like the beta-helix in pectate lyase or the (beta-alpha)-helix in the ribonuclease inhibitor . The 1, 6-anhydromuropeptide product binds in a shallow groove adjacent to the peptidoglycan-binding groove of the catalytic domain . The groove is formed by conserved residues at the interface of the catalytic domain and the alpha-superhelix . The structure of the Slt70-1, 6-anhydromuropeptide complex confirms the presence of a specific binding-site for the peptide moieties of the peptidoglycan and it substantiates the notion that Slt70 starts the cleavage reaction at the anhMurNAc end of the peptidoglycan .

J Surg Res, 1999 Sep, 86(1), 103 - 7
Fish oil augments macrophage cyclooxygenase II (COX-2) gene expression induced by endotoxin; Lo CJ et al.; BACKGROUND: Fish oil-supplemented diets have anti-inflammatory and immunomodulating effects . Although fish oil is readily incorporated into the cell membrane and influences the production of eicosanoids, the exact mechanism is not clear . This study was designed to investigate the effects of eicosapentaenoic acid (EPA), a major component of fish oil, on macrophage (Mphi) cyclooxygenase (COX) gene expression induced by LPS . METHODS: RAW 264.7 cells, a mouse Mphi cell line, were grown in EPA-rich media for 24 h . Mphi were washed and exposed to Escherichia coli LPS (10 microg/ml) . Membrane lipid profile was determined by gas chromatographic analysis . COX-1 and COX-2 mRNA expressions were determined by Northern blot assays with mouse-specific cDNA probes . PGE(2) production of Mphi was measured by ELISA . Mphi production of COX-2 protein was determined by Western blot assays with an anti-COX-2 antibody . RESULTS: Incubation in EPA-rich media increased membrane EPA and decreased arachidonic acid (AA) composition . COX-2 mRNA expression was induced by EPA and further augmented by LPS stimulation . EPA also augmented Mphi production of COX-2 protein . In comparison, COX-1 mRNA expression was not affected by either LPS stimulation or EPA incubation . EPA reduced PGE(2) production by LPS-stimulated Mphi . To further support that COX-2 mRNA was regulated by COX product, exogenous PGE(2) was added to Mphi prior to LPS stimulation . PGE(2) reduced COX-2 mRNA of LPS-stimulated Mphi . CONCLUSION: EPA displaces AA and reduces PGE(2) production by LPS-stimulated Mphi . Fish oil inhibition of Mphi PGE(2) production induces COX-2 mRNA expression through a COX-2 product-mediated feedback mechanism .

Protein Sci, 1999 Aug, 8(8), 1668 - 74
Escherichia coli maltose-binding protein is uncommonly effective at promoting the solubility of polypeptides to which it is fused; Kapust RB et al.; Although it is usually possible to achieve a favorable yield of a recombinant protein in Escherichia coli, obtaining the protein in a soluble, biologically active form continues to be a major challenge . Sometimes this problem can be overcome by fusing an aggregation-prone polypeptide to a highly soluble partner . To study this phenomenon in greater detail, we compared the ability of three soluble fusion partners--maltose-binding protein (MBP), glutathione S-transferase (GST), and thioredoxin (TRX)--to inhibit the aggregation of six diverse proteins that normally accumulate in an insoluble form . Remarkably, we found that MBP is a far more effective solubilizing agent than the other two fusion partners . Moreover, we demonstrated that in some cases fusion to MBP can promote the proper folding of the attached protein into its biologically active conformation . Thus, MBP seems to be capable of functioning as a general molecular chaperone in the context of a fusion protein . A model is proposed to explain how MBP promotes the solubility and influences the folding of its fusion partners.

Protein Sci, 1999 Aug, 8(8), 1623 - 35
The progressive development of structure and stability during the equilibrium folding of the alpha subunit of tryptophan synthase from Escherichia coli; Gualfetti PJ et al.; The urea-induced equilibrium unfolding of the alpha subunit of tryptophan synthase (alphaTS), a single domain alpha/beta barrel protein, displays a stable intermediate at approximately 3.2 M urea when monitored by absorbance and circular dichroism (CD) spectroscopy (Matthews CR, Crisanti MM, 1981, Biochemistry 20:784-792) . The same experiment, monitored by one-dimensional proton NMR, shows another cooperative process between 5 and 9 M urea that involves His92 (Saab-Rincon G et al., 1993, Biochemistry 32:13,981-13,990) . To further test and quantify the implied four-state model, N <--> I1 <--> I2 <--> U, the urea-induced equilibrium unfolding process was followed by tyrosine fluorescence total intensity, tyrosine fluorescence anisotropy and far-UV CD . All three techniques resolve the four stable states, and the transitions between them when the FL total intensity and CD spectroscopy data were analyzed by the singular value decomposition method . Relative to U, the stabilities of the N, I1, and I2 states are 15.4, 9.4, and 4.9 kcal mol(-1), respectively . I2 partially buries one or more of the seven tyrosines with a noticeable restriction of their motion; it also recovers approximately 6% of the native CD signal . This intermediate, which is known to be stabilized by the hydrophobic effect, appears to reflect the early coalescence of nonpolar side chains without significant organization of the backbone . I1 recovers an additional 43% of the CD signal, further sequesters tyrosine residues in nonpolar environments, and restricts their motion to an extent similar to N . The progressive development of a higher order structure as the denaturant concentration decreases implies a monotonic contraction in the ensemble of conformations that represent the U, I2, I1, and N states of alphaTS.

FEBS Lett, 1999 Jul 30, 456(1), 211 - 4
ATP-dependent degradation of SulA, a cell division inhibitor, by the HslVU protease in Escherichia coli; Seong IS et al.; HslVU is an ATP-dependent protease consisting of two multimeric components, the HslU ATPase and the HslV peptidase . To gain an insight into the role of HslVU in regulation of cell division, the reconstituted enzyme was incubated with SulA, an inhibitor of cell division in Escherichia coli, or its fusion protein with maltose binding protein (MBP) . HslVU degraded both proteins upon incubation with ATP but not with its nonhydrolyzable analog, ATPgammaS, indicating that the degradation of SulA requires ATP hydrolysis . The pulse-chase experiment using an antibody raised against MBP-SulA revealed that the stability of SulA increased in hsl mutants and further increased in lon/hsl double mutants, indicating that SulA is an in vivo substrate of HslVU as well as of protease La (Lon) . These results suggest that HslVU in addition to Lon plays an important role in regulation of cell division through degradation of SulA.

FEBS Lett, 1999 Jul 30, 456(1), 13 - 6
Knock-out of the cyaY gene in Escherichia coli does not affect cellular iron content and sensitivity to oxidants; Li DS et al.; Friedreich ataxia is a recessively inherited neurodegenerative disease caused by deficiency of a highly conserved mitochondrial protein, frataxin . Frataxin deficiency results in mitochondrial iron accumulation and oxidative stress . Frataxin shows homology with the CyaY proteins of gamma-purple bacteria, whose function is unknown . We knocked out the CyaY gene in Escherichia coli MM383 by homologous recombination and we generated an E . coli MM383 strain overexpressing CyaY . Bacterial growth, iron content and survival after exposure to H2O2 did not differ among these strains, suggesting that, despite structural similarities, cyaY proteins in bacteria may have a different function from frataxin homologues in mitochondria.

Hear Res, 1999 Aug, 134(1-2), 1 - 8
Gene transfer into the mammalian inner ear using HSV-1 and vaccinia virus vectors; Derby ML et al.; The introduction of foreign genes into cells has become an effective means of achieving intracellular expression of foreign proteins, both for therapeutic purposes and for experimental manipulation . Gene delivery to the nervous system has been extensively studied, primarily using viral vectors . However, to date less work has focused on gene delivery to the inner ear, and existing studies have primarily used adenovirus and adeno-associated virus . Using two recombinant viral vectors, herpes simplex type 1 (HSV-1), and vaccinia virus, bearing the Escherichia coli lacZ gene, we tested gene delivery to the guinea pig cochlea in vivo with beta-galactosidase staining as an assay . The HSV-1 and vaccinia virus vectors were both found to infect and elicit transgene expression successfully in many cells in the guinea pig cochlea, including cells in the organ of Corti . These data demonstrate the feasibility of gene delivery to the inner ear using these two viral vectors . Such techniques may facilitate study of the auditory systems, and might be used to develop gene therapy strategies for some forms of hearing loss.

Pediatr Nephrol, 1999 Aug, 13(6), 487 - 92
Markers of endothelial cell activation and injury in childhood haemolytic uraemic syndrome; Nevard CH et al.; Diarrhoea-associated haemolytic uraemic syndrome (D+ HUS) is usually caused by verotoxin-producing Escherichia coli . Histology shows endothelial swelling with localised thrombus . Activation of coagulation and fibrinolysis also occurs . These facts, combined with the knowledge that recovery usually follows within weeks, led us to hypothesise that verotoxin causes localised endothelial cell activation but not injury . Markers of endothelial cell activation and injury were measured serially in 30 children with acute D+ HUS, healthy children, and children receiving chronic dialysis . Interpretation of markers was complicated by the renal dysfunction characteristic of D+ HUS . Nevertheless there was no evidence for endothelial cell injury, as soluble tissue factor levels were not increased and soluble thrombomodulin levels were lower than dialysed controls (P<0.001) . In the acute phase, soluble vascular cell adhesion molecule levels were raised above normal (P<0.001), but were lower than dialysed controls (P<0.001), and soluble E-selectin levels were not significantly increased compared with normal controls (P=0.2) . Hence, there was no evidence for endothelial cell damage or endothelial cell activation by the time children reached hospital; but this study did not exclude the possibility that endothelial cell activation occurred prior to hospital admission.

Zhonghua Nei Ke Za Zhi, 1997 Nov, 36(11), 759 - 63
{Clinical evaluation and immunomodulatory study of cefodizime}; Li G et al.; To investigate the efficacy, safety and immunomodulating activities of cefodizime in immunocompromised patients with infections, we carried out a randomized controlled prospective study of cefodizime vs ceftizoxime in 107 patients . The total effective cure rate and bacterial eradication rate were 87.3%, 61.8% and 89.3% in cefodizime group and 82.7%, 59.6% and 90.6% in ceftizoxime group . Drug tolerance was similar in the two groups, and side effect was mild and transient, mainly gastrointestinal reactions . Cefodizime had effect on both phagocyte and lymphocyte functions: enhancing the phagocytie rate, phagocytic index and bacterial killing activity, increasing the number of CD4+ lymphacyte and the ratio of CD4+/CD8+, stimulating NK cell activity and enhancing expression of IL-2R of active lymphocyte . Meanwhile ceftizoxime had no effect on any of the parameter mentioned above . The result showed that cefodizime is effective and safe in the treatment of LRTI, upper and complicated UTI in immunocompromised patients, as well as possessed immunomodulating activities.

Proc Natl Acad Sci U S A, 1999 Aug 17, 96(17), 9654 - 9
Integrin alphaIIb promoter-targeted expression of gene products in megakaryocytes derived from retrovirus-transduced human hematopoietic cells; Wilcox DA et al.; Megakaryocyte-specific expression of the platelet-adhesion receptor, integrin alphaIIbbeta3, is caused by the presence of regulatory elements of the alphaIIb promoter that direct high-level, selective gene transcription early in megakaryocytopoiesis . To develop methods for targeted expression of transgenes, we transduced human CD34+ peripheral blood cells with a murine leukemia virus (MuLV) vector controlled by the human integrin alphaIIb promoter (nucleotides -889 to +35) . A naturally occurring cDNA encoding the Pl(A2) alloantigen form (Pro(33)) of the integrin beta3 subunit was subcloned into this construct (-889Pl(A2)beta3) and transduced into cells that endogenously synthesized Pl(A1)beta3 (Leu(33)) as a marker for detection of provirus-derived beta3 . The ability of this vector to target expression of Pl(A2)beta3 to megakaryocytes was first examined in cell lines . Immunoblot analysis with human anti-Pl(A2) alloserum detected synthesis of Pl(A2)beta3 in transduced promegakaryocytic cells; however, Pl(A2)beta3 protein was not detected in transduced epithelial cells . Human hematopoietic CD34+ cells were transduced with -889Pl(A2)beta3 virions and induced to differentiate with megakaryocyte growth and development factor . A hybrid alphaIIbbeta3 complex was formed in progeny megakaryocytes where provirus-derived Pl(A2)beta3 was detected associated with endogenous alphaIIb subunit . Another alphaIIb promoter-driven MuLV vector (-889nlacZ) encoding Escherichia coli beta-galactosidase was used to demonstrate that transgene expression was selectively targeted to the megakaryocyte progeny of transduced CD34+ cells . These studies demonstrate the feasibility of using alphaIIb promoter-driven MuLV vectors for gene transfer of hematopoietic CD34+ cells to target transgene expression in developing megakaryocytes and platelets and indicate potential applications toward human gene therapy for platelet disorders.

Biochemistry, 1999 Aug 17, 38(33), 10730 - 42
Expression and spectroscopic analysis of soluble nicotinic acetylcholine receptor fragments derived from the extracellular domain of the alpha-subunit; Grant MA et al.; To facilitate structural studies of the ligand binding region from the nicotinic acetylcholine receptor (nAChR), we have developed methods for the high-level expression and purification of an important functional portion of the N-terminal extracellular domain (ECD) of the alpha-subunit . Two soluble receptor fragments comprising residues 143-210 of the Torpedo californica alpha-subunit were expressed in E . coli: alphaT68His6, which contains a histidine tag, and alphaT68M1, which includes the first transmembrane region, M1, of the alpha-subunit . Both proteins demonstrate saturable, high-affinity alpha-bungarotoxin (Bgtx) binding with an apparent equilibrium KD (3 nM) that is comparable to the affinities reported for preparations comprising the entire alpha-subunit ECD . These results demonstrate that the ECD determinants required for Bgtx recognition of the alpha-subunit are entirely specified by residues 143-210 . The binding of small ligands was demonstrated in competition assays with 125I-Bgtx yielding KI values of 58 and 105 microM for d-tubocurarine and nicotine, respectively . Circular dichroism (CD) analysis of monomeric alphaT68His6 protein revealed considerable secondary structure . Furthermore, a cooperative, two-state folding transition was observed upon urea denaturation . To circumvent concentration-dependent aggregation of the alphaT68His6 protein at the millimolar concentrations needed for NMR study, we utilized the M1 transmembrane domain to anchor the recombinant receptor fragment onto membrane-mimicking micelles . Monodispersed preparations of alphaT68M1 in dodecylphosphocholine micelles demonstrate high-affinity Bgtx binding and considerable secondary structure by CD . The structural features revealed in the CD profile appear to undergo a cooperative, two-state folding transition upon thermal denaturation . Initial NMR studies suggest that micellar preparations of the alphaT68M1 fragment are amenable to further high-resolution heteronuclear NMR analysis.

Biochemistry, 1999 Aug 17, 38(33), 10707 - 13
Interaction of the soluble recombinant PsaD subunit of spinach photosystem I with ferredoxin I; Pandini V et al.; Photosystem I of higher plants functions in photosynthesis as a light-driven oxidoreductase producing reduced ferredoxin . Its peripheral subunit PsaD has been identified as the docking site for ferredoxin I . With the aim of elucidating the structure-function relationship and the role of this subunit, a recombinant form of the spinach protein was produced by heterologous expression in Escherichia coli . The PsaD protein was synthesized in soluble form and purified to homogeneity . The interaction of the PsaD subunit with ferredoxin I was investigated using three different approaches: chemical cross-linking between the two purified proteins in solution, affinity chromatography of the PsaD subunit on a ferredoxin-coupled resin, and titration with ferredoxin of the protein fluorescence of the subunit . All these studies indicated that the isolated PsaD in solution has a definite conformation and maintains the ability to bind ferredoxin I with high affinity and specificity . The Kd value of the complex of PsaD and ferredoxin is in the nanomolar range, which is consistent with reported Km values for ferredoxin I photoreduction by thylakoid membranes . The ionic strength dependence of the K(d) suggests that the protein-protein interaction is at least partially electrostatic in nature . Nevertheless, none of the glutamate residues of the acidic cluster of residues 92-94 of ferredoxin I, which have been reported to be involved in the interaction with the subunit, seems to be essential for PsaD binding, as borne out by experiments using ferredoxin I mutants in positions 92-94.

Biochemistry, 1999 Aug 17, 38(33), 10649 - 59
Atomic mutations at the single tryptophan residue of human recombinant annexin V: effects on structure, stability, and activity; Minks C et al.; The single tryptophan residue (Trp187) of human recombinant annexin V, containing 320 residues and 5328 atoms, was replaced with three different isosteric analogues where hydrogen atoms at positions 4, 5, and 6 in the indole ring were exchanged with fluorine . Such single atom exchanges of H --> F represent atomic mutations that result in slightly increased covalent bond lengths and inverted polarities in the residue side-chain structure . These minimal changes in the local geometry do not affect the secondary and tertiary structures of the mutants, which were identical to those of wild-type protein in the crystal form . But the mutants exhibit significant differences in stability, folding cooperativity, biological activity, and fluorescence properties if compared to the wild-type protein . These rather large global effects, resulting from the minimal local changes, have to be attributed either to the relatively strong changes in polar interactions of the indole ring or to differences in the van der Waals radii or to a combination of both facts . The changes in local geometry that are below resolution of protein X-ray crystallographic studies are probably of secondary importance in comparison to the strong electronegativity introduced by the fluorine atom . Correspondingly, these types of mutations provide an interesting approach to study cooperative functions of integrated residues and modulation of particular physicochemical properties, in the present case of electronegativity, in a uniquely structured and hierarchically organized protein molecule.

J Rheumatol, 1999 Aug, 26(8), 1769 - 74
Control of delivered gene expression in chondrocytes using heat shock protein 70B promoter; Arai Y et al.; OBJECTIVE: To investigate whether the expressions of delivered Escherichia coli beta-galactosidase (LacZ) gene and transforming growth factor-beta1 (TGF-beta1) gene are regulated by the stress response of human chondrocyte-like cells (HCS-2/8) when heat shock protein 70B (HSP70B) promoter is inserted into the adenovirus vector . METHODS: Two adenovirus vectors that contain either LacZ gene or TGF-beta1 gene regulated by HSP70B promoter were constructed . One of the adenovirus vectors was added to the culture of HCS-2/8 and gene transduced cells were produced . We applied heat stress (43 degrees C) to the transduced cells for 2 h and examined whether the expression of transduced LacZ and TGF-beta1 genes is affected by the stress, using 5 bromo-4-chloro-3-indolyl-beta-D-galactopyranoside (X-gal) staining, measurement of beta-galactosidase activity, Northern blotting, and ELISA . RESULTS: The percentage of X-gal positive stained cells in LacZ gene-delivered cells with heat stress was significantly higher than in controls (no heat stress) . With heat stress, beta-galactosidase activity increased significantly, and the band of exogenous TGF-beta1 mRNA became more apparent and the expression was maintained during the 24 h monitoring period . TGF-beta1 level in culture supernatant of TGF-beta1 gene-delivered cells with heat stress (5477.3+/-321.1 pg/ml) was significantly higher than in the controls (853.2+/-29.2 pg/ml) . CONCLUSION: HSP70B promoter could regulate the expression of delivered genes according to the intensity of heat stress.

Oncol Res, 1999, 11(1), 33 - 9
Verotoxin induces apoptosis and the complete, rapid, long-term elimination of human astrocytoma xenografts in nude mice; Arab S et al.; Verotoxin 1 (VT1) is an E . coli elaborated subunit toxin active only against (tumor) cell lines that express the VT1 receptor, globotriaosyl ceramide-Gb3 . Astrocytomas can be highly malignant brain tumors that remain refractory to clinical treatment . Some human astrocytoma cell lines are particularly sensitive to VT1 in vitro . To address whether this represents a feasible approach to the elimination of these tumors in man, human astrocytoma tumor xenografts in nude mice were treated with verotoxin . Following a single low-dose intratumoral injection of VT1, complete regression of a 1-cm-diameter tumor within 10 days was observed in all treated animals, without reoccurrence (up to 60 days) . Apoptosis was demonstrated in both tumor and vascular cells within the treated xenograft . Verotoxin binding to tumor cells and blood vessels in sections of primary glioblastoma multiforme was found.

Proteins, 1999 Sep 1, 36(4), 436 - 46
Effects of core-packing on the structure, function, and mechanics of a four-helix-bundle protein ROP; Ceruso MA et al.; The effects of core-packing on the structure, function and mechanics of the RNA-binding 4-helix-bundle Rop have been studied by molecular dynamics simulations . The structural, dynamical and geometrical properties of the Rop homodimer, (formed by the antiparallel juxtaposition of two helix-turn-helix motifs), have been compared with those of three protein variants described by Munson et al . (Protein Sci, 5:1584-1593, 1996), where the core of the native protein has been systematically repacked using a two-amino acid alphabet: Ala(2)Leu(2)-8, Ala(2)Leu(2)-8-rev, and Leu(2)Ala(2)-8 . The results showed that it was possible to readily distinguish the inactive protein Leu(2)Ala(2)-8 from the other functionally active systems based on tertiary and quaternary structure criteria . Structural properties such as native secondary structure content did not correlate with biological activity . Biological activity was related in part to the relative arrangement of the residues within the binding site . But, more global aspects, related to the overall topology of the helical bundle, accounted for the small functional differences between Ala(2)Leu(2)-8 and Ala(2)Leu(2)-8-rev . Mechanically, the 4-helix-bundle absorbed core mutations by altering the local structure at the sequence termini and in the turns that join the two helices of each monomer, and by changing the overall orientation and separation of the extremely rigid helices . Proteins 1999;36:436-446 .

Eur Surg Res, 1999, 31(4), 314 - 23
Effects of selective nitric oxide synthase inhibition in hyperdynamic endotoxemia in dogs; Wolfard A et al.; OBJECTIVES: Our aims were to investigate the systemic hemodynamic effects of constitutive endothelial nitric oxide synthase (eNOS) and inducible NOS (iNOS) inhibitors in hyperdynamic endotoxemia . PATIENTS AND METHODS: Group 1 comprised sham-operated controls, while in group 2, 3 and 4, a hyperdynamic circulatory reaction was elicited by a 2-hour infusion of Escherichia coli endotoxin (ETX) in a dose of 5.3 microg/kg . The animals in group 3 were treated with 12 . 5 mg/kg nonselective NOS inhibitor N-omega-nitro-L-arginine methyl ester (L-NAME), and those in group 4 with 2 mg/kg of the specific iNOS inhibitor S-methyl-isothiourea (SMT) . Mean arterial pressure (MAP), cardiac output (CO) and myocardial contractility (MC) were measured, and total peripheral resistance (TPR) was calculated . The eNOS and iNOS activities were determined in myocardial biopsy samples taken after 8 h of endotoxemia . RESULTS: ETX induced significant decreases in TPR and MAP, a transient myocardial depression, and increased the myocardial eNOS and iNOS activities . L-NAME decreased the activities of both isoenzymes, increased MC but induced a fall in CO . SMT inhibited iNOS by 60%, without influencing the eNOS activity, increased MAP and contractility in the early phase of endotoxemia, and induced only a slight decrease in CO . CONCLUSIONS: Nonselective NOS inhibition restores the arterial pressure and exerts a positive inotropic effect, but decreases CO . SMT selectively decreases the iNOS activation without disturbing the vasoregulatory function of the eNOS-derived nitric oxide in hyperdynamic endotoxemia in the dog.

Proc Natl Acad Sci U S A, 1999 Aug 17, 96(17), 9954 - 9
Protochlorophyllide oxidoreductase B-catalyzed protochlorophyllide photoreduction in vitro: insight into the mechanism of chlorophyll formation in light-adapted plants; Lebedev N et al.; The mechanism of the protochlorophyllide (PChlide) photoreduction reaction operating in light-adapted plants and catalyzed by NADPH:protochlorophyllide oxidoreductase B (PORb) has been analyzed by low-temperature fluorescence spectroscopy by using purified barley PORb overexpressed heterologously in Escherichia coli as a fusion protein with the maltose-binding protein . We show that the PORb-catalyzed PChlide reduction reaction consists of two steps, one photochemical and the other nonphotochemical . The initial photochemical reaction follows a single quantum mechanism and leads to the formation of an unstable intermediate with mixed pigment electronic structure and an EPR spectrum that suggests the presence of a free electron . The second step involves the spontaneous conversion of the unstable intermediate into chlorophyllide as defined by its spectroscopic characteristics and migration on an HPLC column . Both steps of the reaction can be performed at subzero temperatures in frozen samples, suggesting that they do not include major changes in enzyme conformation or pigment rearrangement within the active site . The rate of the reaction at room temperature depends linearly on enzyme and substrate (PChlide) concentration, and the kinetic parameters are consistent with one molecule of substrate bound per active monomer in solution . The PORb-catalyzed reaction in vitro is spectroscopically similar to that identified in leaves of light-adapted plants, suggesting that the same reaction sequence observed operates in planta.

Proc Natl Acad Sci U S A, 1999 Aug 17, 96(17), 9775 - 80
SLAP, a dimeric adapter protein, plays a functional role in T cell receptor signaling; Tang J et al.; Engagement of the T cell antigen receptor (TCR) leads to rapid activation of protein tyrosine kinases, which in turn phosphorylate downstream enzymes and adapter proteins . Some adapter proteins, such as SLP-76, Vav, and LAT, positively regulate TCR-mediated signal transduction, whereas others, such as Cbl, play an inhibitory role . SLAP (Src-like adapter protein), an adapter protein containing a Src homology 3 and a Src homology 2 domain, was isolated from a yeast interacting screen by using N-terminal Cbl as bait . N-terminal Cbl interacts with SLAP in vivo and in vitro in a tyrosine phosphorylation-independent manner . We observed that SLAP is expressed in T cells, and upon TCR activation, SLAP interacts with ZAP-70, Syk, LAT, and TCRzeta chain in Jurkat T cells . In transiently transfected COS-7 cells, SLAP forms separate complexes with ZAP-70, Syk, and LAT through its Src homology 2 domain . Overexpression of a C-terminal-truncated SLAP mutant down-regulates nuclear factor of activated T cells-AP1 activity . We have evidence that SLAP forms homodimers through its C-terminal region . Serial truncations and mutations in the C terminus of SLAP demonstrate that there is a correlation between the loss of dimerization and the inhibition of nuclear factor of activated T cells-AP1 activity . The in vivo association of SLAP with key signaling molecules and its inhibition of T cell activation suggests that SLAP plays an important role in TCR-mediated signal transduction.

Proc Natl Acad Sci U S A, 1999 Aug 17, 96(17), 9628 - 32
Basonuclin, a zinc finger protein of keratinocytes and reproductive germ cells, binds to the rRNA gene promoter; Iuchi S et al.; Basonuclin is a protein containing three pairs of C(2)H(2) zinc fingers . The protein has been found in the basal (germinal) cell layer of stratified squamous epithelia, such as the epidermis, and in germ cells of the testis and ovary . We show here that the human protein has specific affinity for a segment of the promoter of the gene for rRNA . Basonuclin interacts with two separate parts of the promoter, each possessing dyad symmetry . The upstream part, but not the downstream part, is known to bind UBF1, a transcription factor for rDNA . Basonuclin is likely to be a cell-type-specific regulatory protein for rDNA transcription.

Proc Natl Acad Sci U S A, 1999 Aug 17, 96(17), 9580 - 5
The Tyr-265-to-Cys mutator mutant of DNA polymerase beta induces a mutator phenotype in mouse LN12 cells; Clairmont CA et al.; DNA polymerase beta functions in both base excision repair and meiosis . Errors committed by polymerase beta during these processes could result in mutations . Using a complementation system, in which rat DNA polymerase beta substitutes for DNA polymerase I of Escherichia coli, we previously isolated a DNA polymerase beta mutant in which Tyr-265 was altered to Cys (Y265C) . The Y265C mutant is dominant to wild-type DNA polymerase beta and possesses an intrinsic mutator activity . We now have expressed the wild-type DNA polymerase and the Y265C mutator mutant in mouse LN12 cells, which have endogenous DNA polymerase beta activity . We demonstrate that expression of the Y265C mutator mutant in the LN12 cells results in an 8-fold increase in the spontaneous mutation frequency of lambdacII mutants compared with expression of the wild-type protein . Expression of Y265C results in at least a 40-fold increase in the frequency of deletions of three bases or more and a 7-fold increase in point mutations . Our results suggest that the mutations we observe in vivo result directly from the action of the mutator polymerase . To our knowledge, this is the first demonstration of a mutator phenotype resulting from expression of a DNA polymerase mutator mutant in mammalian cells . This work raises the possibility that variant polymerases may act in a dominant fashion in human cells, leading to genetic instability and carcinogenesis.

Proc Natl Acad Sci U S A, 1999 Aug 17, 96(17), 9551 - 6
Regions on adenylyl cyclase that are necessary for inhibition of activity by beta gamma and G(ialpha) subunits of heterotrimeric G proteins; Wittpoth C et al.; The two large cytoplasmic domains (C1 and C2) of adenylyl cyclases (AC), when expressed separately and mixed together, reconstitute enzyme activity that can be regulated by various modulators . Therefore, we have used the C1 or its C1a subdomain and C2 regions from type I AC (ACI) and type V AC (ACV) to identify the region on ACI that interacts with beta gamma subunits of heterotrimeric G proteins . In addition, we also used a chimeric C1 domain (VC1aIC1b) in which the C1a region was derived from ACV and the C1b region was from ACI . By mixing the C1 or C1a or VC1aIC1b domains with C2 regions of ACI or ACV, we have shown that the C1a region (amino acids 236-471) of ACI is sufficient to observe beta gamma-mediated inhibition of enzyme activity, which is stimulated by either constitutively active G(salpha) (G(salpha)*) or Ca(2+)/calmodulin (CaM) . Although the C1b region and C2 domain of ACI were by themselves not sufficient for inhibition of activity by beta gamma subunits, the presence of both of these regions formed another beta gamma interaction site that was sufficient to observe G(salpha)*- or Ca(2+)/CaM-stimulated activity . Inhibition of AC activity attributable to interaction of beta gamma subunits at either of the two sites was blocked by a peptide (QEHA) that has previously been shown to inhibit the effects of beta gamma on various effectors . Moreover, the C1 region of ACI was sufficient to observe G(ialpha1)-elicited inhibition of Ca(2+)/CaM-stimulated activity . Although the C1a region of ACV was sufficient for inhibition of activity by G(ialpha1), the presence of C1b region from either ACI or ACV increased sensitivity to inhibition by the inhibitory G protein . Thus, the inhibitory influences of G(ialpha1) are mediated on the C1 regions of both ACI and ACV . The effects of beta gamma on ACI can be mediated by interactions with the C1a region and a beta gamma interacting site formed by the C1b and C2 domains of this enzyme.

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