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***&phi;***-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, Delta&phi; . With EF(0)F(1), a rate of 80 s(-1) is observed at DeltapH=4.1 and Delta&phi; approximately 140 mV . The rate decreases sigmoidally with Delta&phi; and at Delta&phi; 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 Delta&phi; 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 Delta&phi; . EF(0)F(1)-catalyzed ATP synthesis was measured as a function of DeltapH at a constant Delta&phi; . The rate depends sigmoidally on DeltapH reaching a maximal rate which cannot be further increased by increasing DeltapH . However, this maximal rate depends on Delta&phi;, i.e . DeltapH and Delta&phi; 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 Delta&phi;, whereas for CF(0)F(1), the activation depends on DeltapH and Delta&phi;. 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