Escherichia coli

Protein Sci, 2000 Aug, 9(8), 1428 - 38
Recombinant decorsin: dynamics of the RGD recognition site; Krezel AM et al.; Decorsin is an antagonist of integrin alphaIIbbeta3 and a potent platelet aggregation inhibitor . A synthetic gene encoding decorsin, originally isolated from the leech Macrobdella decora, was designed, constructed, and expressed in Escherichia coli . The synthetic gene was fused to the stII signal sequence and expressed under the transcriptional control of the E . coli alkaline phosphatase promoter . The protein was purified by size-exclusion filtration of the periplasmic contents followed by reversed-phase high-performance liquid chromatography . Purified recombinant decorsin was found to be indistinguishable from leech-derived decorsin based on amino acid composition, mass spectral analysis, and biological activity assays . Complete sequential assignments of 1H and proton bound 13C resonances were established . Stereospecific assignments of 21 of 25 nondegenerate b-methylene groups were determined . The RGD adhesion site recognized by integrin receptors was found at the apex of a most exposed hairpin loop . The dynamic behavior of decorsin was analyzed using several independent NMR parameters . Although the loop containing the RGD sequence is the most flexible one in decorsin, the conformation of the RGD site itself is more restricted than in other proteins with similar activities.

Protein Sci, 2000 Aug, 9(8), 1423 - 7
Crystal structure of viral serpin crmA provides insights into its mechanism of cysteine proteinase inhibition; Simonovic M et al.; CrmA is an unusual viral serpin that inhibits both cysteine and serine proteinases involved in the regulation of host inflammatory and apoptosis processes . It differs from other members of the serpin superfamily by having a reactive center loop that is one residue shorter, and by its apparent inability to form SDS-stable covalent complexes with cysteine proteinases . To obtain insight into the inhibitory mechanism of crmA, we determined the crystal structure of reactive center loop-cleaved crmA to 2.9 A resolution . The structure, which is the first of a viral serpin, suggests that crmA can inhibit cysteine proteinases by a mechanism analogous to that used by other serpins against serine proteinases . However, one striking difference from other serpins, which may be significant for in vivo function, is an additional highly charged antiparallel strand for b sheet A, whose sequence and length are unique to crmA.

Gene, 2000 Aug 22, 254(1-2), 37 - 44
Transposon Tn7 gene insertion into an evolutionarily conserved human homolog of Escherichia coli attTn7; Cleaver SH et al.; Escherichia coli transposon Tn7 can integrate into its target DNA sequence, attTn7 at the 3' end of glmS, with high specificity and efficiency . Remarkably, the insertional recognition sequence in the E . coli genome displays a high degree of identity with the corresponding region at the 3' end of the corresponding human gene for glutamine-fructose-6-phosphate transaminase (GFPT), located at 2p13 . It was therefore of interest to determine whether Tn7 could recognize the corresponding human sequence, and transpose at that site . Strains of E . coli DH5alpha were prepared carrying the tnsA-E genes on one plasmid, and attTn7 or the human equivalent on a second recipient plasmid within the alpha-complementation fragment of the lacZ gene . Each strain was transformed with a donor plasmid carrying a gentamycin resistance gene within the Tn7L and Tn7R cassettes . Restriction mapping and sequence analysis of recipient plasmids isolated from white colonies demonstrated that Tn7 inserted the gentamycin resistance gene both into the E . coli attTn7 sequence, and into its human counterpart . No nonspecific insertion was observed in a control plasmid containing only the lacZ fragment . These results provide a basis to investigate whether TnsA-D proteins can mediate gene insertion into comparably conserved sites in eukaryotic chromosomes.

Gene, 2000 Aug 22, 254(1-2), 19 - 28
Identification and partial characterization of a novel hemolysin from Leptospira interrogans serovar lai; Lee SH et al.; It has been suggested that leptospiral hemolysins are important in the virulence and pathogenesis of leptospirosis . We have isolated an Escherichia coli clone carrying the 7.8kb DNA insert from a genomic library of Leptospira interrogans serovar lai by plaque hybridization using a sequence derived from the sphingomyelinase C gene (sphA) of L . borgpetersenii . The clone showed a clear beta-hemolytic zone on sheep blood agar and high hemolytic activities on both human and sheep erythrocytes in liquid assays . The clone carried at least two genes responsible for the hemolytic activities, encoded by two open reading frames of 1662 and 816 nucleotides, which are named sphH and hap-1 (hemolysis associated protein-1), respectively . The SphH showed 75% homology to the SphA at the amino acid level, and the Hap-1 showed no significant homology in major databases . Interestingly, however, E . coli cells harboring sphH did not show sphingomyelinase or phospholipase activities . Moreover, SphH-mediated hemolysis was osmotically protected by polyethylene glycol 5000, suggesting that the hemolysis is likely to be caused by pore formation on the membrane . The SphH was successfully expressed in E . coli as a histidine (His)-SphH fusion protein . Both sphH and hap-1 were highly conserved among the Leptospira species, except for the absence of sphH in non-pathogenic L . biflexa serovar patoc . We concluded that the SphH is a novel hemolysin of a pathogenic Leptospira species, which may be a putative pore-forming protein.

Biochem Pharmacol, 2000 Oct 1, 60(7), 947 - 53
Role of disulfiram in the in vitro inhibition of rat liver mitochondrial aldehyde dehydrogenase; Shen ML et al.; The alcohol aversion therapy drug disulfiram has been shown to inhibit hepatic aldehyde dehydrogenase (ALDH), one of the key enzymes involved in ethanol metabolism . It is believed by some that disulfiram could be one of the active inhibitors in vivo . However, the actual interaction between disulfiram and ALDH remains ambiguous . We report here that when disulfiram inhibited recombinant rat liver mitochondrial ALDH (rlmALDH) in vitro, no significant molecular mass increase was detected during the first 30 min as determined by on-line HPLC-electrospray ionization mass spectrometry (LC-MS) . This indicated that the inhibition in vitro was not caused directly by covalent adduct formation on the enzyme . We subsequently subjected both control and disulfiram-inhibited rlmALDH to Glu-C proteolytic digestion . LC-MS analysis of the Glu-C digestion of disulfiram-inhibited enzyme revealed that one peptide of M(r) = 4821, which contained the putative active site of the enzyme, exhibited a mass decrease of 2 amu as compared with the same peptide found in the Glu-C digestion of the control (M(r) = 4823) . We believe that the loss of 2 amu indicated that inhibition of rlmALDH in vitro was due to formation of an intramolecular disulfide bond between two of the three adjacent cysteines in the active site, possibly via a very rapid and unstable mixed disulfide interchange reaction . Further confirmation of the intramolecular disulfide bond formation came from the fact that by adding dithiothreitol (DTT) we were able to recover partial enzyme activity . In addition, the peptide of M(r) = 4821 observed in the Glu-C digestion of the disulfiram-treated ALDH reverted to M(r) = 4823 after treatment with DTT, which indicated that the disulfide bond was reduced . We, thereby, conclude that disulfiram inhibited rlmALDH by forming an intramolecular disulfide, possibly via a fast intermolecular disulfiram interchange reaction.

Microbiology, 2000 Sep, 146 ( Pt 9), 2277 - 82
Metal-ion tolerance in Escherichia coli: analysis of transcriptional profiles by gene-array technology; Brocklehurst KR et al.; Escherichia coli was adapted to grow in medium containing substantially elevated concentrations of either Zn(II), Cd(II), Co(II) or Ni(II) . Whole-genome transcriptional profiles were generated from adapted strains and analysed for significant alteration in transcript abundance with reference to a wild-type strain . Similar alterations in specific message levels were observed for strains adapted to the four metal ions . One unexpected trend was the increase in transcript level of genes involved in transposition of IS elements, particularly insA . Subsequent expression of insA-7 from a heterologous promoter in E . coli conferred tolerance to Zn(II).

Microbiology, 2000 Sep, 146 ( Pt 9), 2267 - 75
Mosaic plasmids and mosaic replicons: evolutionary lessons from the analysis of genetic diversity in IncFII-related replicons; Osborn AM et al.; The alpha replicons of the multi-replicon plasmids pGSH500 and pLV1402 have been characterized by DNA sequence analysis . Analysis of the DNA sequence of a 3672 bp HIN:dIII fragment from pFDT100, which contains the pGSH500 alpha replicon, revealed similarity to a number of replicons belonging to, or related to, those of the IncFII family . The replicon region contains copA, tapA, repA and oriR, and replication initiation and termination sites are related to those from the IncFII replicon of R1 . A copB gene was found to lie upstream of the HIN:dIII site in the parental plasmid pGSH500 . Downstream of oriR, a 707 bp region shows 72.6% identity to a region of the Escherichia coli chromosome at 43.3', suggesting this region of pGSH500 may have been incorporated into the plasmid during a past chromosomal recombination event . Oligonucleotide primers homologous to consensus regions in the copB and repA genes, and the oriR regions from a number of IncFII-related replicons were used to amplify replication regions from pLV1402 . Analysis of the amplified regions has shown the presence of copB, copA, tapA and repA genes . Phylogenetic analysis of Rep protein sequences from the RepFIIA family of antisense-control-regulated replicons revealed the presence of three distinct subgroups of Rep proteins . Comparative analysis of DNA and protein sequences from members of the RepFIIA family provides evidence supporting the roles of both non-selective divergence in co-integrate (multi-replicon) plasmids and Chi-mediated-recombination in replicon evolution, and in particular, that such processes may have been widespread in the evolution of the RepFIIA family.

J Biol Chem, 2000 Nov 24, 275(47), 36769 - 74
Phosphatidylinositol 3-kinase activation and interaction with focal adhesion kinase in Escherichia coli K1 invasion of human brain microvascular endothelial cells; Reddy MA et al.; Invasion of brain microvascular endothelial cells (BMEC) is a prerequisite for successful crossing of the blood-brain barrier by Escherichia coli K1 . We have previously demonstrated the requirement of cytoskeletal rearrangements and activation of focal adhesion kinase (FAK) in E . coli K1 invasion of human BMEC (HBMEC) . The current study investigated the role of phosphatidylinositol 3-kinase (PI3K) activation and PI3K interaction with FAK in E . coli invasion of HBMEC . PI3K inhibitor LY294002 blocked E . coli K1 invasion of HBMEC in a dose-dependent manner, whereas an inactive analogue LY303511 had no such effect . In HBMEC, E . coli K1 increased phosphorylation of Akt, a downstream effector of PI3K, which was completely blocked by LY294002 . In contrast, non-invasive E . coli failed to activate PI3K . Overexpression of PI3K mutants Deltap85 and catalytically inactive p110 in HBMEC significantly inhibited both PI3K/Akt activation and E . coli K1 invasion of HBMEC . Stimulation of HBMEC with E . coli K1 increased PI3K association with FAK . Furthermore, PI3K/Akt activation was blocked in HBMEC-overexpressing FAK dominant-negative mutants (FRNK and Phe397FAK) . These results demonstrated the involvement of PI3K signaling in E . coli K1 invasion of HBMEC and identified a novel role for PI3K interaction with FAK in the pathogenesis of E . coli meningitis.

J Biol Chem, 2000 Dec 8, 275(49), 38160 - 9
An Lrp-like transcriptional regulator from the archaeon Pyrococcus furiosus is negatively autoregulated; Brinkman AB et al.; The archaeal transcriptional initiation machinery closely resembles core elements of the eukaryal polymerase II system . However, apart from the established basal archaeal transcription system, little is known about the modulation of gene expression in archaea . At present, no obvious eukaryal-like transcriptional regulators have been identified in archaea . Instead, we have previously isolated an archaeal gene, the Pyrococcus furiosus lrpA, that potentially encodes a bacterial-like transcriptional regulator . In the present study, we have for the first time addressed the actual involvement of an archaeal Lrp homologue in transcription modulation . For that purpose, we have produced LrpA in Escherichia coli . In a cell-free P . furiosus transcription system we used wild-type and mutated lrpA promoter fragments to demonstrate that the purified LrpA negatively regulates its own transcription . In addition, gel retardation analyses revealed a single protein-DNA complex, in which LrpA appeared to be present in (at least) a tetrameric conformation . The location of the LrpA binding site was further identified by DNaseI and hydroxyl radical footprinting, indicating that LrpA binds to a 46-base pair sequence that overlaps the transcriptional start site of its own promoter . The molecular basis of the transcription inhibition by LrpA is discussed.

J Biol Chem, 2000 Dec 8, 275(49), 38645 - 53
The critical role of the conserved Thr247 residue in the functioning of the osmosensor EnvZ, a histidine Kinase/Phosphatase, in Escherichia coli; Dutta R et al.; The histidine kinase/phosphatase EnvZ helps Escherichia coli adapt to osmotic shock by controlling the phosphorylation state of the transcription factor OmpR, which regulates the levels of the outer membrane porin proteins OmpF and OmpC . We examined the effects of mutating the highly conserved Thr(247) residue in EnvZ . Using purified C-terminal domains of wild-type and mutant EnvZ proteins, we demonstrate that Thr(247) plays a vital role in EnvZ function, variously affecting its autokinase and phosphotransferase activities, but mostly its function as a phosphatase . The cytoplasmic domain of EnvZ (EnvZc) is composed of three segments: the linker domain (residues 180-222), domain A (residues 223-289), and domain B (residues 290-450) . It has been shown that the isolated domain A itself can dephosphorylate phosphorylated OmpR . Here we show that mutating Thr(247) to Arg in domain A abolishes its phosphatase activity . Furthermore, using an in vivo beta-galactosidase activity assay of Taz1-1 (hybrid of the aspartate receptor Tar and EnvZ) constructs of the Thr(247) mutants in RU1012 cells expressing ompC-lacZ, we demonstrate that the external signal primarily down-regulates the phosphatase activity of EnvZ . Of the nine EnvZc(T247X) mutants (X = Ser, Ala, Cys, Lys, Asn, Glu, Gln, Tyr, or Arg) analyzed, only Ser functionally substituted for Thr at this position, whereas all the others displayed constitutive expression of beta-galactosidase.

FASEB J, 2000 Sep, 14(12), 1801 - 13
Definition of endotoxin binding sites in horseshoe crab factor C recombinant sushi proteins and neutralization of endotoxin by sushi peptides; Tan NS et al.; Three truncated fragments, harboring different sushi domains, namely, sushi123, sushi1, and sushi3 domains, of Factor C were produced as biologically active secreted recombinant proteins . Sushi1 and 3 each has a high-affinity LPS binding site with K:(d) of 10(-9) to 10(-10) M . Positive cooperativity in sushi123 resulted in a 1000-fold increase in K:(d)2 . The core LPS binding region of sushi1 and 3 reside in two 34-mer peptides, S1 and S3 . A rigidly held disulfide-bonded structure is not essential but is important for LPS binding, as confirmed by a 100- to 10000-fold decrease in affinity . Both S1 and S3 can inhibit LAL reaction and LPS-induced hTNF-alpha secretion with different potency . LAL assay revealed that at least two molecules of S1 bind cooperatively to one LPS molecule, with Hill's coefficient of 2.42 . The LPS binding by S3 is independent and noncooperative . The modified SDelta1 and SDelta3 peptides exhibited increased LPS neutralization potential although its LPS binding affinities indicated only a 10-fold improvement . Hence, the structural difference of the four sushi peptides conferred different efficiencies in LPS neutralization without altering their binding affinity for LPS . Circular dichroism spectrometry revealed that the four peptides underwent conformational change in the presence of lipid A, transitioning from a random coil to either an alpha-helical or beta-sheet structure . Two factors are critical for the sensitivity of Factor C to LPS: 1) the presence of multiple binding sites for LPS on a single Factor C molecule; and 2) high positive cooperativity in LPS binding . The results showed that in the design of an improved LPS binding and neutralizing peptide, charge balance of the peptide is a critical parameter in addition to its structure.

Biochem Biophys Res Commun, 2000 Sep 7, 275(3), 936 - 45
Cloning and characterization of ftsZ and pyrF from the archaeon Thermoplasma acidophilum; Yaoi T et al.; To characterize cytoskeletal components of archaea, the ftsZ gene from Thermoplasma acidophilum was cloned and sequenced . In T . acidophilum ftsZ, which is involved in cell division, was found to be in an operon with the pyrF gene, which encodes orotidine-5'-monophosphate decarboxylase (ODC), an essential enzyme in pyrimidine biosynthesis . Both ftsZ and pyrF from T . acidophilum were expressed in Escherichia coli and formed functional proteins . FtsZ expression in wild-type E . coli resulted in the filamentous phenotype characteristic of ftsZ mutants . T . acidophilum pyrF expression in an E . coli mutant lacking pyrF complemented the mutation and rescued the strain . Sequence alignments of ODCs from archaea, bacteria, and eukarya reveal five conserved regions, two of which have homology to 3-hexulose-6-phosphate synthase (HPS), suggesting a common substrate recognition and binding motif .

Biochem Biophys Res Commun, 2000 Sep 7, 275(3), 924 - 30
Expression of a kallikrein-like protease from the snake venom: engineering of autocatalytic site in the fusion protein to facilitate protein refolding; Hung CC et al.; In order to circumvent the difficulty encountered in the expression and purification of the recombinant products in E . coli system, we have developed a novel and facile method of removing the polyhistidine tag from target proteins after heterologous gene expression . The expression of a serine protease (Tm-5) from Taiwan habu (Trimeresurus mucrosquamatus) is taken as an exemplar to illustrate the basic rationales and protocols involved . In place of an enterokinase recognition site, a polyhistidine tag linked to an autocatalyzed site based on cleavage specificity of the serine protease flanking on the 5'-end of Tm-5 clone sequence was engineered before protein expression in E . coli system . Renaturation of the fusion protein after expression revealed that the recombinant protease had refolded successfully from the inclusion bodies . Upon autocleavage of the expressed protease, the polyhistidine tag with additional amino acid residues appended to the N-terminus of the coding sequence is found to be removed accordingly . The protein expressed and purified by this new strategy possesses a molecular weight of approximately 28,000 in accord with the expected value for this venom protease . Further characterization of the recombinant protein employing a variety of techniques which include immunoblot analysis, RP-HPLC, ESI-MS, and N-terminal amino acid sequencing all shows indistinguishable properties to those of the isolated native protease . Most noteworthy is that the recombinant Tm-5 protease also exhibits amidase activity against N-benzoyl-Pro-Phe-Arg-p-nitroanilide, a unique and strict substrate for native Tm proteases reported previously .

Biochem Biophys Res Commun, 2000 Sep 7, 275(3), 817 - 20
A chimeric mini-trypsin inhibitor derived from the oil rape proteinase inhibitor type III; Trovato M et al.; The design of chimeric proteins is a major field of interest in structural biology and biotechnology . The successful design of the chimeric protein composed by the minimized reactive site domain of the low-molecular-mass trypsin inhibitor from Brassica napus (var . oleifera) seed (Ser3-Lys35; mini-RTI-III) and murine dihydrofolate reductase (DHFR) is reported here . The DHFR-mini-RTI-III chimeric protein was expressed in Escherichia coli, purified by metal-chelate affinity chromatography and oxidatively refolded . The affinity of the purified and refolded DHFR-mini-RTI-III for bovine trypsin (K = 5.0 x 10(-10) M) was closely similar to that determined for native RTI-III (K = 2.9 x 10(-10) M), at pH 8.2 and 22.0 degrees C . DHFR-mini-RTI-III may be regarded as a tool in structure-function studies and for developing multifunctional and multidomain proteinase inhibitors .

Biochem Biophys Res Commun, 2000 Sep 7, 275(3), 725 - 30
Evidence for catalytic cysteine-histidine dyad in chalcone synthase; Suh DY et al.; Chalcone and stilbene synthases (CHS and STS) catalyze condensation reactions of p-coumaroyl-CoA and three C(2)-units from malonyl-CoA, but catalyze different cyclization reactions to produce naringenin chalcone and resveratrol, respectively . Condensing activities of wild-type CHS and STS as well as STS-C60S mutant were inhibited by iodoacetamide (Idm) and diethyl pyrophosphate (DPC) . DPC also inhibited malonyl-CoA decarboxylation activity of wild-type and C164S mutants of CHS and STS . Meanwhile, Idm treatment enhanced (two- to fourfold) malonyl decarboxylase activity of wild-type enzymes and STS-C60S, whereas this priming effect was not observed with C164S mutants of CHS and STS, indicating that the cysteine residue being modified by Idm is the catalytic Cys164 of CHS and STS . DPC inhibition of decarboxylation activity of wild-type CHS was pH-independent in the range of pH 5.8 to 7.8; however, its inhibitory effect on CHS-C164S increased as pH increased from 6.2 to 7.4 with a midpoint of 6.4 . Based on the 3-D structure of CHS and the observed shift in microscopic pK(a), it was concluded that the histidine residue being modified by DPC in CHS is likely the catalytic His303 and that His303 forms an ionic pair (catalytic dyad) with Cys164 in wild-type CHS . In addition, our results showed that Cys60 in STS is not essential for the activity and only a single cysteine (Cys164) participates in the catalysis as in CHS .

Exp Eye Res, 2000 Sep, 71(3), 255 - 65
A lens glutathione S-transferase, class mu, with thiol-specific antioxidant activity; Jimenez-Asensio J et al.; A protein that protected against the thiol-mediated metal-catalysed oxidative inactivation of enzymes but did not protect against the ascorbate-dependent oxidation system was extensively purified from bovine lens . The protein was a homodimer (pI 7) of 26 kDa subunits . Sixty per cent of the protein sequence was obtained by Edman sequencing and by sequence comparison was determined to be a class mu glutathione S-transferase (GST) . The sequence of the enzyme is homologous to, but not identical to, that of any other class mu GST in the databanks . The complete protein sequence was derived from sequencing the cDNA and is the first complete sequence of a class mu GST from a bovine tissue . The enzyme was cloned and expressed in E . coli . The recombinant GST also protected against the thiol-mediated oxidative inactivation of enzymes but with lower activity than the native enzyme did and the recombinant GST had a comparable higher K(m)for GSH . The native and recombinant enzymes possessed similar low level peroxidase activity utilizing alkyl and cumene peroxides as substrates, but exhibited little activity against hydrogen peroxide .

Trends Genet, 2000 Sep, 16(9), 404 - 9
Learning new tricks from an old dog: MalT of the Escherichia coli maltose system is part of a complex regulatory network; Boos W et al.; The regulation of the maltose system in Escherichia coli has traditionally been viewed as a simple positive feedback loop . Today, we know that there are cross connections to several, seemingly unrelated, metabolic pathways . MalT, the central activator of the mal genes, is the key element in this complex regulatory network and integrates the different signals to give an appropriate transcriptional response.

Trends Biochem Sci, 2000 Sep, 25(9), 419 - 21
On the conservation of protein sequences in evolution; Kisters-Woike B et al.; The amino acid sequences of enzymes like alcohol dehydrogenase and glyceraldehyde-3-phosphate dehydrogenase are strongly conserved across all phyla . We suggest that the amino acid conservation of such enzymes might be a result of the fact that they function as part of a multi-enzyme complex . The specific interactions between the proteins involved would hinder evolutionary change of their surfaces.

J Cell Biochem, 2000 Sep 7, 79(3), 386 - 94
Binding of Escherichia coli lipopolysaccharide to fasciculata-reticularis and glomerulosa cells evaluated by flow cytometry; Enriquez de Salamanca A et al.; Binding of Escherichia coli lipopolysaccharide (LPS) to the two cell types of the adrenal cortex: fasciculata-reticularis and glomerulosa cells has been studied by flow cytometry and using fluorescein-labeled lipopolysaccharide (FITC-LPS) . The binding characteristics were different in relation to time course and number of binding sites . Both fasciculata-reticularis and glomerulosa cells bound LPS in a specific and saturable process . Fasciculata-reticularis cells showed a higher affinity for LPS binding than glomerulosa cells as deduced from Hill plots . Unlabeled LPS decreased FITC-LPS binding in both fasciculata-reticularis and glomerulosa cells, suggesting competition of both ligands for a limited number of binding sites . Lipid A seemed not to be essential for binding of LPS to fasciculata-reticularis cells . However, serum constituents inhibited FITC-LPS binding to both cell types, possibly due to cell interaction with HDL . The exposure of cells to LPS during cell culture did not modify the number of binding sites, but revealed cell size and surfaces structure changes .

Parasite Immunol, 2000 Aug, 22(8), 415 - 24
Immunogenicity and immunolocalization of the 22.6 kDa antigen of Schistosoma japonicum; Li Y et al.; The 22.6 kDa tegumental-associated antigens of Schistosoma mansoni (Sm22.6) and Schistosoma japonicum (Sj22.6) are of recognized interest in schistosomiasis vaccine development, although no direct vaccination/challenge experiments using either Sm22.6 or Sj22.6 had been previously described . We report that Escherichia coli-expressed reSj22.6 failed to protect mice or water buffaloes against subsequent challenge with S . japonicum cercariae . This was despite the fact that specific IgG (buffaloes) and IgG and IgE (CBA mice) antibodies were generated against the 22.6 kDa molecule, observations consistent with some of our earlier findings . We could find no evidence from immunolocalization studies that Sj22.6 is expressed or exposed on the surface of the adult parasite since it appears to be restricted to the apical cytoplasm of the tegument and is not associated with the apical or basal membrane or any membrane-bound structures in the apical cytoplasm . Nevertheless, Sj22.6 must be released to the immune system during the course of infection because specific anti-Sj22.6 IgG antibodies were present in the sera of nonvaccinated but challenged mice . We conclude that it may be necessary to produce reSj22.6 in a more relevant expression system, such as baculovirus, to further establish its vaccine potential and that detailed immunochemical and immunolocalization studies of early developmental stages may be necessary to determine how Sj22.6 is released or shed in S . japonicum infections.

Parasite Immunol, 2000 Aug, 22(8), 373 - 80
Differential ability of specific regions of Plasmodium falciparum sexual-stage antigen, Pfs230, to induce malaria transmission-blocking immunity; Bustamante PJ et al.; Antibodies raised against an Escherichia coli-produced recombinant protein encoding a 76-kDa section (region C) of malaria transmission-blocking vaccine candidate, Pfs230, have previously been shown to significantly reduce the ability of Plasmodium falciparum parasites to infect mosquitoes (71.2-89.8%) . To further define the region of the Pfs230 required for transmission-blocking activity, four recombinant proteins each encoding a section of region C (Pfs230 amino acids 443-1132) were produced using the same E . coli expression system and tested for immunogenicity in mice: (i) r230/MBP.C5' encodes the first half of region C (amino acids 443-791, six cysteines); (ii) r230/MBP.CM1 encodes only cysteine motif (CM) 1 (amino acids 583-913, eight cysteines); (iii) r230/MBP.C1.6 (amino acids 453-913, eight cysteines) also includes all of CM1; and (iv) r230/MBP.C2 encodes only CM2 (amino acids 914-1268, 11 cysteines) . All the recombinant proteins induced antibodies that recognized parasite-produced Pfs230, but the titre of the Pfs230 specific-antibodies generated varied, C = C1.6 = C5' > CM1 > CM2 . Two recombinants, r230/MBP.C5' and r230/MBP.C1.6, induced antibody titres that were equivalent to or greater than the titre generated by r230/MBP.C . However, in contrast to r230/MBP.C, none of the recombinants induced antibodies that effectively blocked parasite infectivity to mosquitoes . This suggests that the inclusion of amino acids 914-1132 is important for the production of the transmission-blocking epitope present in region C.

Mol Microbiol, 2000 Sep, 37(5), 1270 - 9
The interaction domains of the DnaA and DnaB replication proteins of Escherichia coli; Seitz H et al.; The initiation of chromosome replication in Escherichia coli requires the recruitment of the replicative helicase DnaB from the DnaBC complex to the unwound region within the replication origin oriC, supported by the oriC-bound initiator protein DnaA . We defined physical contacts between DnaA and DnaB that involve residues 24-86 and 130-148 of DnaA and residues 154-210 and 1-156 of DnaB respectively . We propose that contacts between DnaA and DnaB occur via two interaction sites on each of the proteins . Interaction domain 24-86 of DnaA overlaps with its N-terminal homo-oligomerization domain (residues 1-86) . Interaction domain 154-210 of DnaB overlaps or is contiguous with the domains known to interact with plasmid initiator proteins . Loading of the DnaBC helicase in vivo can only be performed by DnaA derivatives containing (in addition to residues 24-86 and the DNA-binding domain 4) a structurally intact domain 3 . Nucleotide binding by domain 3 is, however, not required . The parts of DnaA required for replication of pSC101 were clearly different from those used for helicase loading . Domains 1 and 4 of DnaA, but not domain 3, were found to be involved in the maintenance of plasmid pSC101.

Mol Microbiol, 2000 Sep, 37(5), 1258 - 69
Suppression of FNR-dependent transcription activation at the Escherichia coli nir promoter by Fis, IHF and H-NS: modulation of transcription initiation by a complex nucleo-protein assembly; Browning DF et al.; Expression from the Escherichia coli nir promoter is co-dependent on both the FNR protein (an anaerobically triggered transcription activator) and the NarL or NarP proteins (transcription activators triggered by nitrite and nitrate) . Under anaerobic conditions, FNR binds to a site centred between positions -41 and -42, activating transcription of the nir operon . In previous work, we showed that this activation is suppressed by the binding of Fis protein, and at least one other protein, to sequence elements located upstream of the nir promoter . We proposed that the binding of NarL or NarP to a site centred between positions -69 and -70 counteracts this suppression, resulting in increased transcription in response to nitrite or nitrate . Here we have further investigated the different proteins that downregulate the nir promoter . We show that the nir promoter is repressed by three DNA binding proteins, Fis, IHF and H-NS . We demonstrate that, in addition to binding to its previously characterized upstream site located at position -142, Fis also binds to a second downstream site located at position +23 . A second suppressing factor is IHF, that binds to a site located at position -88 . Finally, the nucleoid associated protein, H-NS, preferentially binds to upstream sequences at the nir promoter and represses promoter activity . The association of Fis, IHF and H-NS suggests that nir promoter DNA is sequestrated into a highly ordered nucleo-protein structure that represses FNR-dependent transcription activation . NarL and NarP can relieve both IHF- and Fis-mediated repression, but are unable to counteract H-NS-mediated repression.

Mol Microbiol, 2000 Sep, 37(5), 1186 - 97
Tethering of CpxP to the inner membrane prevents spheroplast induction of the cpx envelope stress response; Raivio TL et al.; The Cpx envelope stress response of Escherichia coli is controlled by a two-component regulatory system that senses misfolded proteins in extracytoplasmic compartments and responds by inducing the expression of envelope protein folding and degrading factors . We have proposed that in the absence of envelope stress the pathway is maintained in a downregulated state, in part through interactions between the periplasmic inhibitor molecule CpxP and the sensing domain of the histidine kinase CpxA . In this study, we show that depletion of the periplasmic contents of the cell by spheroplast formation does indeed lead to induction of the Cpx envelope stress response . Further, removal of CpxP is an important component of this induction because tethering an MBP-CpxP fusion protein to the spheroplast inner membranes prevents full activation by this treatment . Spheroplast formation has previously been demonstrated to induce the expression of a periplasmic protein of unknown function, Spy . Analysis of spy expression in response to spheroplast formation by Western blot analysis and by lacZ operon fusion in various cpx mutant backgrounds demonstrated that spy is a member of the Cpx regulon . Interestingly, although the only known spy homologue is cpxP, Spy does not appear to perform the same function as CpxP as it is not involved in inhibiting the Cpx envelope stress response . Rather, deletion of spy leads to activation of the sigmaE stress response . Because the sigmaE response is specifically affected by alterations in outer membrane protein biogenesis, we think it possible that Spy may be involved in this process.

Mol Microbiol, 2000 Sep, 37(5), 1087 - 93
Rifampicin-resistant initiation of chromosome replication from oriC in ihf mutants; Von Freiesleben U et al.; IHF (integration host factor) mutants exhibit asynchronous initiation of chromosome replication from oriC as determined from flow cytometric analysis of cultures where RNA synthesis was inhibited with rifampicin . However, the run-out kinetics of chromosome replication in ihf mutants shows that they continue to produce oriCs for some time in the absence of RNA synthesis resulting in a twofold increase in the oriC per mass ratio . An ihf dnaA double mutant did not exhibit this continued increase of the oriC per mass ratio . This indicates that ihf mutants can initiate replication from oriC in a rifampicin-resistant initiation mode but requires fully functional DnaA protein . The origin per mass ratio, determined by a quantitative Southern blotting technique, showed that the ihf mutants had an origin per mass ratio that was 60% of the wild type although it had a normal DnaA protein concentration . This shows that the initiation mass was substantially higher in the ihf mutants . The oriC per terminus ratio, which was also determined by Southern blotting, was very low in the ihf mutant, although it grew with the same doubling times as the wild-type strain . This indicates that cells lacking IHF replicate their chromosome(s) very fast.

Mol Microbiol, 2000 Sep, 37(5), 1066 - 74
Overcoming the restriction barrier to plasmid transformation of Helicobacter pylori; Donahue JP et al.; Helicobacter pylori strains demonstrate substantial variability in the efficiency of transformation by plasmids from Escherichia coli, and many strains are completely resistant to transformation . Among the barriers to transformation are numerous strain-specific restriction-modification systems in H . pylori . We have developed a method to protect plasmid DNA from restriction by in vitro site-specific methylation using cell-free extracts of H . pylori before transformation . In two cases, plasmid DNA treated with cell-free extracts in vitro acquired the restriction pattern characteristic of genomic DNA from the source strain . Among three strains examined in detail, the transformation frequency by treated plasmid shuttle and suicide vectors was significantly increased compared with mock-treated plasmid DNA . The results indicate that the restriction barrier in H . pylori can be largely overcome by specific DNA methylation in vitro . The approach described should significantly enhance the ability to manipulate gene function in H . pylori and other organisms that have substantial restriction barriers to transformation.

Mol Microbiol, 2000 Sep, 37(5), 1052 - 65
Restriction-modification system differences in Helicobacter pylori are a barrier to interstrain plasmid transfer; Ando T et al.; Helicobacter pylori cells are naturally competent for the uptake of both plasmid and chromosomal DNA . However, we demonstrate that there are strong barriers to transformation of H . pylori strains by plasmids derived from unrelated strains . We sought to determine the molecular mechanisms underlying these barriers . Transformation efficiency was assessed using pHP1, an Escherichia coli-H . pylori shuttle vector conferring kanamycin resistance . Transformation of 33 H . pylori strains was attempted with pHP1 purified from either E . coli or H . pylori, and was successfully introduced into only 11 strains . Digestion of H . pylori chromosomes with different restriction endonucleases (REs) showed that DNA methylation patterns vary substantially among strains . The strain most easily transformed, JP26, was found to have extremely low endogenous RE activity and to lack a restriction-modification (R-M) system, homologous to MboI, which is highly conserved among H . pylori strains . When we introduced this system to JP26, pHP1 from MboI.M+ JP26, but not from wild-type JP26, transformed MboI R-M+ JP26 and heterologous MboI R-M+ wild-type H . pylori strains . Parallel studies with pHP1 from dam+ and dam- E . coli strains confirmed these findings . These data indicate that the endogenous REs of H . pylori strains represent a critical barrier to interstrain plasmid transfer among H . pylori.

Mol Microbiol, 2000 Sep, 37(5), 1032 - 40
Analysis of interactions between Activating Region 1 of Escherichia coli FNR protein and the C-terminal domain of the RNA polymerase alpha subunit: use of alanine scanning and suppression genetics; Lee DJ et al.; Activating Region 1 of Escherichia coli FNR protein is proposed to interact directly with the C-terminal domain of the RNA polymerase alpha subunit (alphaCTD) during transcription activation at FNR-regulated promoters . Using an alphaCTD alanine scan mutant library, we have identified the residues of alphaCTD that are important for FNR-dependent transcription activation . Residues Asp-305, Gly-315, Arg-317, Leu-318 and Asp-319 are proposed to be the key residues in the contact site on alphaCTD for Activating Region 1 of FNR . In previous work, it had been shown that Activating Region 1 of FNR is a large surface-exposed patch and that the two crucial amino acid residues are Thr-118 and Ser-187 . In this work, we have constructed Arg-118 FNR and Arg-187 FNR and shown that both FNR derivatives are defective in transcription activation . However, the activity of FNR carrying Arg-118 can be partially restored by substitutions of Lys-304 in alphaCTD . Similarly, the activity of FNR carrying Arg-187 can be partially restored by substitutions of Arg-317 or Leu-318 in alphaCTD . The specificity of the restoration suggests that, during transcription activation by FNR, the side-chain of residue 118 in Activating Region 1 of FNR is located close to Lys-304 and Asp-305 in alphaCTD . Similarly, the side-chain of residue 187 in Activating Region 1 of FNR is located close to Arg-317 and Leu-318 in alphaCTD . These results can be used to model the interface between Activating Region 1 of FNR and its contact target in alphaCTD, and permit comparison of this interface with the interface between Activating Region 1 of the related transcription activator, CRP and alphaCTD.

Mol Microbiol, 2000 Aug, 37(4), 856 - 68
The gcvB gene encodes a small untranslated RNA involved in expression of the dipeptide and oligopeptide transport systems in Escherichia coli; Urbanowski ML et al.; The Escherichia coli gcvB gene encodes a small RNA transcript that is not translated in vivo . Transcription from the gcvB promoter is activated by the GcvA protein and repressed by the GcvR protein, the transcriptional regulators of the gcvTHP operon encoding the enzymes of the glycine cleavage system . A strain carrying a chromosomal deletion of gcvB exhibits normal regulation of gcvTHP expression and glycine cleavage enzyme activity . However, this mutant has high constitutive synthesis of OppA and DppA, the periplasmic-binding protein components of the two major peptide transport systems normally repressed in cells growing in rich medium . The altered regulation of oppA and dppA was also demonstrated using oppA-phoA and dppA-lacZ gene fusions . Although the mechanism(s) involving gcvB in the repression of these two genes is not known, oppA regulation appears to be at the translational level, whereas dppA regulation occurs at the mRNA level.

Mol Microbiol, 2000 Aug, 37(4), 788 - 99
The Myxococcus xanthus socE and csgA genes are regulated by the stringent response; Crawford EW Jr et al.; Disruption of the Myxococcus xanthus socE gene bypasses the requirement for the cell contact-dependent C-signalling system mediated by CsgA and restores fruiting body morphogenesis and spore differentiation . The socE gene has been identified by genetic complementation, cloned and sequenced . SocE is highly basic, unique and is predicted to be a soluble protein with a molecular size of 53 . 6 kDa . The socE and csgA genes have opposite transcription patterns during the M . xanthus life cycle . socE expression is high in growing cells and declines during the early stages of development . Expression of csgA is low in vegetative cells and increases during development . socE transcription is negatively regulated by the stringent response, the major amino acid-sensing pathway in M . xanthus . A relA null mutation, which eliminates the stringent response, prevents the decline in socE expression normally observed at the onset of development . CsgA is positively regulated by the stringent response and is negatively regulated by socE . A relA mutation virtually eliminates developmental csgA expression . Expression of socE in Escherichia coli leads to a rapid loss of viability in relA- cells during stationary phase, suggesting a relationship with the stringent response.

Mol Microbiol, 2000 Aug, 37(4), 740 - 51
Localization of components of the chemotaxis machinery of Escherichia coli using fluorescent protein fusions; Sourjik V et al.; We prepared fusions of yellow fluorescent protein {the YFP variant of green fluorescent protein (GFP)} with the cytoplasmic chemotaxis proteins CheY, CheZ and CheA and the flagellar motor protein FliM, and studied their localization in wild-type and mutant cells of Escherichia coli . All but the CheA fusions were functional . The cytoplasmic proteins CheY, CheZ and CheA tended to cluster at the cell poles in a manner similar to that observed earlier for methyl-accepting chemotaxis proteins (MCPs), but only if MCPs were present . Co-localization of CheY and CheZ with MCPs was CheA dependent, and co-localization of CheA with MCPs was CheW dependent, as expected . Co-localization with MCPs was confirmed by immunofluorescence using an anti-MCP primary antibody . The motor protein FliM appeared as discrete spots on the sides of the cell . These were seen in wild-type cells and in a fliN mutant, but not in flhC or fliG mutants . Co-localization with flagellar structures was confirmed by immunofluorescence using an antihook primary antibody . Surprisingly, we did not observe co-localization of CheY with motors, even under conditions in which cells tumbled.

Mol Microbiol, 2000 Aug, 37(4), 696 - 702
A SeqA hyperstructure and its interactions direct the replication and sequestration of DNA; Norris V et al.; A level of explanation in biology intermediate between macromolecules and cells has recently been proposed . This level is that of hyperstructures . One class of hyperstructures comprises the genes, mRNA, proteins and lipids that assemble to fulfil a particular function and disassemble when no longer required . To reason in terms of hyperstructures, it is essential to understand the factors responsible for their formation . These include the local concentration of sites on DNA and their cognate DNA-binding proteins . In Escherichia coli, the formation of a SeqA hyperstructure via the phenomenon of local concentration may explain how the binding of SeqA to hemimethylated GATC sequences leads to the sequestration of newly replicated origins of replication.

Lett Appl Microbiol, 2000 Sep, 31(3), 223 - 7
Elimination of volatile chemicals in disinfectant evaluation procedures by freeze drying; Quinn PJ et al.; Standard methods for the removal or inactivation of disinfectants from reaction mixtures are often unsatisfactory when dealing with high concentrations of particulate organic matter . A method employing freeze drying was developed to eliminate residual disinfectant activity from reaction mixtures . Following the addition of volatile chemicals to sterilized cattle slurry, samples were removed from the mixture, freeze dried, and the dried material was reconstituted with diluent containing Escherichia coli as the test micro-organism to determine if the volatile chemicals had been removed . Twelve chemicals and one commercial disinfectant (Virkon S) were evaluated at different concentrations . The chemicals selected were acetone, ammonium hydroxide, carbon tetrachloride, chloroform, dichloromethane, ethyl alcohol, formalin, hydrogen peroxide, isopropyl alcohol, peracetic acid, sodium hypochlorite and xylene . Eight chemicals, acetone, ammonium hydroxide, carbon tetrachloride, chloroform, dichloromethane, ethyl alcohol, isopropyl alcohol and xylene were removed by freeze drying . The remaining four chemicals and the commercial disinfectant, which were not removed by freeze drying, could not be evaluated by this method.

Lett Appl Microbiol, 2000 Sep, 31(3), 203 - 8
Shiga-toxin-producing Escherichia coli in faeces of healthy dairy cows, sheep and goats: prevalence and virulence properties; Zschock M et al.; The prevalence of Shiga-toxin-producing Escherichia coli (STEC) in healthy dairy ruminants was investigated between 1996 and 1998 by a multiplex polymerase chain reaction (mPCR) technique . A total of 13 552 E . coli colonies from 726 cows, 28 sheep and 93 goats out of 112 randomly selected dairy farms in Hessia, Germany were analysed . STEC strains were recovered from 131 (18.0%) cows, nine (32.1%) sheep and 70 (75.3%) goats . Further characterization of the STEC isolates showed that 89 (0.66% of the investigated colonies) of animal field strains carried stx1 gene, 64 (0.47%) stx2 gene and 57 (0.42%) stx1 and stx2 gene . Sixty (93.8%) out of 64 stx2 field strains were harboured by cows . In contrast, 74 (83.1%) out of 89 stx1 dairy animal field strains were from ovine or caprine origin . Only 17 (8 . 1%) stx-positive isolates (13 from cattle, three from sheep and only one from goat) were positive for eaeA gene . Eight (9.0%) of the stx1, five (7.8%) of the stx2 and four (7.0%) of the stx1/stx2 gene-positive field strains carried the eaeA gene . The prevalence of EHEC-haemolysin (EHEC-hlyA) gene sequence was 88.8% (79 isolates) of the stx1 and 68.8% (44 isolates) of the stx2 isolates . Out of 57 stx1- and stx2-positive field-strains, 34 (59.6%) carried the EHEC-hlyA gene . E . coli O serovars O:157 and O:111 were not found . Only one isolate was positive with O26 antiserum.

Nature, 2000 Aug 24, 406(6798), 855 - 62
Structural and biochemical basis of apoptotic activation by Smac/DIABLO; Chai J et al.; Apoptosis (programmed cell death), an essential process in the development and homeostasis of metazoans, is carried out by caspases . The mitochondrial protein Smac/DIABLO performs a critical function in apoptosis by eliminating the inhibitory effect of IAPs (inhibitor of apoptosis proteins) on caspases . Here we show that Smac/DIABLO promotes not only the proteolytic activation of procaspase-3 but also the enzymatic activity of mature caspase-3, both of which depend upon its ability to interact physically with IAPs . The crystal structure of Smac/DIABLO at 2.2 A resolution reveals that it homodimerizes through an extensive hydrophobic interface . Missense mutations inactivating this dimeric interface significantly compromise the function of Smac/DIABLO . As in the Drosophila proteins Reaper, Grim and Hid, the amino-terminal amino acids of Smac/DIABLO are indispensable for its function, and a seven-residue peptide derived from the amino terminus promotes procaspase-3 activation in vitro . These results establish an evolutionarily conserved structural and biochemical basis for the activation of apoptosis by Smac/DIABLO.

Extremophiles, 2000 Aug, 4(4), 215 - 25
Cloning, expression, and characterization of DNA polymerase I from the hyperthermophilic archaea Thermococcus fumicolans; Cambon-Bonavita MA et al.; The DNA polymerase I gene of a newly described deep-sea hydrothermal vent Archaea species, Thermococcus fumicolans, from IFREMERS's collection of hyperthermophiles has been cloned in Escherichia coli . As in Thermococcus litoralis, the gene is split by two intervening sequences (IVS) encoding inteins inserted in sites A and C of family B DNA polymerases . The entire DNA polymerase gene, containing both inteins, was expressed at 30 degrees C in E . coli strain BL21(DE3)pLysS using the pARHS2 expression vector . The native polypeptide precursor of 170kDa was obtained, and intein splicing as well as ligation of the three exteins was observed in vitro after heat exposure . The recombinant enzyme was purified and some of its activities were characterized: polymerization, thermostability, exonuclease activities, and fidelity.

Scand J Gastroenterol, 2000 Jul, 35(7), 711 - 8
Treatment of enterotoxigenic and enteropathogenic Escherichia coli-induced diarrhoea in children with bovine immunoglobulin milk concentrate from hyperimmunized cows: a double-blind, placebo-controlled, clinical trial; Casswall TH et al.; BACKGROUND: Enterotoxigenic Escherichia coli (ETEC) and enteropathogenic Escherichia coli (EPEC) are important causes of diarrhoea in young children and are associated with significant mortality rates . Passive immunization with antibodies from immunized cows has previously been shown to be effective as prophylaxis against E . coli-induced diarrhoea and therapeutically against rotavirus and cryptosporidia-induced diarrhoea . METHODS: We tested the therapeutic efficacy of an oral bovine immunoglobulin milk concentrate (BIC) from cows hyperimmunized with ETEC and EPEC strains, in a randomized, placebo-controlled study in children with E . coli-induced diarrhoea . Eighty-six children between 4-24 months of age attending the International Centre for Diarrhoeal Disease Research, Bangladesh (ICDDR, B) with E . coli-induced diarrhoea (63 EPEC/ETEC and 23 with other diarrhoeagenic E . coli) were randomly assigned to receive orally administered BIC (20 g) containing anti-ETEC/EPEC antibodies or a placebo preparation daily for 4 consecutive days . Daily stool output, intake of oral rehydration solution (ORS), stool frequency, and presence of diarrhoeagenic E . coli strains in the stool were monitored for 4 days . RESULTS: Children in the treatment group tolerated the BIC with no side effects . There were no significant differences between the two groups with regard to ORS intake, stool output, frequency of diarrhoea, or clearance of pathogen . Nor was there any significant alteration in the duration of diarrhoea . CONCLUSIONS: In contrast to the prophylactic efficacy of anti-E . coli BIC and the therapeutic efficacy of a similarly prepared anti-rotavirus BIC, antibodies from hyperimmunized cows appear to have no significant therapeutic benefit in the treatment of acute diarrhoea due to EPEC/ETEC.

J Viral Hepat, 2000 Sep, 7(5), 335 - 42
RNA-dependent RNA polymerase activity encoded by GB virus-B non-structural protein 5B; Zhong W et al.; Phylogenetic analysis and polyprotein organization comparison have shown that GB virus-B (GBV-B) is closely related to hepatitis C virus (HCV) . In this study, the coding region for GBV-B non-structural protein 5B (NS5B) was isolated by reverse transcription-polymerase chain reaction (RT-PCR) from pooled serum of GBV-B-infected tamarins . Expression of soluble GBV-B NS5B protein in Escherichia coli was achieved by removal of a 19-amino acid hydrophobic domain at the C-terminus of the protein . The truncated GBV-B NS5B (NS5BDeltaCT19) was purified to homogeneity and shown to possess an RNA-dependent RNA polymerase (RdRp) activity in both gel-based and scintillation proximity assays . NS5BDeltaCT19 required the divalent cation Mn2+ for enzymatic activity, at an optimal concentration of 15 mM . Interestingly, Mg2+, at concentrations up to 20 mM, did not support the GBV-B NS5B activity . This differs from HCV NS5B where both Mn2+ and Mg2+ can support RdRp activity . Zn2+ was found to inhibit the activity of GBV-B NS5B, with a 50% inhibitory concentration (IC50) of 5-10 microM . Higher concentrations of monovalent salts (NaCl or KCl > 100 mM) and glycerol (> 3%) were also inhibitory . NS5BDeltaCT19 was able to bind to RNA homopolymers, but utilized most efficiently poly(C), the one with the lowest binding affinity for RNA synthesis . Mutational analysis of GBV-B NS5B demonstrated the importance of several conserved sequence motifs for enzymatic activity . Based on sequence homology ( approximately 37% identity and 52% similarity) between GBV-B and HCV NS5B proteins, the active GBV-B RdRp provides a good surrogate assay system for HCV polymerase studies.

Genes Cells, 2000 Sep, 5(9), 689 - 98
The role of tightly bound ATP in Escherichia coli tRNA nucleotidyltransferase; Tomari Y et al.; BACKGROUND: The CCA-adding enzyme {ATP(CTP): tRNA nucleotidyltransferase (EC . 2.7.7.25)} catalyses the addition of the conserved CCA sequence to the 3'-terminus of tRNAs . All CCA-adding enzymes are classified into the nucleotidyltransferase superfamily . In the absence of ATP, the Escherichia coli CCA-adding enzyme displays anomalous poly(C) polymerase activity . RESULTS: We show that CCA-adding enzyme over-expressed in E . coli exists in an ATP-bound form . The affinities of ATP and CTP towards the enzyme were estimated by several methods, and the dissociation constants for ATP and CTP were determined to be 6.3 and 188 microM, respectively . AMP-incorporation terminated the nucleotidyltransferase reaction, while in the absence of ATP, the enzyme continued poly(C) polymerization . In the case of a tRNA substrate with a mutation in the T-loop region, normal CC was added at a much slower rate compared with the wild-type, but anomalous poly(C) polymerization occurred at the same rate as in the wild-type . CONCLUSION: Based on the findings outlined above, we concluded that the E . coli CCA-adding enzyme possesses at least two distinct nucleotide binding sites, one responsible for ATP binding and the other(s) for CTP binding . The addition of ATP from the tight ATP binding site terminates nucleotide incorporation, thus limiting poly(C) polymerization to CCA . It is also suggested that during anomalous poly(C) polymerization, tRNA translocates from the tRNA binding site upon the third C addition.

Eur J Biochem, 2000 Sep, 267(18), 5751 - 7
The LCCL module; Trexler M et al.; Here we show that Lgl1 protein, cub-1-related proteins, coch-5b2-related proteins, coagulation factor C of horse-shoe crab and a predicted protein of Plasmodium falciparum share a homologous domain . Since this domain-type was first identified in Limulus factor C, Coch-5b2 and Lgl1 we propose the name LCCL for this domain-family . The LCCL module of coch-5b2 is of special biological interest because it has been shown recently that mutations affecting this module cause the deafness disorder DFNA9 in humans . With a view to defining the structure and function of the LCCL domain of human coch-5b2 protein, we have expressed it in Escherichia coli and subjected it to preliminary structural characterization . Structure prediction and circular dichroism studies on the recombinant protein indicate that the domain possesses both alpha helices and beta strands . It is shown that the mutations which cause hearing loss in humans affect residues that are critical for the integrity of the LCCL module of the coch-5b2 protein.

Eur J Biochem, 2000 Sep, 267(18), 5699 - 710
Characterization of human HtrA2, a novel serine protease involved in the mammalian cellular stress response; Gray CW et al.; Human HtrA2 is a novel member of the HtrA serine protease family and shows extensive homology to the Escherichia coli HtrA genes that are essential for bacterial survival at high temperatures . HumHtrA2 is also homologous to human HtrA1, also known as L56/HtrA, which is differentially expressed in human osteoarthritic cartilage and after SV40 transformation of human fibroblasts . HumHtrA2 is upregulated in mammalian cells in response to stress induced by both heat shock and tunicamycin treatment . Biochemical characterization of humHtrA2 shows it to be predominantly a nuclear protease which undergoes autoproteolysis . This proteolysis is abolished when the predicted active site serine residue is altered to alanine by site-directed mutagenesis . In human cell lines, it is present as two polypeptides of 38 and 40 kDa . HumHtrA2 cleaves beta-casein with an inhibitor profile similar to that previously described for E . coli HtrA, in addition to an increase in beta-casein turnover when the assay temperature is raised from 37 to 45 degrees C . The biochemical and sequence similarities between humHtrA2 and its bacterial homologues, in conjunction with its nuclear location and upregulation in response to tunicamycin and heat shock suggest that it is involved in mammalian stress response pathways.

Clin Exp Allergy, 2000 Sep, 30(9), 1285 - 92
Recombinant Hev b 1: large-scale production and immunological characterization; Rihs HP et al.; BACKGROUND: Hev b 1 represents one of the most important allergens in Hevea brasiliensis latex . It is difficult to get an appropriate amount of native Hev b 1 (nHev b 1) for research purposes . OBJECTIVE: The aim of this study was to produce sufficient amounts of Hev b 1 by recombinant methods to prove its suitability for latex allergy diagnostics . METHODS: We isolated total RNA of Hevea brasiliensis leaves and synthesized cDNA by RT PCR . Recombinant Hev b 1 (rHev b 1) as well as three fragments (amino acid residues 29-137, 48-137, 78-137) were subcloned and expressed as fusion proteins with Maltose-binding protein (MBP) in Escherichia coli . The MBP-rHev b 1 fusion protein was examined by RAST with the CAP method, histamine release test and immunoblots with human sera from spina bifida patients as well as from health care workers with latex allergy and monoclonal antibodies . RESULTS: Histamine release test and immunoblots revealed the high allergenicity of the MBP-rHev b 1 construct . By the CAP method, 54 out of 58 serum samples (93%) from latex-sensitized spina bifida patients previously showing immunoglobulin (Ig) E to nHev b 1 exhibited IgE-binding to rHev b 1 . Among 71 latex-allergic health care workers tested, 16 (22.5%) had IgE antibodies to rHev b 1 . The analysis of the fusion proteins carrying rHev b 1 fragments revealed that the loss of the N-terminal 28 amino acid residues did not affect IgE-binding . In contrast, the lack of the first 47 amino acid residues led to decreased IgE-binding reactivity in two out of four sera tested, whereas the absence of the N-terminal 77 residues abolished IgE-binding in these two sera . CONCLUSION: The MBP-rHev b 1 fusion protein exhibits a corresponding IgE-binding reactivity to nHev b 1 and may therefore substitute natural Hev b 1 for both in vitro diagnostics and research purposes.

Br J Haematol, 2000 Aug, 110(2), 379 - 84
Pegylated asparaginase (Oncaspar) in children with ALL: drug monitoring in reinduction according to the ALL/NHL-BFM 95 protocols; Muller HJ et al.; Hypersensitivity reactions are relevant adverse effects of asparaginase therapy . Therefore, children treated with native Escherichia coli asparaginase in induction therapy of acute lymphoblastic leukaemia (ALL) or non-Hodgkin's lymphoma (NHL) were switched to the pegylated enzyme for reinduction under drug monitoring . Seventy children, including four patients with allergic reactions during induction, were given one dose of Oncaspar 1,000 U/m2 intravenously . Activity was determined every third or fourth day until it dropped below the limit of quantification . In current reinduction protocols {ALL/NHL-Berlin-Frankfurt-Munster (BFM) 95 trials}, four doses of 10,000 U/m2 E . coli asparaginase deplete asparagine for about 2-3 weeks, therefore activities of >/= 100 U/l up to day 14 and >/= 50 U/l up to day 21 were targeted . In 66 patients without an allergic reaction during induction, the mean activity was 606 +/- 313 U/l, 232 +/- 211 U/l and 44 +/- 50 U/l after 1, 2 and 3 weeks respectively . In 44/66 patients, activity was >/= 100 U/l after 14 d . A rapid decline in activity was seen in the remaining 22 patients, including 8/22 patients who showed no activity after 1 week . Toxicity was low and comparable to the native enzymes but, in contrast to about 30% of hypersensitivity reactions with conventional reinduction therapy, no allergic reaction was seen . Substituting 4 x 10,000 U/m2 asparaginase medac for one dose of 1,000 U/m2 Oncaspar was safe and well tolerated . Comparable pharmacokinetic treatment intensity was achieved in about two-thirds of patients.

J Biomed Sci, 2000 Sep-Oct, 7(5), 420 - 8
The action mode of the ribosome-inactivating protein alpha-sarcin; Hwu L et al.; Based on the tertiary structure of the ribosome-inactivating protein alpha-sarcin, domains that are responsible for hydrolyzing ribosomes and naked RNA have been dissected . In this study, we found that the head-to-tail interaction between the first amino beta-strand and the last carboxyl beta-strand is not involved in catalyzing the hydrolysis of ribosomes or ribonucleic acids . Instead, a four-strand pleated beta-sheet is indispensable for catalyzing both substrates, suggesting that alpha-sarcin and ribonuclease T1 (RNase T1) share a similar catalytic center . The integrity of an amino beta-hairpin and that of the loop L3 in alpha-sarcin are crucial for recognizing and hydrolyzing ribosomes in vitro and in vivo . However, a mutant protein without the beta-hairpin structure, or with a disrupted loop L3, is still capable of digesting ribonucleic acids . The functional involvement of the beta-hairpin and the loop L3 in the sarcin stem/loop RNA of ribosomes is demonstrated by a docking model, suggesting that the two structures are in essence naturally designed to distinguish ribosome-inactivating proteins from RNase T1 to inactivate ribosomes .

J Biol Chem, 2000 Nov 24, 275(47), 36758 - 65
Self-association of human apolipoprotein E3 and E4 in the presence and absence of phospholipid; Perugini MA et al.; Human apolipoprotein E (apoE) exists as three main isoforms, differing by single amino acid substitutions, with the apoE4 isoform strongly linked to the incidence of late onset Alzheimer's disease . We have expressed and purified apoE3 and apoE4 from Escherichia coli and compared their hydrodynamic properties by gel permeation liquid chromatography, capillary electrophoresis, circular dichroism, and sedimentation methods . Sedimentation velocity experiments, employing a new method for determining the size distribution of polydisperse macromolecules in solution (Schuck, P . (2000) Biophys . J . 78, 1606-1619), provide direct evidence for the heterogeneous solution structures of apoE3 and apoE4 . In a lipid-free environment, apoE3 and apoE4 exist as a slow equilibrium mixture of monomer, tetramer, octamer, and a small proportion of higher oligomers . Both sedimentation velocity and equilibrium experiments indicate that apoE4 has a greater propensity to self-associate . We also demonstrate that apoE3 and apoE4 oligomers dissociate significantly in the presence of dihexanoylphosphatidylcholine micelles (20 mm) and to a lesser extent at submicellar concentrations (4 mm) . The alpha-helical content for both isoforms was almost identical (50%) in the presence and absence of dihexanoylphosphatidylcholine . These results reveal that apoE oligomers undergo phospholipid-induced dissociation to folded monomers, suggesting the monomeric form prevails on the lipoprotein surface in vivo.

J Biol Chem, 2000 Nov 24, 275(47), 36665 - 70
FAD insertion is essential for attaining the assembly competence of the dihydrolipoamide dehydrogenase (E3) monomer from Escherichia coli; Lindsay H et al.; Dihydrolipoamide dehydrogenase (E3) from Escherichia coli, an FAD-linked homodimer, can be fully reconstituted in vitro following denaturation in 6 m guanidinium chloride . Complete restoration of activity occurs within 1-2 h in the presence of FAD, dithiothreitol, and bovine serum albumin . In the absence of FAD, the dihydrolipoamide dehydrogenase monomer forms a stable folding intermediate, which is incapable of dimerization . This intermediate displays a similar tryptic resistance to the native enzyme but is less heat-stable, because its ability to form native E3 is lost after incubation at 65 degrees C for 15 min . The presence of FAD promotes slow, additional conformational rearrangements of the E3 subunit as observed by cofactor-dependent decreases in intrinsic tryptophan fluorescence . However, after 2 h, the tryptophan fluorescence spectrum and far UV CD spectrum of E3, refolded in the absence of FAD, are similar to that of the native enzyme, and full activity can still be recovered on addition of FAD . Cross-linking studies show that FAD insertion is necessary for the monomeric folding intermediate to attain an assembly competent state leading to dimerization . Thus cofactor insertion represents a key step in the assembly of this enzyme, although its initial presence appears not to be required to promote the correct folding pathway.

Genes Dev, 2000 Sep 1, 14(17), 2242 - 55
Promoter opening by sigma(54) and sigma(70) RNA polymerases: sigma factor-directed alterations in the mechanism and tightness of control; Guo Y et al.; Transcription control at the melting step is not yet understood . Here, band shift, cross-linking, and transcription experiments on diverse DNA probes were used with two bacterial RNA polymerase holoenzymes that differ in how they regulate melting . Data indicated that both sigma(54) and sigma(70) holoenzymes assume a default closed form that cannot establish single-strand binding . Upon activation the enzymes are converted to an open form that can bind simultaneously to the upstream fork junction and to the melted transcription start site . The key difference is that sigma(54) imposes tighter regulation by creating a complex molecular switch at -12/-11; the current data show that this switch can be thrown by activator . In this case an ATP-bound enhancer protein causes sigma(54) to alter its cross-linking pattern near -11 and also causes a reorganization of holoenzyme: DNA interactions, detected by electrophoretic mobility-shift assay . At a temperature-dependent sigma(70) promoter, elevated temperature alone can assist in triggering conformational changes that enhance the engagement of single-strand DNA . Thus, the two sigma factors modify the same intrinsic opening pathway to create quite different mechanisms of transcriptional regulation.

Genes Dev, 2000 Sep 1, 14(17), 2206 - 15
Promotion of Rad51-dependent D-loop formation by yeast recombination factor Rdh54/Tid1; Petukhova G et al.; The first DNA joint formed in homologous recombination processes is a D-loop . Saccharomyces cerevisiae RDH54/TID1-encoded product, a Swi2/Snf2-like factor involved in recombination, is shown here to promote D-loop formation with Rad51 recombinase . Physical interaction between Rdh54 and Rad51 is functionally important because Rdh54 does not enhance the recombinase activity of the Escherichia coli RecA protein . Robust dsDNA-activated ATPase activity in Rdh54 generates unconstrained negative and positive supercoils in DNA . Efficient D-loop formation occurs with even topologically relaxed DNA, suggesting that via specific protein-protein interactions, the negative supercoils produced by Rdh54 are used by Rad51 for making DNA joints.

EMBO J, 2000 Sep 1, 19(17), 4838 - 45
Structure of the ArsA ATPase: the catalytic subunit of a heavy metal resistance pump; Zhou T et al.; Active extrusion is a common mechanism underlying detoxification of heavy metals, drugs and antibiotics in bacteria, protozoa and mammals . In Escherichia coli, the ArsAB pump provides resistance to arsenite and antimonite . This pump consists of a soluble ATPase (ArsA) and a membrane channel (ArsB) . ArsA contains two nucleotide-binding sites (NBSs) and a binding site for arsenic or antimony . Binding of metalloids stimulates ATPase activity . The crystal structure of ArsA reveals that both NBSs and the metal-binding site are located at the interface between two homologous domains . A short stretch of residues connecting the metal-binding site to the NBSs provides a signal transduction pathway that conveys information on metal occupancy to the ATP hydrolysis sites . Based on these structural features, we propose that the metal-binding site is involved directly in the process of vectorial translocation of arsenite or antimonite across the membrane . The relative positions of the NBS and the inferred mechanism of allosteric activation of ArsA provide a useful model for the interaction of the catalytic domains in other transport ATPases.

Biochem J, 2000 Sep 15, 350 Pt 3, 917 - 23
Kinetics and thermodynamics of activation of quinoprotein glucose dehydrogenase apoenzyme in vivo and catalytic activity of the activated enzyme in Escherichia coli cells; Iswantini D et al.; Apo-glucose dehydrogenase existing in Escherichia coli is converted to the holoenzyme with exogenous pyrroloquinoline quinone (PQQ) and Mg(2+) . Catalytic behaviour of the E . coli cells with the holoenzyme is characterized by a Michaelis-Menten-type equation with a catalytic constant of the cell and apparent Michaelis constants for D-glucose and an artificial electron acceptor added to the E . coli suspension . The catalytic constant is expressed as the product of the number of molecules of the enzyme contained in an E . coli cell (z) and the catalytic constant of the enzyme (k(cat)), which were determined to be 2.2x10(3) and 6.8+/-0.8x10(3) s(-1) (phenazine methosulphate as an electron acceptor) respectively . Kinetics of the in vivo holoenzyme formation can be followed by an enzyme-electrochemical method developed by us . The rate constants for the reactions of apoenzyme with PQQ (k(f,PQQ)) and with Mg(2+) (k(f,Mg)) were determined to be 3.8+/-0.4x10(4) M(-1).s(-1) and 4 . 1+/-0.9 M(-1).s(-1) respectively . Equilibrium constants for the binding of apoenzyme to PQQ and Mg(2+) were determined as the dissociation constants K(d,PQQ(Mg)) and K(d,Mg) to be 1.0+/-0.1 nM and 0.14+/-0.01 mM respectively . The dissociation constants for Ca(2+) were also determined . The holoenzyme, once formed in E . coli, returns gradually to the apoenzyme in the absence of PQQ and/or Mg(2+) in solution . EDTA was effective to remove Mg(2+) from the enzyme in the cells to deactivate the enzyme completely, while PQQ remained in the E . coli cells.

Biochem J, 2000 Sep 15, 350 Pt 3, 815 - 22
Photoaffinity labelling with P3-(4-azidoanilido)uridine 5'-triphosphate identifies gpi3p as the UDP-GlcNAc-binding subunit of the enzyme that catalyses formation of GlcNAc-phosphatidylinositol, the first glycolipid intermediate in glycosylphosphatidylinositol synthesis; Kostova Z et al.; Glycosylphosphatidylinositols (GPIs) are made by all eukaryotes . The first step in their synthesis is the transfer of GlcNAc from UDP-GlcNAc to phosphatidylinositol (PI) . Four proteins in mammals and at least three in yeast make up a complex that carries out this reaction . Three of the proteins are highly conserved between yeast and mammals: the Gpi1 protein, the Pig-C/Gpi2 protein and the Pig-A/Gpi3 protein . The function of the individual subunits is not known, but of the three, the Pig-A/Gpi3 proteins resemble members of a large family of nucleotide-sugar-utilizing glycosyltransferases . To establish whether Gpi3p is the UDP-GlcNAc-binding subunit of the yeast GlcNAc-PI synthetic complex, we tested its ability to become cross-linked to the photoactivatable substrate analogue P(3)-(4-azidoanilido)-uridine 5'-triphosphate (AAUTP) . We report that Gpi3p bearing the FLAG epitope at its C-terminus becomes cross-linked to AAUTP{alpha-(32)P}, but that Gpi2p-FLAG does not . Furthermore, Gpi3p-FLAG expressed in Escherichia coli is also cross-linked . These results indicate that Gpi3p is the UDP-GlcNAc-binding and probable catalytic subunit of the GlcNAc-PI synthetic complex.

J Mol Biol, 2000 Sep 15, 302(2), 411 - 26
Mutational analysis of Escherichia coli PepA, a multifunctional DNA-binding aminopeptidase; Charlier D et al.; Escherichia coli PepA is a hexameric aminopeptidase that is also endowed with a DNA-binding activity that functions in transcription control and plasmid dimer resolution . To gain further insight into the functioning of PepA, mutants were selected on the basis of reduced repressibility of a genomic carA-lacZ fusion and studied for the various cellular processes requiring PepA, i.e . repression of the carAB operon, autoregulation, resolution of ColE1 multimers, and peptide proteolysis . The methylation status of the carAB control region was analysed in several pepA mutants and purified proteins were assayed in vitro for car operator DNA binding . This study provides a critical test of predictions advanced on the basis of the structural analysis of PepA and demonstrates the importance for DNA binding of several secondary structural elements in the N-terminal domain and near the very C terminus . By analysis of single amino acid substitutions, we could distinguish the mode of PepA action in car regulation from its action in plasmid resolution . We demonstrate that mere binding of PepA to the car control region is not sufficient to explain its role in pyrimidine-specific regulation; protein-protein interactions appear to play an important role in transcriptional repression . The multifunctional character of PepA and of an increasing number of transcriptional regulators that combine catalytic and regulatory properties, of which several participate in the metabolism of arginine and of the pyrimidines, suggests that enzymes and DNA (RNA) binding proteins fulfilling an essential primeval function may have been recruited in evolution to fulfil an additional regulatory task .

Mutagenesis, 2000 Sep, 15(5), 379 - 83
Mutation frequency in the lacI gene of liver DNA from lambda/lacI transgenic mice following the interaction of PCBs with iron causing hepatic cancer and porphyria; Davies R et al.; The synergistic interaction of iron overload, AHR: genotype and exposure to a mixture of polychlorinated biphenyls (PCBs) (Aroclor 1254) in mice leads to hepatic porphyria, oxidative DNA damage and cancer . In humans, hepatocellular cancer is associated with iron overload and hepatic porphyria . Neither the mechanism of hepatic carcinogenesis induced by PCBs in rodents nor hepatocellular cancer induced by iron and porphyria in humans are understood . To test the hypothesis that chronic interaction of iron and PCBs may induce mutagenesis in liver DNA, lambda /lacI transgenic C57BL/6 mice were given iron dextran (600 mg iron/kg) and then administered Aroclor 1254 in the diet (0.01%) for 7 weeks . Hepatic iron, CYP1A activity and CYP1A1/1A2 protein were elevated >20-fold as a result of iron or Aroclor treatments, respectively, but porphyria with associated histological changes only developed in the combined iron/Aroclor treatment group . lambda/lacI shuttle vectors were isolated from liver genomic DNA and the mutational frequency (MF) in the lacI gene determined . Both iron and Aroclor treatments alone caused significant small increases in MF (1.5- and 1.4-fold, respectively), however, the MF following the combined iron and Aroclor treatment (1 . 6-fold) was not greater than the additive effects . In contrast, the MF was significantly elevated (4.7-fold) in liver DNA of mice 2 weeks following five daily doses of N-nitrosodimethylamine (4 mg/kg) . These studies demonstrate that neither PCBs nor iron overload caused marked point mutations even in a combination regime that leads to oxidative damage and cancer . There was also no strong evidence either that porphyrins or chronic CYP1A1 expression induced by the PCBs after this period caused marked point mutagens or simple deletions . Hence, to understand the PCBs-iron synergism more complex scenarios than point mutations or simple deletions must be invoked.

Bioorg Med Chem Lett, 2000 Aug 21, 10(16), 1835 - 7
Synthesis and cytotoxic activity of a glucuronylated prodrug of nornitrogen mustard; Papot S et al.; A new glucuronylated prodrug of nornitrogen mustard, incorporating the same spacer group as the doxorubicin prodrug HMR 1826, has been prepared . Upon exposure to E . coli beta-glucuronidase, fast hydrolysis occurs but a lower cytotoxicity against LoVo cancer cells is observed compared to the nornitrogen mustard alone . This is explained by cyclization of the intermediate carbamic acid to the inactive chloroethyl oxazolidinone.

Med J Malaysia, 1997 Dec, 52(4), 412 - 5
Parasitic infections among aborigine children at Post Brooke, Kelantan, Malaysia; Rahmah N et al.; This study investigated the prevalence of parasitic infections among aborigine children at Post Brooke, Kelantan . Eighty-four formalin-fixed specimens and 78 PVA-fixed specimens were obtained . 79.8% and 35.9% of the samples were positive for helminth ova and protozoa respectively . The parasites detected (single plus mixed infections) were A . lumbricoides (50/84, 59.5%), T . trichiura (35/84, 41.7%), hookworm (5/84, 6.0%), S stercoralis (1/84, 1.2%), G . intestinalis (18/78, 23.1%), E . histolytica (7/78, 9.0%) and E . coli (7/78, 9.0%) . Two hundred thick blood film examinations detected only one case of Plasmodium falciparum infection . A high prevalence of intestinal parasitic infections among the children at Post Brooke was demonstrated in this study; thus there is an urgent need to improve the hygiene, education and living standards of this population.

Int Immunol, 2000 Sep, 12(9), 1255 - 65
Reconstitution of conformationally dependent epitopes on the N-terminal extracellular domain of the human muscle acetylcholine receptor alpha subunit expressed in Escherichia coli: implications for myasthenia gravis therapeutic approaches; Tsouloufis T et al.; Myasthenia gravis (MG) is an autoimmune disease, caused by autoantibodies against the muscle acetylcholine receptor (AChR), an oligomeric transmembrane glycoprotein composed of alpha(2)beta gamma delta subunits . The alpha subunit carries in its N-terminal extracellular domain the main immunogenic region (MIR), a group of conformationally dependent epitopes that seems to be a major target for the anti-AChR antibodies in MG patients . Detailed epitope studies on pathogenic anti-AChR antibodies have been hindered because the binding of most of these antibodies is conformationally dependent, which precludes the use of denatured AChR fragments . The N-terminal extracellular fragment, residues 1-207, of the human AChR alpha subunit was expressed in Escherichia coli in a denatured form, solubilized in a guanidinium hydrochloride-containing buffer, purified, and renatured using a refolding approach which employs a detergent and a cyclodextrin as 'artificial chaperones' . Compared with the non-refolded protein, the refolded molecule exhibited a dramatic improvement in terms of the binding of all anti-MIR mAb tested . Anti-MIR mAb that normally bind weakly to the denatured alpha subunit bound approximately 30-100 times better to the refolded polypeptide and other anti-MIR mAb that bind exclusively to completely conformationally dependent epitopes also bound quite efficiently . These results, in addition to providing a means for the thorough investigation of the antigenic structure of the AChR, show that the conformationally dependent MIR epitopes do not require the participation of the oligosaccharide moiety of the alpha subunit nor the contribution of neighboring subunits for antibody binding . Such AChR fragments may be used in structural studies of the AChR autoantigen, and should prove valuable in the understanding and development of therapeutic approaches for MG.

J Mol Biol, 2000 Sep 1, 301(5), 1257 - 66
"Open" structures of MurD: domain movements and structural similarities with folylpolyglutamate synthetase; Bertrand JA et al.; UDP-N-acetylmuramoyl-l-alanine:d-glutamate (MurD) ligase catalyses the addition of d-glutamate to the nucleotide precursor UDP-N-acetylmuramoyl-l-alanine (UMA) . The crystal structures of Escherichia coli in the substrate-free form and MurD complexed with UMA have been determined at 2.4 A and 1.88 A resolution, respectively . The MurD structure comprises three domains each of a topology reminiscent of nucleotide-binding folds . In the two structures the C-terminal domain undergoes a large rigid-body rotation away from the N-terminal and central domains . These two "open" structures were compared with the four published "closed" structures of MurD . In addition the comparison reveals which regions are affected by the binding of UMA, ATP and d-Glu . Also we compare and discuss two structurally characterized enzymes which belong to the same ligase superfamily: MurD and folylpolyglutamate synthetase (FGS) . The analysis allows the identification of key residues involved in the reaction mechanism of FGS . The determination of the two "open" conformation structures represents a new step towards the complete elucidation of the enzymatic mechanism of the MurD ligase .

J Mol Biol, 2000 Aug 25, 301(4), 1003 - 17
NMR solution structure of hPar14 reveals similarity to the peptidyl prolyl cis/trans isomerase domain of the mitotic regulator hPin1 but indicates a different functionality of the protein; Sekerina E et al.; The 131-amino acid residue parvulin-like human peptidyl-prolyl cis/trans isomerase (PPIase) hPar14 was shown to exhibit sequence similarity to the regulator enzyme for cell cycle transitions human hPin1, but specificity for catalyzing pSer(Thr)-Pro cis/trans isomerizations was lacking . To determine the solution structure of hPar14 the (1)H, (13)C, and (15)N chemical shifts of this protein have been assigned using heteronuclear two and three-dimensional NMR experiments on unlabeled and uniformly (15)N/(13)C-labeled recombinant protein isolated from Escherichia coli cells that overexpress the protein . The chemical shift assignments were used to interpret the NOE data, which resulted in a total of 1042 NOE restraints . The NOE restraints were used along with 71 dihedral angle restraints and 38 hydrogen bonding restraints to produce 50 low-energy structures . The hPar14 folds into a betaalpha(3)betaalphabeta(2) structure, and contains an unstructured 35-amino acid basic tail N-terminal to the catalytic core that replaces the WW domain of hPin1 homologs . The three-dimensional structures of hPar14 and the PPIase domain of human hPin1 reveal a high degree of conservation . The root-mean-square deviations of the mean atomic coordinates of the heavy atoms of the backbone between residues 38 to 45, 50 to 58, 64 to 70, 81 to 86, 115 to 119 and 122 to 128 of hPar14 were 0.81(+/-0.07) A . The hPar14 model structure provides insight into how this class of PPIases may select preferential secondary catalytic sites, and also allows identification of a putative DNA-binding motif in parvulin-like PPIases .

J Mol Biol, 2000 Aug 25, 301(4), 851 - 67
DNA polymerase lambda (Pol lambda), a novel eukaryotic DNA polymerase with a potential role in meiosis; Garcia-Diaz M et al.; A new gene (POLL) encoding a novel DNA polymerase (Pol lambda) has been identified at mouse chromosome 19 . Murine Pol lambda, consisting of 573 amino acid residues, has a 32% identity to Pol beta, involved in nuclear DNA repair in eukaryotic cells . It is interesting that Pol lambda contains all the critical residues involved in DNA binding, nucleotide binding and selection, and catalysis of DNA polymerization, that are conserved in Pol beta and other DNA polymerases belonging to family X . Murine Pol lambda, overproduced in Escherichia coli, displayed intrinsic DNA polymerase activity when assessed by in situ gel analysis . Pol lambda also conserves the critical residues of Pol beta required for its intrinsic deoxyribose phosphate lyase (dRPase) activity . The first 230 amino acid residues of Pol lambda, that have no counterpart in Pol beta, contain a BRCT domain, present in a variety of cell-cycle check-point control proteins responsive to DNA damage and proteins involved in DNA repair . Northern blotting, in situ hybridization analysis and immunostaining showed high levels of Pol lambda specifically expressed in testis, being developmentally regulated and mainly associated to pachytene spermatocytes . These first evidences, although indirect, suggest a potential role of Pol lambda in DNA repair synthesis associated with meiosis .

J Mol Biol, 2000 Aug 25, 301(4), 839 - 50
Functional interactions of Mycobacterium leprae RuvA with Escherichia coli RuvB and RuvC on holliday junctions; Arenas-Licea J et al.; The Mycobacterium leprae RuvA homologue (MlRuvA) was over-expressed in Escherichia coli and purified to homogeneity . The DNA-binding specificity and the functional interactions of MlRuvA with E . coli RuvB and RuvC (EcRuvB and EcRuvC) were examined using synthetic Holliday junctions . MlRuvA bound specifically to Holliday junctions and produced similar band-shift patterns as EcRuvA . Moreover, MlRuvA formed functional DNA helicase and branch-migration enzymes with EcRuvB, although the heterologous enzyme had a lower efficiency . These results demonstrate that the RuvA homologue of M . leprae is a functional branch-migration subunit.Whereas MlRuvA promoted branch-migration in combination with EcRuvB, it was unable to stimulate branch-migration-dependent resolution in a RuvABC complex . The inability to stimulate RuvC was not due to its failure to form heterologous RuvABC complexes on junctions, since such complexes were detected by co-immunoprecipitation . Most likely, the stability of the heterologous RuvABC complex and, possibly, the interactions between RuvA and RuvC were impaired, as gel-shift experiments failed to show mixed MlRuvA-EcRuvC-junction complexes . These results demonstrate that branch-migration per se and the assembly of a RuvABC complex on the Holliday junction are insufficient for RuvAB-dependent resolution of the junction by RuvC, suggesting that specific and intimate interactions between all three proteins are required for the function of a RuvABC "resolvasome" .

J Mol Biol, 2000 Aug 25, 301(4), 827 - 37
Identification of residues involved in the specificity and regulation of the highly efficient multisubstrate deoxyribonucleoside kinase from Drosophila melanogaster; Knecht W et al.; In contrast to all known deoxyribonucleoside kinases, a single highly efficient deoxyribonucleoside kinase from Drosophila melanogaster (Dm-dNK) is able to phosphorylate all precursor nucleosides for DNA synthesis . Dm-dNK was mutated in vitro by high-frequency random mutagenesis, expressed in the thymidine kinase-deficient Escherichia coli strain KY895 and clones were selected for sensitivity to the nucleoside analogs 1-beta-d-arabinofuranosylcytosine (AraC, Cytarabine), 3'-azido-2', 3'-dideoxythymidine (AZT, Zidovudine, Retrovir, 2', 3'-dideoxyadenosine (ddA) and 2',3'-dideoxycytidine (ddC, Zalcitabine, Hivid . Thirteen mutants with increased sensitivity compared to the wild-type Dm-dNK were isolated from a relatively small pool of less than 10,000 clones . Eight mutant Dm-dNKs increased the sensitivity of KY895 to more than one analog, and two of these mutants even to all four nucleoside analogs . Surprisingly, the mutations did not map to the five regions which are highly conserved among deoxyribonucleoside kinases . The molecular background of improved sensitivity was characterized for the double-mutant MuD (N45D, N64D), where the LD(100) value of transformed KY895 decreased 316-fold for AZT and more than 11-fold for ddC when compared to wild-type Dm-dNK . Purified recombinant MuD displayed higher K(m) values for the native substrates than wild-type Dm-dNK and the V(max) values were substantially lower . On the other hand, the K(m) and V(max) values for AZT and the K(m) value for ddC were nearly unchanged between MuD and wild-type Dm-dNK . Additionally, a decrease in feedback inhibition of MuD by thymidine triphosphate (TTP) was found . This study demonstrates how high-frequency mutagenesis combined with a parallel selection for desired properties provides an insight into the structure-function relationships of the multisubstrate kinase from D . melanogaster . At the same time these mutant enzymes exhibit properties useful in biotechnological and medical applications .

J Mol Biol, 2000 Aug 25, 301(4), 817 - 26
A ribozyme derived from the catalytic subunit of RNase P from Escherichia coli is highly effective in inhibiting replication of herpes simplex virus 1; Trang P et al.; A sequence-specific ribozyme (M1GS RNA) derived from the catalytic RNA subunit of RNase P from Escherichia coli was used to target the mRNA encoding human herpes simplex virus 1 (HSV-1) major transcription activator, ICP4 . A reduction of more than 80% in the expression level of ICP4 and a reduction of about 1000-fold in viral growth were observed in cells that stably expressed the ribozyme . In contrast, a reduction of less than 10 % in ICP4 expression and viral growth was observed in cells that either did not express the ribozyme or produced a catalytically inactive ribozyme mutant . Thus, M1GS ribozyme is highly effective in inhibiting HSV-1 growth and can be used as a general gene-targeting agent for anti-HSV applications .

J Mol Biol, 2000 Aug 18, 301(3), 737 - 47
Multiple roles of prolyl residues in structure and folding; Eyles SJ et al.; To explore the ways that proline residues may influence the conformational options of a polypeptide backbone, we have characterized Pro-->Ala mutants of cellular retinoic acid-binding protein I (CRABP I) . While all three Xaa-Pro bonds are in the trans conformation in the native protein and the equilibrium stability of each mutant is similar to that of the parent protein, each has distinct effects on folding and unfolding kinetics . The mutation of Pro105 does not alter the kinetics of folding of CRABP I, which indicates that the flexible loop containing this residue is passive in the folding process . By contrast, replacement of Pro85 by Ala abolishes the observable slow phase of folding, revealing that correct configuration of the 84-85 peptide bond is prerequisite to productive folding . Substitution of Pro39 by Ala yields a protein that folds and unfolds more slowly . Removal of the conformational constraint imposed by the proline ring likely raises the transition state barrier by increasing the entropic cost of narrowing the conformational ensemble . Additionally, the Pro-->Ala mutation removes a helix-termination signal that is important for efficient folding to the native state .

Nat Struct Biol, 2000 Sep, 7(9), 762 - 5
The structure of the pro-apoptotic protease granzyme B reveals the molecular determinants of its specificity; Waugh SM et al.; Granzyme B is a serine protease of the chymotrypsin fold that mediates cell death by cytotoxic lymphocytes . It is a processing enzyme, requiring extended peptide substrates containing an Asp residue . The determinants that allow for this substrate specificity are revealed in the three-dimensional structure of granzyme B in complex with a macromolecular inhibitor . The primary specificity for Asp occurs through a side-on interaction with Arg 226, a buried Arg side chain of granzyme B . An additional nine amino acids make contact with the substrate and define the granzyme B extended substrate specificity profile . The substrate determinants found in this structure are shared by other members of this protein class and help to reveal the properties that define substrate specificity.

Nat Struct Biol, 2000 Sep, 7(9), 735 - 9
Identifying conformational changes with site-directed spin labeling; Hubbell WL et al.; Site-direct spin labeling combined with electron paramagnetic resonance (EPR) spectroscopy is a powerful tool for detecting structural changes in proteins . This review provides examples that illustrate strategies for interpreting the data in terms of specific rearrangements in secondary and tertiary structure . The changes in the mobility and solvent accessibility of the spin label side chains, and in the distances between spin labels, report (i) rigid body motions of alpha-helices and beta-strands (ii) relative movements of domains and (iii) changes in secondary structure . Such events can be monitored in the millisecond time-scale, making it possible to follow structural changes during function . There is no upper limit to the size of proteins that can be investigated, and only 50-100 picomoles of protein are required . These features make site-directed spin labeling an attractive approach for the study of structure and dynamics in a wide range of systems.

Proteins, 2000 Nov 1, 41(2), 238 - 47
The synthetase domains of cobalamin biosynthesis amidotransferases cobB and cobQ belong to a new family of ATP-dependent amidoligases, related to dethiobiotin synthetase; Galperin MY et al.; Phosphotransacetylases of Escherichia coli and several other bacteria contain an additional 350-aa N-terminal fragment that is not required for phosphotransacetylase activity . Sequence analysis of this fragment revealed that it is closely related to a family of ATP-dependent enzymes that also includes dethiobiotin synthetase and the synthetase domains of two amidotransferases involved in cobalamin biosynthesis, cobyrinic acid a,c-diamide synthase (CobB) and cobyric acid synthase (CobQ) . Further database searches showed that this enzyme family is also related to the MinD family of ATPases involved in regulation of cell division in bacteria and archaea . Analysis of sequence conservation in the members of this enzyme family using the structure of dethiobiotin synthetase active site as a guide allowed us to suggest a model for the interaction of CobB and CobQ with their respective substrates . CobB and CobQ were also found to contain unusual Triad family (class I) glutamine amidotransferase domains with conserved Cys and His residues, but lacking the Glu residue of the catalytic triad . These results should help in understanding the enzymology of cobalamin biosynthesis and in resolving the role of phosphotransacetylase in regulation of the carbon flow to and from acetate.

Proteins, 2000 Nov 1, 41(2), 164 - 72
Glucose-6-phosphate transporter and receptor functions of the glucose 6-phosphatase system analyzed from a consensus defined by multiple alignments; Mechin MC et al.; The cDNA encoding the protein (P46) that is mutated in glycogen storage disease type-1b (GSD-1b) has been previously cloned by homology with bacterial sequences of the uhp (upper hexose phosphate) system . Hydropathic profiles, transmembrane-prediction analysis, and a multiple alignment of 14 sequences related to P46 (with percentage of identity around 30%) allowed to identify two large domains in the proteins linked by a large variable loop . Highly conserved transmembrane (TM) segments, TM1 and TM4 in the first domain and TM5 in the second one, were identified almost in all the integral proteins related to P46 . The multiple alignment allowed definition of a consensus involving the 14 sequences related to P46 . The detailed comparison of the consensus with the UhpT (the bacterial G6P transporter) and with UhpC (the bacterial G6P receptor) sequences reveals that the P46 protein could carry both G6P receptor and transporter functions.

Appl Environ Microbiol, 2000 Sep, 66(9), 3960 - 5
Overexpression of protein disulfide isomerase DsbC stabilizes multiple-disulfide-bonded recombinant protein produced and transported to the periplasm in Escherichia coli; Kurokawa Y et al.; Dsb proteins (DsbA, DsbB, DsbC, and DsbD) catalyze formation and isomerization of protein disulfide bonds in the periplasm of Escherichia coli . By using a set of Dsb coexpression plasmids constructed recently, we analyzed the effects of Dsb overexpression on production of horseradish peroxidase (HRP) isozyme C that contains complex disulfide bonds and tends to aggregate when produced in E . coli . When transported to the periplasm, HRP was unstable but was markedly stabilized upon simultaneous overexpression of the set of Dsb proteins (DsbABCD) . Whereas total HRP production increased severalfold upon overexpression of at least disulfide-bonded isomerase DsbC, maximum transport of HRP to the periplasm seemed to require overexpression of all DsbABCD proteins, suggesting that excess Dsb proteins exert synergistic effects in assisting folding and transport of HRP . Periplasmic production of HRP also increased when calcium, thought to play an essential role in folding of nascent HRP polypeptide, was added to the medium with or without Dsb overexpression . These results suggest that Dsb proteins and calcium play distinct roles in periplasmic production of HRP, presumably through facilitating correct folding . The present Dsb expression plasmids should be useful in assessing and dissecting periplasmic production of proteins that contain multiple disulfide bonds in E . coli.

Appl Environ Microbiol, 2000 Sep, 66(9), 3856 - 67
Characterization of the minimal replicon of a cryptic Deinococcus radiodurans SARK plasmid and development of versatile Escherichia coli-D . radiodurans shuttle vectors; Meima R et al.; The nucleotide sequence of a 12-kb fragment of the cryptic Deinococcus radiodurans SARK plasmid pUE10 was determined, in order to direct the development of small, versatile cloning systems for Deinococcus . Annotation of the sequence revealed 12 possible open reading frames . Among these are the repU and resU genes, the predicted products of which share similarity with replication proteins and site-specific resolvases, respectively . The products of both genes were demonstrated using an overexpression system in Escherichia coli . RepU was found to be required for replication, and ResU was found to be required for stable maintenance of pUE10 derivatives . Gel shift analysis using purified His-tagged RepU identified putative binding sites and suggested that RepU may be involved in both replication initiation and autoregulation of repU expression . In addition, a gene encoding a possible antirestriction protein was found, which was shown to be required for high transformation frequencies . The arrangement of the replication region and putative replication genes for this plasmid from D . radiodurans strain SARK is similar to that for plasmids found in Thermus but not to that for the 45.7-kb plasmid found in D . radiodurans strain R1 . The minimal region required for autonomous replication in D . radiodurans was determined by sequential deletion of segments from the 12-kb fragment . The resulting minimal replicon, which consists of approximately 2.6 kb, was used for the construction of a shuttle vector for E . coli and D . radiodurans . This vector, pRAD1, is a convenient general-purpose cloning vector . In addition, pRAD1 was used to generate a promoter probe vector, and a plasmid containing lacZ and a Deinococcus promoter was shown to efficiently express LacZ.

Crit Care Med, 2000 Aug, 28(8), 2943 - 8
Leukocyte-independent plasma extravasation during endotoxemia; Walther A et al.; OBJECTIVES: To determine the meaning of leukocyte-endothelial interactions for the development of endotoxin-induced vascular leakage . DESIGN: Randomized, blinded, controlled trial . SETTING: Experimental laboratory . SUBJECTS: Twenty-four male Wistar rats . INTERVENTIONS: After application of fucoidin to prevent leukocyte rolling and adherence (25 mg/kg; n = 8; fucoidin/LPS group) or saline 0.9% (n = 8; LPS group), animals were given an intravenous infusion of endotoxin (Escherichia coli lipopolysaccharide 026:B6; 2 mg/kg/hr) over 120 mins . Animals in the control group (n = 8) received an equivalent volume of saline 0.9% . MEASUREMENTS AND MAIN RESULTS: Leukocyte rolling and leukocyte adherence, red cell velocity, vessel diameters, venular wall shear rate, volumetric blood flow, and macromolecular leakage were determined in mesenteric postcapillary venules using in vivo videomicroscopy at baseline, 60 mins, and 120 mins after start of a continuous endotoxin infusion . Fucoidin prevented leukocyte rolling (baseline, 3+/-2 rollers; 120 mins, 3+/-1 rollers; not significant vs . baseline; p < .01 vs . LPS group) and reduced the adherence of leukocytes at baseline and during endotoxemia and showed only a slight increase in adherent leukocytes (baseline, 100+/-38 cells/mm2; 120 mins, 244+/-68 cells/mm2; p < .05 vs . baseline; p < .01 vs . LPS group) . In the LPS group, endotoxin exposure induced a marked increase in adherent leukocytes (baseline, 248+/-24 cells/mm2; 120 mins, 560+/-57 cells/mm2; p < .01) . Leukocyte adherence in control animals (control group) did not increase significantly . Macromolecular leakage, expressed as the ratio of perivenular to intravenular fluorescence intensity after injection of fluorescence-labeled albumin, increased from 0.16+/-0.03 to 0.49+/-0.04 (p < .01 vs . baseline; p < .05 vs . control) during the infusion of endotoxin in the LPS group . Fucoidin application did not diminish the extravasation of albumin (baseline, 0.09+/-0.03; 120 mins, 0.61+/-0.10; p < .01 vs . baseline; p < .01 vs . control) . CONCLUSIONS: These results demonstrate that despite a significant reduction of adherent leukocytes to the endothelium by fucoidin, there is no reduction in macromolecular leakage, indicating that leukocyte-endothelial interactions only play a minor role for the development of macromolecular leakage and microvascular damage in the early phase of endotoxemia.

Crit Care Med, 2000 Aug, 28(8), 2851 - 7
Impact of endothelin-1 in endotoxin-induced pulmonary vascular reactions; Schmeck J et al.; OBJECTIVES: Elevated endothelin-1 (ET-1) levels have been detected during sepsis . The aim of the study was to examine the role of thromboxane A2 (TXA2) and ET-1 in pulmonary vascular reactions after endotoxin (LPS) challenge . DESIGN: Prospective experimental study in rabbits . SETTING: Experimental laboratory in a university teaching hospital . SUBJECTS: Twenty-four adult rabbits of either sex . INTERVENTIONS: Experiments were performed on 30 isolated and ventilated rabbit lungs, which were perfused with a saline solution containing 10% autologous blood . MEASUREMENTS AND MAIN RESULTS: Pulmonary arterial pressure and lung weight gain were continuously registered . Perfusate samples were drawn intermittently to determine ET-1, TXA2, and prostacyclin (PGI2) concentrations . LPS isolated from Escherichia coli (0.5 mg/mL; n = 6) was added to the perfusate . A marked pulmonary arterial pressure increase followed by massive edema formation after 60 mins was observed after LPS injection . At the same time, elevated TXA2 and PGI2 levels in the perfusate were measured . ET-1 was detected 30 mins after LPS infusion (13.4+/-2.6 fmol/L) . Pretreatment with the ET(A) receptor antagonist LU135252 (10(-6) M; n = 6) almost completely suppressed the pressure reaction after endotoxin injection (p < .01 at 50 and 60 mins) and reduced edema formation (p < .05) . The cyclooxygenase inhibitor diclofenac (10 microg/mL; n = 6) was as effective as LU135252 in preventing vascular reactions after LPS injection . CONCLUSIONS: Pretreatment with the ET(A) receptor antagonist LU135252 and the cyclooxygenase inhibitor diclofenac reduced pulmonary vascular reactions after LPS challenge . Based on the current data, we conclude that the pulmonary arterial pressure increase and edema formation after LPS injection are related to an ET-1- and TXA2-dependent mechanism.

Crit Care Med, 2000 Aug, 28(8), 2685 - 9
Interleukin-10 gene transfer improves the survival rate of mice inoculated with Escherichia coli; Takakuwa T et al.; OBJECTIVE: To determine whether administration of recombinant adenovirus vectors encoding the interleukin (IL)-10 protein (AxCAmIL-10) decreases the mortality of septic mice . DESIGN: Prospective, randomized, controlled study . SETTING: University research laboratory . SUBJECTS: Adult male C57B/6 mice . INTERVENTIONS: Untreated mice and those injected intraperitoneally with 1 x 10(9) pfu of AxCAmIL-10 were used as control 1 and 2, respectively . Double-capsules without Escherichia coli were intraperitoneally embedded in another group (control 3) . Mice embedded with capsules containing E . coli were divided into the following groups: simultaneous administration of 0.5 mL of saline (group 1), and administration of AxCAmIL-10 3 hrs before embedding (group 2) or 1 hr after embedding (group 3) . Histopathologic changes together with expression concentrations of IL-10 and tumor necrosis factor (TNF) in various organs and plasma were examined 18 hrs after each treatment . Observation periods were 5-8 days . Survival rates were compared between these groups . MEASUREMENTS AND MAIN RESULTS: The plasma IL-10 concentrations were increased in control 2, group 2, and group 3 but not in control 1, control 3, or group 1, indicating successful adenovirus gene transfer . Plasma TNF values were significantly reduced in groups 2 and 3 as compared with group 1, with no significant differences in endotoxin concentrations . Survival rates were significantly better in groups 2 and 3 than in group 1 (p < .05) . CONCLUSION: These findings suggested that IL-10 has a favorable effect on survival of septic mice via inhibition of TNF production or endotoxin stimulation.

J Med Microbiol, 2000 Sep, 49(9), 823 - 6
Unidentified serogroups of enteropathogenic Escherichia coli (EPEC) associated with diarrhoea in infants in Londrina, Parana, Brazil; Vidotto MC et al.; Digoxigenin-labelled DNA probes were used to characterise enteropathogenic Escherichia coli (EPEC) isolated in Londrina (Brazil) from faeces samples of 102 children with diarrhoea, and the results were compared with those obtained by serogrouping and adherence to HEp-2 cells . The probes employed detect the gene coding EPEC adherence factor (EAF) and the virulence genes for bundle-forming pilus (bfp) and entero-attaching-effacing (eae) factor . Twenty-one isolates hybridised with at least one probe, and 11 of them were classified as typical EPEC because they hybridised with all three probes, showed a pattern of localised adherence (LA) and carried no genes for enterotoxins (ST and LT) or invasion as detected by PCR . Six of the typical EPEC strains belonged to the classical serotype 0119:H6 and one to O111:H6; O antigens could not be determined in four strains with antisera against 01-0173 . All typical EPEC strains carried a 70-MDa plasmid plus two other large plasmids . These data showed that typical EPEC virulence traits may be found in strains not belonging to classical serogroups/serotypes and that molecular identification is required for studying the epidemiology of diarrhoea in children.

Plant Cell Physiol, 2000 Jul, 41(7), 874 - 80
Organ and cellular localization of asparagine synthetase in rice plants; Nakano K et al.; DNA gel blot analysis suggested that asparagine synthetase (AS; EC 6.3.5.4) occurred as a single gene in rice . A fusion protein consisting of 17 kDa tagged-region from pET32a(+) expression plasmid and 42 kDa N-terminal region of rice AS was first expressed in Escherichia coli . The resulting polypeptide was purified and a mono-specific antibody for rice AS was prepared after affinity-purification with the antigen . Immunoblotting revealed a high content of AS protein in the leaf sheath at the second position from the fully expanded top leaf and in grains at the middle stage of ripening . Accumulation of mRNA for AS was also observed in these organs . During the ripening of the spikelets, the AS protein contents increased during the first 21 days after flowering, then declined rapidly . Immunolocalization analysis revealed signals for AS protein in the companion cells of vascular bundles of leaf sheath and phloem-parenchyma cells, nucellar projection, and nucellar epidermis of dorsal vascular bundles of grains.

Nippon Hinyokika Gakkai Zasshi, 2000 Jul-Aug, 91(7-8), 595 - 8
{A case of embolization to a large polycystic kidney with infection}; Takaiwa M et al.; 75 year old female who was hospitalized for abdominal pain and fever up on 12th May 1998 . She had been followed as a polycystic kidney patient since few years . The swelling of the right kidney and her general condition became gradually worse . On 18th May, the embolization to the right kidney using pure alcohol and gelatin sponge was performed . Within a month, CT scan showed the reduced volume of the right kidney and her blood examination data as well as her general condition became gradually well . And on 17th June, she left our hospital without any complication.

Neuroimmunomodulation, 2000, 8(2), 59 - 69
Blood interleukin-6 and tumor necrosis factor-alpha elevation after intracerebroventricular injection of Escherichia coli endotoxin in the rat is determined by two opposing factors: peripheral induction by LPS transferred from brain to blood and inhibition of peripheral response by a brain-mediated mechanism; Chen G et al.; Following intracerebroventricular injection of LPS in rats, IL-6 and TNF-alpha appear in peripheral blood . To determine whether these changes are mediated by passage of the injected LPS from the brain to the blood, the time course of appearance in blood of bioactive LPS after intracerebroventricular injection was compared with the time course of appearance of IL-6 and of TNF-alpha in blood . Bioactive LPS was detected 30 min after intracerebroventricular injection, the first time interval tested . TNF-alpha appeared in peripheral blood at 30 min, IL-6 at 60 min and both cytokines as well as LPS achieved highest levels at 120 min . To determine pharmacokinetics of LPS transfer from brain to blood more precisely, radioiodinated LPS was injected intracerebroventricularly . (125)I-LPS was detected in blood as early as 5 min after intracerebroventricular injection, reached peak levels at about 2 h, and was transferred from brain to blood at a rate corresponding to bulk flow (% of brain content per min was 1.40 +/- 0.58 and 1.00 +/- 0.21% in series 1 and 2, respectively) . 70.0% of total injected LPS had entered blood by 4 h . However, when administered intravenously (by a programmed pump) at the same rate that it enters the blood after intracerebroventricular injection LPS induced a much greater cytokine response than when given intracerebroventricularly . This paradoxical response was shown in further studies to be due to the simultaneous central inhibitory effect of LPS; coinjection of intracerebroventricular LPS markedly reduced the peripheral cytokine response to intravenous LPS infusion .

J Biochem (Tokyo), 2000 Sep, 128(3), 481 - 91
Common architecture of the primary galactose binding sites of Erythrina corallodendron lectin and heat-labile enterotoxin from Escherichia coli in relation to the binding of branched neolactohexaosylceramide; Teneberg S et al.; The heat-labile enterotoxin from Escherichia coli (LT) is responsible for so-called traveller's diarrhea and is closely related to the cholera toxin (CT) . Toxin binding to GM1 at the epithelial cell surface of the small intestine initiates the subsequent diarrheal disease . However, LT has a broader receptor specificity than CT in that it also binds to N-acetyllactosamine-terminated structures . The unrelated lectin from Erythrina corallodendron (ECorL) shares this latter binding property . The findings that both ECorL and porcine LT (pLT) bind to lactose as well as to neolactotetraosylceramide suggests a common structural theme in their respective primary binding sites . Superimposing the terminal galactose of the lactoses in the respective crystal structures of pLT and ECorL reveals striking structural similarities around the galactose despite the lack of sequence and folding homology, whereas the interactions of the penultimate GlcNAcb3 in the neolactotetraosylceramide differ . The binding of branched neolactohexaosylceramide to either protein reveals an enhanced affinity relative to neolactotetraosylceramide . The b3-linked branch is found to bind to the primary Gal binding pocket of both proteins, whereas the b6-linked branch outside this site provides additional interactions in accordance with the higher binding affinities found for this compound . While the remarkable architectural similarities of the primary galactose binding sites of pLT and ECorL point to a convergent evolution of these subsites, the distinguishing structural features determining the overall carbohydrate specificities are located in extended binding site regions . In pLT, Arg13 is thus found to play a crucial role in enhancing the affinity not only for N-acetyllactosamine-terminated structures but also for GM1 as compared to human LT (hLT) and CT . The physiological relevance of the binding of N-acetyllactosamine-containing glycoconjugates to LT and ECorL is briefly discussed.

J Biochem (Tokyo), 2000 Sep, 128(3), 377 - 81
Ligand-binding properties of annexin from Caenorhabditis elegans (annexin XVI, Nex-1); Satoh A et al.; Annexins are structurally related proteins that bind phospholipids in a calcium-dependent manner . Recently, we showed that annexins IV, V, and VI also bind glycosaminoglycans in a calcium-dependent manner . Annexins are widely distributed from lower to higher eukaryotes, and the nematode Caenorhabditis elegans has been found to contain Nex-1, an annexin homologue . Here, we characterize the ligand-binding properties of Nex-1 using recombinant Nex-1 . Nex-1 binds to liposomes containing phosphatidylserine . The apparent K(d) was calculated by Biacore to be 4.4 nM . Compared to mammalian annexins, the Nex-1 phospholipid-binding specificities were similar whereas the K(d) values were one order of magnitude larger . The Nex-1 glycosaminoglycan-binding specificities were investigated by affinity chromatography and solid-phase assays . Nex-1 binds to heparin, heparan sulfate, and chondroitin sulfate but not to chondroitin and chemically N- or O-desulfated heparin . Besides phospholipids, heparan sulfate and/or chondroitin (sulfate), probably on perlecan, could be endogenous ligands of Nex-1.

J Biochem (Tokyo), 2000 Sep, 128(3), 363 - 9
States of tryptophyl residues and stability of recombinant human matrix metalloproteinase 7 (matrilysin) as examined by fluorescence; Inouye K et al.; States of tryptophyl residues and stability of human matrilysin were studied . The activation energy for the thermal inactivation of matrilysin was determined to be 237 kJ/mol, and 50% of the activity was lost upon incubation at 69 degrees C for 10 min . The activity was increased by adding NaCl, and was doubled with 3 M NaCl . Denaturation of matrilysin by guanidine hydrochloride (GdnHCl) and urea was monitored by fluorescence change of tryptophyl residues . Half of the change was observed at 2.2-2.7 M GdnHCl, whereas no change was observed even with 8 M urea . Half of the inactivation was induced at 0.8 M GndHCl and at 2 M urea . The presence of an inactive intermediate with the same fluorescence spectrum as the native enzyme was suggested in the denaturation . Matrilysin contains four tryptophyls, and their states were examined by fluorescence-quenching with iodide and cesium ions and acrylamide . No tryptophyls in the native enzyme were accessible to I(-) and Cs(+), and 2.4 residues were accessible to acrylamide . Based on the crystallographic study, Trp154 is water-accessible, but it should be in a crevice not to contact with I(-) and Cs(+) . All tryptophyls in the GdnHCl-denatured enzyme were exposed to the quenchers, while a considerable part was inaccessible in the urea-denatured one.

J Biochem (Tokyo), 2000 Sep, 128(3), 355 - 61
Disulfide bonds in rat cutaneous fatty acid-binding protein; Odani S et al.; Unlike other fatty acid-binding proteins, cutaneous (epidermal) fatty acid-binding proteins contain a large number of cysteine residues . The status of the five cysteine residues in rat cutaneous fatty acid-binding protein was examined by chemical and mass-spectrometric analyses . Two disulfide bonds were identified, between Cys-67 and Cys-87, and between Cys-120 and Cys-127, though extent of formation of the first disulfide bond was rather low in another preparation . Cys-43 was free cysteine . Homology modeling study of the protein indicated the close proximity of the sulfur atoms of these cysteine pairs, supporting the presence of the disulfide bonds . These disulfide bonds appear not to be directly involved in fatty acid-binding activity, because a recombinant rat protein expressed in Escherichia coli in which all five cysteines are fully reduced showed fatty acid-binding activity as examined by displacement of a fluorescent fatty acid analog by long-chain fatty acids . However, the fact that the evolutionarily distant shark liver fatty acid-binding protein also has a disulfide bond corresponding to the one between Cys-120 and Cys-127, and that fatty acid-binding proteins play multiple roles suggests that some functions of cutaneous fatty acid-binding protein might be regulated by the cellular redox state through formation and reduction of disulfide bonds . Although we cannot completely exclude the possibility of oxidation during preparation and analysis, it is remarkable that a protein in cytosol under normally reducing conditions appears to contain disulfide bonds.

Protein Eng, 2000 Aug, 13(8), 583 - 8
Construction of a diabody (small recombinant bispecific antibody) using a refolding system; Takemura S et al.; Diabodies are the recombinant bispecific antibodies (BsAbs), constructed from heterogeneous single-chain antibodies . Usually, diabodies have been prepared from bacterial periplasmic fraction using a co-expression vector (i.e . genes encoding two chains were tandemly located under the same promoter) . Some diabodies, however, cannot be expressed as a soluble material owing to inclusion body formation, which limits the utilization of diabodies in various fields . Here we report an improved method for the construction of diabodies using a refolding system . As a model, a bispecific diabody binding to adenocarcinoma-associated antigen MUC1 and to CD3 on T cells was studied . One chain consisted of a VH specific for MUC1 linked to a VL specific for CD3 with a short polypeptide linker (GGGGS) . The second was composed of a VL specific for MUC1 linked to a VH specific for CD3 . The two hetero scFvs were independently obtained from intracellular insoluble fractions of Escherichia coli, purified, mixed stoichiometrically (at an equivalent molar ratio of 1:1) and refolded . The refolded two hetero scFv has a hetero-dimeric structure, with complete specificity for both target cells {i.e . MUC1 positive cells and CD3 positive lymphokine-activated killer cells with a T cell phenotype (T-LAK)} . Evaluation of the in vitro efficacy of T-LAK with the diabody by growth inhibition assay of cancer cells demonstrated maximum growth inhibition of cancer cells to reach approximately 98% at an effector:target ratio (E:T ratio) of 10, almost identical with that with anti-MUC1xanti-CD3 chemically synthesized BsAbs (c-BsAbs) . This is the first report of the construction of a diabody using a refolding system.

Protein Eng, 2000 Aug, 13(8), 575 - 81
Characterization of diphtheria fusion proteins targeted to the human interleukin-3 receptor; Frankel AE et al.; Diphtheria fusion proteins are chimeric proteins consisting of the catalytic and translocation domains of diphtheria toxin (DT(388)) linked through an amide bond to one of a variety of peptide ligands . The ligand targets the molecule to cells and the toxin enters the cell, inactivates protein synthesis and induces cell death . Diphtheria fusion proteins directed to human myeloid leukemic blasts are a novel class of therapeutics for patients with chemotherapy refractory myeloid leukemia . Because of the presence of interleukin-3 (IL3) receptors on myeloid leukemic progenitors and its absence from mature myeloid cells, we synthesized four bacterial expression vectors encoding DT(388) fused to human IL3 . Different molecules were engineered to assess the effects of modifications on yield, purity and potency of product . The constructs differed in the size of the linker peptide between the DT(388) and IL3 domains and in the presence or absence of an oligohistidine tag on the N- or C-terminus . Escherichia coli were transformed and recombinant protein induced and purified from inclusion bodies . Similar final yields of 3-6 mg of purified protein per liter of bacterial culture were obtained with each of the four molecules . Purity ranged from 70 to 90% after partial purification by anion-exchange, size-exclusion chromatography and/or nickel affinity chromatography . Proteins were soluble and stable at 4 degrees C and -80 degrees C in phosphate-buffered saline at 0.03-0.5 mg/ml . The fusion proteins showed predicted molecular weights by SDS-PAGE, HPLC and tandem mass spectrometry and had full ADP-ribosylating activities . Each was immunoreactive with antibodies to DT(388) and IL3 . Each of the fusion proteins with the exception of the one with an N-terminal oligohistidine tag showed full IL3 receptor binding affinity (K:(d) = 3 nM) and potent and selective cytotoxicity to IL3 receptor positive human myeloid leukemia cell lines (IC(50) = 5-10 pM) . In contrast, the N-terminal histidine-tagged fusion protein bound IL3 receptor with a 10-fold lower affinity and was 10-fold less cytotoxic to IL3 receptor positive blasts . Thus, we report a series of novel, biologically active DT(388)IL3 fusion proteins for potential therapy of patients with receptor positive myeloid leukemias.

Protein Eng, 2000 Aug, 13(8), 565 - 74
ScFv multimers of the anti-neuraminidase antibody NC10: shortening of the linker in single-chain Fv fragment assembled in V(L) to V(H) orientation drives the formation of dimers, trimers, tetramers and higher molecular mass multimers; Dolezal O et al.; Synthetic genes encoding single-chain variable fragments (scFvs) of NC10 anti-neuraminidase antibody were constructed by joining the V(L) and V(H) domains with linkers of fifteen, five, four, three, two, one and zero residues . These V(L)-V(H) constructs were expressed in Escherichia coli and the resulting proteins were characterized and compared with the previously characterized NC10 scFv proteins assembled in V(H)-V(L) orientation . Size-exclusion chromatography and electron microscope images of complexes formed between various NC10 scFvs and anti-idiotype Fab' were used to analyse the oligomeric status of these scFvs . The result showed that as the linker length between V(L) and V(H) was reduced, different patterns of oligomerization were observed compared with those with V(H)-V(L) isomers . As was the case for V(H)-V(L) orientation, the scFv-15 V(L)-V(H) protein existed mainly as a monomer whereas dimer (diabody) was a predominant conformation for the scFv-5, scFv-4 and scFv-3 V(L)-V(H) proteins . In contrast to the V(H)-V(L) isomer, direct ligation of V(L) to V(H) led to the formation of predominantly a tetramer (tetrabody) rather than to an expected trimer (triabody) . Furthermore, the transition between dimers and higher order oligomers was not as distinct as for V(H)-V(L) . Thus reducing the linker length in V(L)-V(H) from three to two residues did not precisely dictate a transition between dimers and tetramers . Instead, two-residue as well as one-residue linked scFvs formed a mixture of dimers, trimers and tetramers.

J Biol Chem, 2000 Nov 17, 275(46), 35908 - 13
A sulfenic acid enzyme intermediate is involved in the catalytic mechanism of peptide methionine sulfoxide reductase from Escherichia coli; Boschi-Muller S et al.; Methionine oxidation into methionine sulfoxide is known to be involved in many pathologies and to exert regulatory effects on proteins . This oxidation can be reversed by a ubiquitous monomeric enzyme, the peptide methionine sulfoxide reductase (MsrA), whose activity in vivo requires the thioredoxin-regenerating system . The proposed chemical mechanism of Escherichia coli MsrA involves three Cys residues (positions 51, 198, and 206) . A fourth Cys (position 86) is not important for catalysis . In the absence of a reducing system, 2 mol of methionine are formed per mole of enzyme for wild type and Cys-86 --> Ser mutant MsrA, whereas only 1 mol is formed for mutants in which either Cys-198 or Cys-206 is mutated . Reduction of methionine sulfoxide is shown to proceed through the formation of a sulfenic acid intermediate . This intermediate has been characterized by chemical probes and mass spectrometry analyses . Together, the results support a three-step chemical mechanism in vivo: 1) Cys-51 attacks the sulfur atom of the sulfoxide substrate leading, via a rearrangement, to the formation of a sulfenic acid intermediate on Cys-51 and release of 1 mol of methionine/mol of enzyme; 2) the sulfenic acid is then reduced via a double displacement mechanism involving formation of a disulfide bond between Cys-51 and Cys-198, followed by formation of a disulfide bond between Cys-198 and Cys-206, which liberates Cys-51, and 3) the disulfide bond between Cys-198 and Cys-206 is reduced by thioredoxin-dependent recycling system process.

J Biol Chem, 2000 Nov 17, 275(46), 36007 - 12
Identification of the putrescine recognition site on polyamine transport protein PotE; Kashiwagi K et al.; The PotE protein can catalyze both uptake and excretion of putrescine . The K(m) values of putrescine for uptake and excretion are 1.8 and 73 microm, respectively . Uptake of putrescine is dependent on the membrane potential, whereas excretion involves putrescine-ornithine antiporter activity . Amino acids involved in both activities were identified using mutated PotE proteins . It was found that Cys(62), Trp(201), Trp(292), and Tyr(425) were strongly involved in both activities, and that Tyr(92), Cys(210), Cys(285), and Cys(286) were moderately involved in the activities . Mutations of Tyr(78), Trp(90), and Trp(422) mainly affected uptake activity, and the K(m) values for putrescine uptake by these PotE mutants increased greatly, indicating that these amino acids are involved in the high affinity uptake of putrescine by PotE . Mutations of Lys(301) and Tyr(308) mainly affected excretion activity (putrescine-ornithine antiporter activity), and excretion by these mutants was not stimulated by ornithine, indicating that these amino acids are involved in the recognition of ornithine . It was found that the putrescine and ornithine recognition site on PotE is located at the cytoplasmic surface and the vestibule of the pore consisting of 12 transmembrane segments . Based on the results of competition experiments with various putrescine analogues and the disulfide cross-linking of PotE between cytoplasmic loops and the COOH terminus, a model of the putrescine recognition site on PotE consisting of the identified amino acids is presented.

J Biol Chem, 2000 Nov 17, 275(46), 35978 - 85
Dm1-MMP, a matrix metalloproteinase from Drosophila with a potential role in extracellular matrix remodeling during neural development; Llano E et al.; We have cloned and characterized a cDNA encoding Dm1-MMP, the first matrix metalloproteinase (MMP) identified in Drosophila melanogaster . The isolated cDNA encodes a protein of 541 residues that has a domain organization identical to that of most vertebrate MMPs including a signal sequence, a prodomain with the activation locus, a catalytic domain with a zinc-binding site, and a COOH-terminal hemopexin domain . Northern blot analysis of Dm1-MMP expression in embryonic and larval adult tissues revealed a strong expression level in the developing embryo at 10-22 h, declining thereafter and being undetectable in adults . Western blot analysis confirmed the presence of pro- and active forms of Dm1-MMP in vivo during larval development . In situ hybridization experiments demonstrated that Dm1-MMP is expressed in a segmented pattern in cell clusters at the midline during embryonic stage 12-13, when neurons of the central nervous system start to arise . Recombinant Dm1-MMP produced in Escherichia coli exhibits a potent proteolytic activity against synthetic peptides used for analysis of vertebrate MMPs . This activity is inhibited by tissue inhibitors of metalloproteinases and by synthetic MMP inhibitors such as BB-94 . Furthermore, Dm1-MMP is able to degrade the extracellular matrix and basement membrane proteins fibronectin and type IV collagen . On the basis of these data, together with the predominant expression of Dm1-MMP in embryonic neural cells, we propose that this enzyme may be involved in the extracellular matrix remodeling taking place during the development of the central nervous system in Drosophila.

Virology, 2000 Sep 1, 274(2), 367 - 77
Oligomerization and cell-binding properties of the avian reovirus cell-attachment protein sigmaC; Grande A et al.; Avian reovirus protein sigmaC, the viral cell-attachment protein, is a minor component of the outer-capsid shell of the viral particle that is synthesized in small amounts in infected cells . We cloned the sigmaC-encoding ORF in vector pIL-2f, expressed it in Escherichia coli, and partially purified the resulting recombinant protein from inclusion bodies . Rabbit polyclonal antibodies raised against the recombinant protein specifically recognized the viral polypeptide in ELISA, immunoprecipitation, and Western blotting . To study the oligomerization capacity and cell-binding affinity of protein sigmaC, the sigmaC-encoding ORF was also expressed in chicken embryo fibroblasts (CEFs) and in reticulocyte lysates . In all three systems protein sigmaC is expressed as a multimer with identical electrophoretic mobility to the naturally occurring protein . Cell-binding experiments show that both in vitro and in vivo expressed protein sigmaC display affinity for CEF receptors, and this property is exclusively associated with the oligomeric form of the protein . The fact that incubation of CEF cells with the recombinant protein expressed in bacterial cells completely blocks the binding of purified reovirions indicates both that binding of this protein to cells is specific and saturable, and that reovirions and protein sigmaC bind to the same class of cell receptor . Saturation binding experiments, performed with the recombinant protein expressed in E . coli and with purified reovirions, showed that the number of cellular receptor sites (CRSs) for avian reovirus S1133 is 1.8 x 10(4) per CEF cell, whereas the number of cellular receptor units (CRUs) for sigmaC is 2.2 x 10(5) per CEF cell . These results are consistent with previous reports on the binding of mammalian reoviruses .

Biochem Biophys Res Commun, 2000 Aug 28, 275(2), 601 - 7
Arabidopsis chloroplast chaperonin 10 is a calmodulin-binding protein; Yang T et al.; Calcium regulates diverse cellular activities in plants through the action of calmodulin (CaM) . By using (35)S-labeled CaM to screen an Arabidopsis seedling cDNA expression library, a cDNA designated as AtCh-CPN10 (Arabidopsis thaliana chloroplast chaperonin 10) was cloned . Chloroplast CPN10, a nuclear-encoded protein, is a functional homolog of E . coli GroES . It is believed that CPN60 and CPN10 are involved in the assembly of Rubisco, a key enzyme involved in the photosynthetic pathway . Northern analysis revealed that AtCh-CPN10 is highly expressed in green tissues . The recombinant AtCh-CPN10 binds to CaM in a calcium-dependent manner . Deletion mutants revealed that there is only one CaM-binding site in the last 31 amino acids of the AtCh-CPN10 at the C-terminal end . The CaM-binding region in AtCh-CPN10 has higher homology to other chloroplast CPN10s in comparison to GroES and mitochondrial CPN10s, suggesting that CaM may only bind to chloroplast CPN10s . Furthermore, the results also suggest that the calcium/CaM messenger system is involved in regulating Rubisco assembly in the chloroplast, thereby influencing photosynthesis .

Biochem Biophys Res Commun, 2000 Aug 28, 275(2), 553 - 7
Affinity maturation of natural antibody using a chain shuffling technique and the expression of recombinant antibodies in Escherichia coli; Park SG et al.; The affinity of natural antibody (Ka = 8 x 10(6) M(-1)) recognizing preS1 of hepatitis B virus (HBV) was improved by replacing the heavy (H) chain gene with repertoires of VH genes, obtained from two nonimmunized donors . Two separate clones, 1C2 and 1E4, showed affinities of 2.3 x 10(7) and 5.2 x 10(7) M(-1), which were increased by factors of 2.8 and 6.5, respectively, compared to the parental clone . Recombinant scFvs (rscFvs) were expressed as fusion protein with minor coat protein, pIII, and secreted into medium after 3 h of induction with 1 mM IPTG . The expression level of functional rscFv capable of binding to preS1 reached a peak after 6-10 h (1C2) and 8-10 h (1E4) of IPTG induction, and afterwards decreased gradually . In order to achieve the overexpression of rscFv in E . coli, gene encoding scFv of 1C2 or 1E4 was inserted into pRSET vector . RscFvs were overexpressed as cytoplasmic inclusion bodies in E . coli BL 21 strain, which were denatured and carefully refolded using a continuous dialysis system . The purified recombinant fragments were pure when analyzed by SDS-PAGE and had the predicted size of 34 kDa . Clone 1E4 used the heavy chain gene belonging to family VII and subgroup III . Chain shuffling offers an alternative to random point mutation for affinity maturation of human antibody in vitro .

Plasmid, 2000 Sep, 44(2), 173 - 82
Generation of an improved luciferase reporter gene plasmid that employs a novel mechanism for high-copy replication; Bert AG et al.; We have engineered the reporter gene plasmid pXPG by incorporating a novel high-copy origin of replication and a modified luciferase gene into a pXP1-derived vector that efficiently blocks read-through transcription in eukaryotic cells . pXPG contains the Luc+ luciferase gene derived from pGL3 that lacks a peroxisomal targeting sequence, thereby allowing accumulation of luciferase protein in the cytoplasm rather than subcellular organelles of transfected eukaryotic cells . pXPG has distinct advantages over pGL3, because it contains SV40 polyadenylation signals that appear to be more efficient at blocking read-through transcription than the synthetic polyadenylation signal present in pGL3 . pXPG contains a novel mutation near the origin of replication that increases plasmid copy number in Escherichia coli . This mutation alters the -10 sequence in the RNA II promoter of the ColE1 origin of replication from TAATCT to TAATAT . As this sequence is a closer match to the consensus -10 element, we suggest that the mutation increases copy number by increasing the rate of transcription of the RNA II replication primer . This novel mechanism for increasing copy number may have more widespread applications than the commonly used pUC high-copy origin of replication mutation . Unlike pUC, which reverts to low copy number at 30 degrees C, the pXPG mutation supports a higher copy number at both 37 and 30 degrees C .

Plasmid, 2000 Sep, 44(2), 138 - 51
Construction and characterization of a highly regulable expression vector, pLAC11, and its multipurpose derivatives, pLAC22 and pLAC33; Warren JW et al.; A number of different expression vectors have been developed to facilitate the regulated overproduction of proteins in Escherichia coli and related bacteria . Some of the more popular ones include pKK223-3, pKK233-2, pTrc99A, and the pET family of expression vectors . These vectors were designed to be regulable and can be grown under conditions that repress protein production or under conditions that induce protein production . Unfortunately, however, numerous researchers have found that these vectors produce significant amounts of protein even when grown under repressed conditions . We describe here a new expression vector, pLAC11, which was designed to be more regulable and thus more tightly repressible when grown under repressed conditions . The tight regulation of pLAC11 was achieved by utilizing the O3 auxiliary operator, CAP binding site, promoter, and O1 operator that occur in the wild-type lac control region . The pLAC11 vector can be used to conduct physiologically relevant studies in which the cloned gene is expressed at levels comparable to that obtainable from the chromosomal copy of the gene in question . In experiments in which a bacterial cell contained both a null allele in the chromosome and a second copy of the wild-type allele on pLAC11, we observed that cells grown under repressed conditions exhibited the null phenotype while cells grown under induced conditions exhibited the wild-type phenotype . Two multipurpose derivatives of pLAC11, pLAC22, and pLAC33 have also been constructed to fulfill different experimental needs .

J Mol Biol, 2000 Sep 8, 302(1), 79 - 91
Stabilization of RNA tertiary structure by monovalent cations; Shiman R et al.; The effects of monovalent cations (Li(+), Na(+), K(+), Rb(+), Cs(+), and NH4(+)) on the thermal stability of RNA tertiary structure were investigated by UV melting . We show that with the RNA used here (nucleotides 1051-1108 of Escherichia coli 23 S rRNA with four base substitutions), monovalent cations and Mg(2+) compete in stabilizing the RNA tertiary structure, and that the competition takes place between two boundaries: one where Mg(2+) concentration is zero and the other where it is maximally stabilizing ("saturating") . The pattern of competition is the same for all monovalent cations and depends on the cation's ability to displace Mg(2+) from the RNA, its ability to stabilize tertiary structure in the absence of Mg(2+), and its ability to stabilize tertiary structure at saturating Mg(2+) concentrations . The stabilizing ability of a monovalent cation depends on its unhydrated ionic radius, and at a low monovalent cation concentration and saturating Mg(2+), there is a (calculated) net release of a single monovalent cation/RNA molecule when tertiary structure is denatured . The implications are that under these conditions there is at least one binding site for monovalent cations on the RNA, the site is specifically associated with formation of stable tertiary structure, K(+) is the most effective of the tested cations, and Mg(2+) appears ineffective at this site . At high ionic strength, and in the absence of Mg(2+), stabilization of tertiary structure is still monovalent-cation specific and ionic-radius dependent, but a larger number of cations ( approximately eight) are released upon RNA tertiary structure denaturation, and NH(4)(+) appears to be the most effective cation in stabilizing tertiary structure under these conditions . In the majority of the experiments, methanol was added as a cosolvent to the buffer . Its use allowed the examination of the behavior of monovalent ions under conditions where their effects would otherwise have been too weak to be observed . Methanol stabilizes tertiary but not secondary structure of the RNA . There was no evidence that it either causes qualitative changes in cation-binding properties of the RNA or a change in the pattern of monovalent cation/Mg(2+) competition .

Exp Neurol, 2000 Sep, 165(1), 27 - 34
Human amniotic epithelial cells produce dopamine and survive after implantation into the striatum of a rat model of Parkinson's disease: a potential source of donor for transplantation therapy; Kakishita K et al.; We have recently found that human amniotic epithelial (HAE) cells synthesize catecholamines including dopamine (DA) . The present study was designed to explore the possibility of HAE cells to serve as a donor for transplantation therapy of Parkinson's disease (PD) . Thus, we investigated their ability to produce DA in vitro and the survival and function of HAE cells grafted into a rat model of PD . RT-PCR and Western blotting revealed that HAE cells express tyrosine hydroxylase (TH) mRNA and protein, respectively . TH-immunohistochemistry on cultured HAE cells demonstrated that around 10% of the total cells are immunopositive for this protein . The production of DA by HAE cells was increased with time in the presence of L-tyrosine and BH(4), and was abolished with a specific TH inhibitor, alpha-methyl-rho-tyrosine . Dissociated HAE cells transduced with the Escherichia coli LacZ marker gene (beta-gal) were implanted into the previously DA-depleted striatum of immunosuppressed rats . Two weeks postgrafting HAE grafts were demonstrated to survive without overgrowth, as evidenced by the presence of beta-gal-positive cells and TH-immunoreactive cells within the grafts . The grafts also provided partial amelioration of apomorphine-induced rotational asymmetry . The results clearly indicate that HAE cells capable of producing DA can survive and function in the brain of a rat model of PD . Although DA replacement therapy of PD could possibly be achieved with implantation of HAE cells, further studies are needed to develop strategies to enhance the ability of HAE cells to produce DA as well as the graft survival .

Anal Biochem, 2000 Sep 10, 284(2), 401 - 5
Detection of beta-amyloid peptide aggregation using DNA electrophoresis; Ahn BW et al.; DNA could readily associate with the aggregated forms of the beta-amyloid peptides beta(1-40) and beta(25-35), giving rise to a shift in the electrophoretic mobility of DNA . As a result, DNA was retained at the top of a 1% agarose gel . In contrast, the electrophoretic mobility of DNA was little influenced by the monomeric forms of beta(1-40) and beta(25-30) . DNA from different sources such as lambda phage, Escherichia coli plasmid, and human gene showed similar results . However, the electrophoretic mobility of RNA was shifted by the monomeric beta(1-40) and beta(25-35) as well as by the aggregated beta(1-40) and beta(25-35) . The association of DNA with the aggregated beta-amyloid peptides could occur at pH 4-9 . The inhibitory action of hemin on beta-amyloid aggregation could be confirmed using the DNA mobility shift assay . These results indicate that the DNA mobility shift assay is useful for kinetic study of beta-amyloid aggregation as well as for testing of agents that might modulate beta-amyloid aggregation .

Anal Biochem, 2000 Sep 10, 284(2), 382 - 7
Implementation of a continuous, enzyme-coupled fluorescence assay for high-throughput analysis of glutamate-producing enzymes; McElroy KE et al.; Enzymatic formation of glutamate is critical to numerous biological pathways . However, current methods for assaying the activities of glutamate-forming enzymes are not particularly suitable for high-throughput screening in drug discovery . We present a continuous-read, fluorometric assay for high-throughput analysis of glutaminases . This assay is adapted to a microplate format and employs glutamate oxidase and horseradish peroxidase to couple glutamate formation to production of the fluorescent reporter molecule, resorufin, for enhancement of sensitivity (M . Zhou, Z . Diwu, N . Panchuk-Voloshina, and R . P . Haughland, 1997, Anal . Biochem . 253, 162-168) . Described herein is the selection of suitable levels of coupling enzymes for optimal kinetic response and lag time of the reporter system, based on the kinetic characteristics of the individual coupling enzymes . Finally, implementation of the assay in a format for high-throughput kinetic analysis of glutaminases is demonstrated for Escherichia coli carbamoyl phosphate synthase . Derived kinetic constants are comparable to literature values determined using a variety of assay techniques .

Anal Biochem, 2000 Sep 10, 284(2), 266 - 78
A method for global analysis of complex proteomes using sample prefractionation by solution isoelectrofocusing prior to two-dimensional electrophoresis; Zuo X et al.; Two-dimensional electrophoresis is a critical technique for proteome research, but currently available methods are not capable of resolving the >10,000 protein components in most eukaryotic proteomes . We have developed and demonstrated the utility of a novel solution isoelectrofocusing device and method that can reproducibly prefractionate cell extracts into well-defined pools prior to 2D PAGE on a scale directly compatible with the high sensitivity of proteome studies . A prototype device was used to separate metabolically radiolabeled Escherichia coli extracts in method optimization and proof-of-principle experiments . Samples were loaded into separation chambers divided by thin polyacrylamide gels containing immobilines at specific pH values and isoelectrically focused for several hours, which resulted in well-resolved fractions . Total recoveries in the fractionated samples were greater than 80% and most protein spots in the original sample were recovered after this prefractionation step . Nonideal behavior (precipitation/aggregation), typically encountered when unfractionated samples at high protein loads were applied directly to either narrow- or broad-range IPG gels, was dramatically reduced . Hence this approach allows increases in overall protein loads, resolution, and dynamic detection range compared with either alternative prefractionation methods or direct use of parallel narrow pH range gels without sample prefractionation . The pH ranges and number of fractions can be readily adapted to the requirements of specific types of samples and projects . This method should allow quantitative comparisons of at least 10,000 protein components on a series of narrow pH range gels, and protein detection limits are estimated to be 1000 molecules per cell when mammalian proteomes are fractionated into five or more pools .

Anal Biochem, 2000 Sep 10, 284(2), 221 - 30
Quantitative measurement of bitagged recombinant proteins using an immunometric assay: application to an anti-substance P recombinant antibody; Boquet D et al.; We have developed two different immunometric assays to directly quantify both the total and the active fractions of a recombinant antibody (single chain fragment variable, or ScFv) as obtained in a crude extract from an Escherichia coli expression system . For total determination, the assay is based on the simultaneous recognition of two different peptide Tag sequences (Ha-Tag and Myc-Tag) at each of the N- and C-terminal extremities of the recombinant protein . A monoclonal antibody (mAb 12CA5, directed against Ha-Tag), coated on microtiter plates, is used for capture, and the mAb 9E10 (directed against Myc-Tag), labeled with acetylcholinesterase (AChE, EC 3.1.1.7), acts as tracer . In parallel, for the determination of the active fraction, the capture is performed using microtiter plates coated with the antigen, while solid-phase-immobilized ScFv is measured using the same 9E10 tracer mAb . A synthetic peptide in which the two Tag sequences were joined was used as a standard, thus avoiding the laborious purification of a recombinant protein as reference . The method was applied to the direct measurement, in periplasmic extracts, of the total and active fractions of an ScFv produced at different induction temperatures .

Endocrinology, 2000 Sep, 141(9), 3194 - 9
Androgen-regulated expression of a novel member of the aldo-keto reductase superfamily in regrowing rat prostate; Nishi N et al.; The rat prostate is dependent on androgen for normal growth and differentiation . In addition, the organ undergoes rapid cell death upon withdrawal of androgen on castration, and the atrophied tissue is capable of regrowth after androgen replacement in adult animals . In our search for novel factor(s) that participate in this androgen-induced proliferation of adult rat prostate cells, we have generated a complementary DNA (cDNA) library enriched in cDNAs transiently up-regulated after androgen stimulation in castrated rat ventral prostate using a PCR-based subtractive hybridization technique . Sequence analysis of about one hundred clones in the library showed that approximately 70% of them are identical or closely related to genes of known function, the remaining ones showing no or very low similarity to any genes characterized previously . Among the former a new member of the rat aldo-keto reductase superfamily that is closely related to aflatoxin, B1 aldehyde reductase has been identified . The newly identified protein (androgen-inducible aldehyde reductase, AIAR) and rat aflatoxin B1 aldehyde reductase (AFAR) exhibit 80% amino acid sequence homology . The enzymatic activity toward 4-nitrobenzaldehyde of recombinant AIAR expressed in Escherichia coli was about 16% of that of rat AFAR . Northern blot analysis revealed AIAR expression in various adult rat tissues in addition to the ventral and dorsolateral prostates, which differs from the highly restricted expression of AFAR in the kidney and liver . The AIAR messenger RNA (mRNA) content of the ventral prostate was low in normal and castrated rats, transiently increased after androgen administration to castrated rats, attaining a peak 12-24 h after the treatment . Although the physiological substrate(s) of AIAR has not been identified, the current results suggest that AIAR expression is associated with some growth-related processes in regrowing rat prostate.

Toxicol In Vitro, 2000 Oct, 14(5), 423 - 8
Mutagenicity and genotoxicity of sorbic acid-amine reaction products; Ferrand C et al.; Sorbic acid as well as potassium and calcium sorbate (E202 and E203) are legally used as preservatives in numerous processed foods . Owing to its system of conjugated double bonds, sorbic acid is likely to undergo a nucleophilic attack, which may turn it into mutagenic products . The cyclic derivatives resulting from a double addition reaction between sorbic acid and various amines at two different temperatures (50 degrees C and 80 degrees C) have been analysed . A genotoxicity study has been performed with HeLa cells and plasmid DNA . A mutagenesis study has been carried out by using the Ames test . A SOS spot test and a cytotoxicity study have been realised as well . The results showed that the products involved exhibited neither mutagenic nor genotoxic activities.

Immunol Lett, 2000 Jul 3, 73(1), 29 - 34
Anti-DNA antibodies exhibit different binding motif preferences for single stranded or double stranded DNA; Wang Y et al.; A common feature for most anti-DNA antibodies (Abs) is their induction in an antigen (Ag)-driven specific clonal expansion pattern though crossreactivity . However, the fine sequences in DNA Ags that interact directly with immune system and the ability of DNA to induce immune responses is poorly understood . In order to define the characteristics of possible antigenic determinants in DNA Ags, we immunized mice with the pBR322 plasmid and used antisera as source of anti-DNA Abs . A systemic evolution of ligands by exponential enrichment (SELEX) procedure was performed on an oligodeoxynucleotide library either in single stranded (ss-) or double stranded (ds-) form . The SELEXed fragments were cloned and sequenced . The resulting sequences were analyzed using the Multiple Alignment Construction and Analysis Workbench program . We show that the fragments of ss- or ds- form bound by a same stock of antibodies were different in their conserved sequences . ss-DNA fragments recognized by anti-DNA Abs were rich in cacc, caccc, accc or cccc blocks, while the same stock of Abs exhibited significant preference for the (5'gcg3'/3'cgc5') motif located in ds-DNA . At the same time sera from unimmunized control mice showed no sequence preference in either ss-DNA or ds-DNA . Future improvement of this work and the potential use of SELEX for studies of DNA Ags are also discussed.

Phytochemistry, 2000 Jul, 54(6), 567 - 72
Purification and characterization of a NAD+-dependent sorbitol dehydrogenase from Japanese pear fruit; Oura Y et al.; NAD+-dependent sorbitol dehydrogenase NAD-SDH, EC 1.1.1.14) from Japanese pear fruit was purified to apparent homogeneity (single band by SDS-PAGE with silver staining), and had a specific activity of 916.7 nKatal/mg protein . The molecular of the native enzyme was calculated to be 160 kDa by gel filtration, whereas SDS-PAGE gave a subunit size of 40 kDa, indicating that the native enzyme is a homotetramer . The protein immunologically reacted with an antibody raised in rabbit against the fusion protein expressed in E . coli harboring an apple NAD-SDH cDNA . The Km, values for sorbitol and fructose were 96.4+/-8.60 and 4239+/-33.5 mM, respectively, and optimum pH for sorbitol oxidation was 9.0 and 7.0 for fructose reduction . Pear NAD-SDH had a very narrow substrate specificity, that is, sorbitol, L-iditol, xylitol and L-threitol were oxidized but not any of the other alcohols tested . These data suggest the structural importance of an S configuration at C-2 and an R configuration at C-4 in the substrate(s) . Its enzymatic activity was strongly inhibited both by heavy metal ions such as mercury, and by thiol compounds, such as L-cysteine . However, the addition of zinc ion reversed the enzyme inactivation caused by addition of L-cysteine.

J Invertebr Pathol, 2000 Jul, 76(1), 33 - 42
Venom from the endoparasitic wasp Pimpla hypochondriaca adversely affects the morphology, viability, and immune function of hemocytes from larvae of the tomato moth, Lacanobia oleracea; Richards EH et al.; During oviposition, the endoparasitic wasp Pimpla hypochondriaca injects its pupal hosts with venom . This complex fluid has toxic properties and recently several venom components were characterized . In addition, it was suggested that venom might be involved in host immune suppression . For this to be the case, venom would have to adversely affect hemocytes and this aspect was further addressed in the current study utilizing the larval stage of the tomato moth Lacanobia oleracea as a model system . Using sublethal venom injections we investigated the effects of venom on encapsulation and hemocyte concentration . Additionally, the effects of venom on hemocyte morphology, viability, and phagocytic capability were determined in vitro . Injection of 16 microg of venom protein into sixth instar larvae was sufficient to reduce the ability of hemocytes to encapsulate Sephadex A25 beads by more than 50% in four of five insects examined . Hemocyte concentration in sixth instar larvae 32 h after injection with 16 microg of venom was reduced by 56% compared to that in controls . Damaged hemocytes and cell debris were also observed in hemolymph from venom-treated insects, suggesting that P . hypochondriaca venom has cytotoxic properties . In vitro incubation of washed hemocytes for 20 h with 500 ng/microl venom resulted in disintegration of a high proportion of hemocytes, leaving only parts of the plasma membrane and nucleus intact . Treatment with low concentrations of venom (1.6 ng/microl) resulted in an absence of spread plasmatocytes, which were abundant on control monolayers . High-resolution microscopy of hemocyte cultures exposed to 320 ng/microl venom for 3.5 h on glass slides indicated that venom induced a variety of effects on cellular morphology, including blebbing of the plasma membrane, degranulation, and the formation of cytoplasmic vacuoles . Incubation of hemocytes with 320, 64, or 3.2 ng/microl venom for 3.5 h reduced cell viability to 70, 90, and 92%, respectively, confirming that venom is cytotoxic to hemocytes . Treatment with 320 ng/microl venom reduced the capacity of hemocytes to phagocytose Escherichia coli by 85% . Together, these results demonstrate that at sublethal doses venom has a potent anti-hemocyte action and can impair hemocyte-mediated immune responses.

Arch Virol, 2000, 145(7), 1437 - 47
Cross-reactive and major virus-specific epitopes are located at the N-terminal halves of the cylindrical inclusion proteins of turnip mosaic and zucchini yellow mosaic potyviruses; Kundu AK et al.; To investigate the antigenic nature of cylindrical inclusion proteins (CIPs) of the potyviruses Turnip mosaic virus (TuMV) and Zucchini yellow mosaic virus (ZYMV), monoclonal antibodies (MAbs) against the two CIPs were produced and epitopes on the CIPs were localized using Escherichia coli-expressed CIP fragments in Western blot analysis . All 23 MAbs against ZYMV CIP reacted only with ZYMV CIP . In contrast out of the 18 MAbs produced against TuMV CIP, 14 MAbs were TuMV CIP-specific while the remaining four MAbs cross-reacted with both CIPs . The four cross-reactive MAbs recognized two distinct epitopes in the N-terminal half of TuMV CIP corresponding to amino acid residues 103-119 and 224-237 . Thirteen out of 14 TuMV CIP-specific MAbs recognized two distinct epitopes within residues 1-102 and 120-214, while the other one recognized an epitope within residues 301-644 . On the other hand, 21 out of 23 ZYMV CIP-specific MAbs recognized epitopes within residues 1-118, while the remaining two recognized epitopes within residues 301-522 . These results suggest that cross-reactive and major virus-specific epitopes are located at the N-terminal half of the respective CIPs.

Biol Pharm Bull, 2000 Aug, 23(8), 941 - 5
Expression and characterization of human rheumatoid factor single-chain Fv; Hashimoto Y et al.; The variable region of heavy chain {V(H)} of human rheumatoid factor (hRF) IgM was connected with the variable region of light chain {V(L)} with the peptide-linker (GGGSGGGSGGGS) by genetic engineering method and the single-chain Fv (scFv) was expressed in E . coli . On design, scFv and scFv (tag) were planned; the latter had a detection marker at the carboxyl-terminal . These scFvs were expressed as inclusion bodies in E . coli, purified in the presence of 8 M urea by gel filtration and renatured to the active form in vitro . As a control, the Fv, non-covalently associated V(H) and V(L) fragments, was also constructed . The 3 derivatives showed almost the same binding activity to rabbit-IgG to which hRF is cross-reactive . ScFv (tag) was the most stable against urea among the 3 derivatives.

Int J Cancer, 2000 Oct 1, 88(1), 28 - 36
Abnormal expression of BRCA1 and BRCA1-interactive DNA-repair proteins in breast carcinomas; Yoshikawa K et al.; Breast cancer is one of the most common malignancies among women . The molecular mechanisms involved in breast carcinogenesis, however, remain to be elucidated . Although somatic mutation of BRCA1 is rare, BRCA1 protein expression is reduced in about 30% of sporadic breast carcinomas (Yoshikawa et al., Clin . Cancer Res., 5:1249-1261, 1999), indicating its possible involvement even in sporadic breast carcinogenesis . Among the BRCA1-interactive proteins are hRAD51 (a human homologue of Escherichia coli rec A protein), BARD1 (BRCA1-associated RING domain 1) and p53, all of which are involved in DNA repair . We have analyzed the expression patterns of the hRAD51, BARD1 and p53 proteins in five breast cancer cell lines, including a BRCA1-deficient cell line, and in 179 breast cancer tissue samples from Japanese women, including 113 sporadic, 47 hereditary (i.e., BRCA1 status unknown), and 19 BRCA1-associated cases . Of the 179 breast carcinomas, fifty-four (30%) exhibited reduced hRAD51 expression, and sixty-two (35%) exhibited p53 overexpression . On the other hand, reduced expression level of BARD1, and of hMSH2 and hMLH1, which are components of DNA mismatch-repair pathway and are involved in colorectal carcinogenesis, was observed respectively in only 10 (6%), 8 (5%) and 3 (2%) cases . The overall frequency of sporadic breast carcinomas with abnormal expression of either BRCA1 or the BRCA1-interactive proteins was 67% (76/113) . These results indicate that there may be an important role for the BRCA1-associated DNA-repair pathway, not only in BRCA1-associated breast carcinomas, but also in sporadic breast carcinomas .

Biochim Biophys Acta, 2000 Aug 31, 1481(1), 167 - 74
Expression and purification of a recombinant DNA-binding domain of ADR6 protein from Escherichia coli and its secondary structure characterization; Tu X et al.; From Saccharomyces cerevisiae, a piece of ADR6 gene that encodes a DNA-binding domain of ADR6 protein was cloned and expressed in Escherichia coli . With Ni-chelating column and high-performance liquid chromatography (HPLC), This recombinant protein (RDB-ADR6) could reach more than 95% purity . The molecular weight (MW) of RDB-ADR6 is 13405 Da with mass spectra technique containing 114 amino acid residues . Structural aspects of RDB-ADR6 were examined by spectroscopic techniques . It contains approximately 25% alpha-helix and 24% beta-turn both with circular dichroism (CD) and Fourier transform infrared spectroscopy (FTIR) . Percent of beta-sheet differs between these two methods in that 22% in CD while 35% in FTIR . RDB-ADR6 contains only one tryptophan residue . Fluorescence studies show that this residue may lie in a hydrophobic circumstance either on or near the surface of the molecule . This was confirmed by a blue shift of 20 nm in the fluorescence emission spectrum as compared to the protein in 6 M guanidine hydrochloride (GuHCl) and by quenching studies with KI . Effects of different pH and SDS in different concentration on the secondary structure of RDB-ADR6 were also studied . A model was obtained by comparative modeling with homologous known structure protein by program Modeller 4.

J Biol Chem, 2000 Dec 1, 275(48), 37887 - 94
Transit of tRNA through the Escherichia coli ribosome . Cross-linking of the 3' end of tRNA to specific nucleotides of the 23 S ribosomal RNA at the A, P, and E sites; Wower J et al.; When bound to Escherichia coli ribosomes and irradiated with near-UV light, various derivatives of yeast tRNA(Phe) containing 2-azidoadenosine at the 3' terminus form cross-links to 23 S rRNA and 50 S subunit proteins in a site-dependent manner . A and P site-bound tRNAs, whose 3' termini reside in the peptidyl transferase center, label primarily nucleotides U2506 and U2585 and protein L27 . In contrast, E site-bound tRNA labels nucleotide C2422 and protein L33 . The cross-linking patterns confirm the topographical separation of the peptidyl transferase center from the E site domain . The relative amounts of label incorporated into the universally conserved residues U2506 and U2585 depend on the occupancy of the A and P sites by different tRNA ligands and indicates that these nucleotides play a pivotal role in peptide transfer . In particular, the 3'-adenosine of the peptidyl-tRNA analogue, AcPhe-tRNA(Phe), remains in close contact with U2506 regardless of whether its anticodon is located in the A site or P site . Our findings, therefore, modify and extend the hybrid state model of tRNA-ribosome interaction . We show that the 3'-end of the deacylated tRNA that is formed after transpeptidation does not immediately progress to the E site but remains temporarily in the peptidyl transferase center . In addition, we demonstrate that the E site, defined by the labeling of nucleotide C2422 and protein L33, represents an intermediate state of binding that precedes the entry of deacylated tRNA into the F (final) site from which it dissociates into the cytoplasm.

Biochem Soc Trans, 2000, 28(4), 520 - 6
Structure-function relationships in an anion-translocating ATPase; Bhattacharjee H et al.; The ArsAB ATPase is an efflux pump located in the inner membrane of Escherichia coli . This transport ATPase confers resistance to arsenite and antimonite by their extrusion from the cells . The pump is composed of two subunits, the catalytic ArsA subunit and the membrane subunit ArsB . The complex is similar in many ways to ATP-binding cassette ('ABC') transporters, which typically have two groups of six transmembrane-spanning helical segments and two nucleotide-binding domains (NBDs) . The 45 kDa ArsB protein has 12 transmembrane-spanning segments . ArsB contains the substrate translocation pathway and is capable of functioning as an anion uniporter . The 63 kDa ArsA protein is a substrate-activated ATPase . It has two homologous halves, A1 and A2, which are clearly the result of an ancestral gene duplication and fusion . Each half has a consensus NBD . The mechanism of allosteric activation of the ArsA ATPase has been elucidated by a combination of molecular genetics and biochemical, structural and kinetic analyses . Conformational changes produced by binding of substrates, activator and/or products could be revealed by stopped-flow fluorescence measurements with single-tryptophan derivatives of ArsA . The results demonstrate that the rate-limiting step in the overall reaction is a slow isomerization between two conformations of the enzyme . Allosteric activation increases the rate of this isomerization such that product release becomes rate-limiting, thus accelerating catalysis . ABC transporters, which exhibit similar substrate activation of ATPase activity, can undergo similar conformational changes to overcome a rate-limiting step . Thus the ArsAB pump is a useful model for elucidating mechanistic aspects of the ABC superfamily of transport ATPases.

Biochem Soc Trans, 2000, 28(4), 410 - 4
Recruitment of chromatin remodelling factors during gene activation via the glucocorticoid receptor N-terminal domain; Wallberg AE et al.; We have shown that yeast mutants with defects in the Ada adaptor proteins are defective in hormone-dependent gene activation by ectopically expressed human glucocorticoid receptor (GR) . Others have shown that the Ada2 protein is required for physical interactions between some activation domains and TBP (TATA-binding protein), whereas the Gcn5 (Ada4) protein has a histone acetyltransferase (HAT) activity . Although all HAT enzymes are able to acetylate histone substrates, some also acetylate non-histone proteins . Taken together, these observations suggest that the Ada proteins have the ability to effect different steps in the process of gene activation . It has recently been shown that the Ada proteins are present in two distinct protein complexes, the Ada complex and a larger SAGA complex . Our recent work has focused on determining (1) which of the Ada-containing complexes mediates gene activation by GR, (2) whether the HAT activity encoded by GCN5 is required for GR-dependent gene activation, (3) whether the Ada proteins contribute to GR-mediated activation at the level of chromatin remodelling and (4) how the role of these HAT complexes is integrated with other chromatin remodelling activities during GR-mediated gene activation . Our results suggest a model in which GR recruits the SAGA complex and that this contributes to chromatin remodelling via a mechanism involving the acetylation of histones . Furthermore, recruitment of the SWI/SNF remodelling complex also has a role in GR-mediated activation that is independent of the role of SAGA . These complexes are similar to analogous mammalian complexes and therefore these results are likely to be relevant to the human system.

Blood, 2000 Sep 1, 96(5), 1709 - 15
Weekly polyethylene glycol conjugated L-asparaginase compared with biweekly dosing produces superior induction remission rates in childhood relapsed acute lymphoblastic leukemia: a Pediatric Oncology Group Study; Abshire TC et al.; The relapse rate in childhood acute lymphoblastic leukemia (ALL) is approximately 30% but few reinduction regimens have investigated the intensive use of polyethylene glycol Escherichia coli asparaginase (PEG-Asp) . Therefore, we assessed the pharmocokinetics and efficacy of PEG-Asp in this setting . Children with B-precursor ALL, in first marrow and/or extramedullary relapse were eligible . Reinduction included doxorubicin on day 1, prednisone for 28 days, vincristine weekly for 4 weeks, and PEG-Asp either weekly or biweekly by randomization . Asparaginase levels and antibody to both E coli asparaginase and PEG-asp were measured weekly just before each PEG-asp dose . Overall, 129 of 144 patients (pts) (90%) achieved a complete remission (CR) . There was a highly significant difference in CR rates between weekly (69 of 71; 97%) and biweekly (60 of 73; 82%) PEG-Asp dosing (P =.003) . Grade 3 or 4 infectious toxicity was common (50%), but only 4 pts died of sepsis during induction . Other toxicities were infrequent and hypersensitivity was rare (6 of 144; 4%) . Low asparaginase levels were associated with high antibody titers to either native (P =.024) or PEG asp (P =.0013) . The CR rate was significantly associated with higher levels of asparaginase (P = . 012) . Patients with ALL in first relapse receiving weekly PEG-Asp had a higher rate of second remission compared with biweekly dosing . Low levels of asparaginase were associated with high antibody titers . Increased asparaginase levels may correlate with an improved CR rate . The use of intensive PEG-Asp should be explored further in the treatment of ALL . (Blood . 2000;96:1709-1715)

Int J Parasitol, 1999 Dec, 29(12), 1925 - 33
Identification and characterisation of three antigenic proteins from Cryptosporidium parvum sporozoites using a DNA library expressing poly-histidine tagged peptides; Tosini F et al.; To identify antigenic peptides of the parasite Cryptosporidium parvum, an expression library that allows for the production of chimeric proteins fused with a 6-histidine tag was made . The library was screened with C . parvum sporozoite rabbit anti-serum, and three positive clones (sa20, sa35, and sa40) were identified . The corresponding recombinant proteins (SA20, SA35, and SA40) were expressed in Escherichia coli and purified by metal-affinity chromatography . The sequence of sa20 and sa35 clones did not show any homology with known genes or proteins, whereas the 5' end of the sa40 clone showed homology with two previously identified C . parvum sequences . Hybridisations to intact chromosomes fractionated by pulsed-field gel electrophoresis revealed that the sa35 and sa40 sequences are localised on chromosome VII, whereas the sa20 sequence is localised on chromosome VI . Reverse transcriptase-PCR experiments showed the presence of mRNAs for sa35 and sa40 in the oocyst, whereas the sa20 mRNA was undetectable in this stage . The serological response to the three proteins was assayed in C . parvum-immunised rabbits and in immunocompetent individuals with cryptosporidiosis . The Western blot results indicated that rabbits, challenged with a sporozoite crude antigen or with an oocyst crude antigen, were highly responsive to these three antigens . Human serum samples showed a response to the three proteins, although the response to SA20 appeared to be unrelated to a recent C . parvum infection . These results suggest that the SA35 and the SA40 proteins may be useful in detecting C . parvum infections.

Int J Parasitol, 1999 Dec, 29(12), 1893 - 905
Differential recognition of Toxoplasma gondii recombinant nucleoside triphosphate hydrolase isoforms by naturally infected human sera; Johnson MS et al.; Toxoplasma gondii possesses a highly active nucleoside triphosphate hydrolase, which has been shown to be an immunodominant antigen in mice and humans . Two isoforms (I and II) which exhibit different activities with respect to hydrolysis of ATP exist . Past studies suggest that all strains of T . gondii contain the less active nucleoside triphosphate hydrolase II, whilst only virulent strains contain the nucleoside triphosphate hydrolase I isoform . In order to further investigate the correlation between nucleoside triphosphate hydrolase isoform and biological significance, we cloned and expressed as glutathione S-transferase fusion proteins the full-length nucleoside triphosphate hydrolase I and II isoforms and two truncations of the nucleoside triphosphate hydrolase I isoform in Escherichia coli . We then used ELISAs with the full-length recombinant nucleoside triphosphate hydrolases as antigens to examine 188 naturally infected T . gondii-positive sera and 83 T . gondii-negative sera for antibody reactivity . All positive sera reacted to T . gondii whole tachyzoite lysate antigen, 31 sera reacted to both nucleoside triphosphate hydrolase isoforms, three sera reacted specifically to nucleoside triphosphate hydrolase I and two sera reacted to only nucleoside triphosphate hydrolase II . Immunoblot analysis of the five sera reacting to either nucleoside triphosphate hydrolase I or II revealed both quantitative and qualitative differences in reactivity to the two isoforms . Comparative immunoblot analysis using the truncations of the nucleoside triphosphate hydrolase I isoform, and one of these positive sera identified a presumptive differential epitope between the nucleoside triphosphate hydrolase I and II isoforms within an 81 amino acid region (amino acids 445-526) at the C-terminus of the nucleoside triphosphate hydrolase I isoform . This differential reactivity was further localised to the 12-residue region of greatest variability between the two isoforms (residues 488-499) using synthetic peptides . This is the first report where naturally infected human sera have been used to identify a differential epitope . Because this region is essential for substrate binding, an antibody response to this region may play some role in inhibition of this highly active enzyme.

Dig Dis Sci, 2000 Jul, 45(7), 1343 - 51
Effects of chronic administration of Helicobacter pylori extracts on rat gastric mucosal microcirculation in vivo; Kalia N et al.; The mechanisms by which Helicobacter pylori contributes to gastroduodenal injury are unclear . We have previously described platelet aggregation within rat gastric mucosal microcirculation following acute administration of H . pylori extracts . However, leukocyte activation was not observed . This study aimed to determine whether chronic administration of H . pylori could induce leukocyte activation . Rats were gavaged with either H . pylori or E . coli extracts or with distilled water three times daily at three-day intervals . Acridine red was used to quantitate gastric mucosal leukocyte/platelet activity using fluorescent in vivo microscopy . Further animals received additional acute H . pylori after 1 hr and recordings were made for a further 1 hr . Significant numbers of "flyers," "rollers," and adherent leukocytes were observed throughout the study in H . pylori animals . Only adherent leukocytes were observed following E . coli . Acute H . pylori induced a further significant increase in adherent leukocytes . Significant platelet thrombi were also present in H . pylori-treated animals . In conclusion, earlier studies demonstrated platelet aggregation but no leukocyte activation, which is in contrast to the current chronic studies . Platelet activation may be the initial response to H . pylori and involved in recruitment of leukocytes . These activated cells may contribute to the development of gastric mucosal damage.

J Pept Res, 2000 Aug, 56(2), 59 - 62
Plasminogen activator inhibitor-1 fused with erythropoietin (EPO) mimetic peptide (EMP) enhances the EPO activity of EMP; Kuai L et al.; Erythropoietin (EPO) mimetic peptide (EMP) encoding sequence was inserted into the gene of plasminogen activator inhibitor-1 (PAI-1) between Ala348 and Pro349 (P2'-P3'), generating a novel gene, PAI-1/EMP (PMP) . This was cloned into pET32a expression vector, fused with TrxA peptide in the vector, and a 63-kDa protein was expressed in inclusion bodies with an expression level >50% . The TrxA/PMP protein was purified by Ni-NTA-agarose metal-ligand affinity chromatography to a purity >90%, showing a single, silver-stained band on SDS-PAGE . Using a reticulocyte counting assay, the EPO activity of PMP was determined to be 5,000 IU/mg, 2,500-fold that of EMP.

J Clin Pathol, 2000 Jul, 53(7), 518 - 24
Immunohistowax processing, a new fixation and embedding method for light microscopy, which preserves antigen immunoreactivity and morphological structures: visualisation of dendritic cells in peripheral organs; Pajak B et al.; AIMS: To describe a new fixation and embedding method for tissue samples, immunohistowax processing, which preserves both morphology and antigen immunoreactivity, and to use this technique to investigate the role of dendritic cells in the immune response in peripheral tissues . METHODS: This technique was used to stain a population of specialised antigen presenting cells (dendritic cells) that have the unique capacity to sensitise naive T cells, and therefore to induce primary immune responses . The numbers of dendritic cells in peripheral organs of mice either untreated or injected with live Escherichia coli were compared . RESULTS: Numbers of dendritic cells were greatly decreased in heart, kidney, and intestine after the inoculation of bacteria . The numbers of dendritic cells in the lung did not seem to be affected by the injection of E coli . However, staining of lung sections revealed that some monocyte like cells acquired morphological and phenotypic features of dendritic cells, and migrated into blood vessles . CONCLUSIONS: These observations suggest that the injection of bacteria induces the activation of dendritic cells in peripheral organs, where they play the role of sentinels, and/or their movement into lymphoid organs, where T cell priming is likely to occur.

J Biol Chem, 2000 Nov 17, 275(46), 36415 - 22
Functional synergism between the most common polymorphism in human alanine:glyoxylate aminotransferase and four of the most common disease-causing mutations; Lumb MJ et al.; The autosomal recessive disorder primary hyperoxaluria type 1 (PH1) is caused by a deficiency of the liver-specific pyridoxal-phosphate-dependent enzyme alanine:glyoxylate aminotransferase (AGT) . Numerous mutations and polymorphisms in the gene encoding AGT have been identified, but in only a few cases has the causal relationship between genotype and phenotype actually been demonstrated . In this study, we have determined the effects of the most common naturally occurring amino acid substitutions (both normal polymorphisms and disease-causing mutations) on the properties, especially specific catalytic activity, of purified recombinant AGT . The results presented in this paper show the following: 1) normal human His-tagged AGT can be expressed at high levels in Escherichia coli and purified in a correctly folded, dimerized and catalytically active state; 2) presence of the common P11L polymorphism decreases the specific activity of purified recombinant AGT by a factor of three; 3) AGTs containing four of the most common PH1-specific mutations (G41R, F152I, G170R, and I244T) are all soluble and catalytically active in the absence of the P11L polymorphism, but in its presence all lead to protein destabilization and aggregation into inclusion bodies; 4) naturally occurring and artificial amino acid substitutions that lead to peroxisome-to-mitochondrion AGT mistargeting in mammalian cells also lead to destabilization and aggregation in E . coli; and 5) the PH1-specific G82E mutation abolishes AGT catalytic activity by interfering with cofactor binding, as does the artificial K209R mutation at the putative site of cofactor Shiff base formation . These results are discussed in the light of the high allelic frequency ( approximately 20%) of the P11L polymorphism and its importance in determining the phenotypic manifestations of mutations in PH1.

J Biol Chem, 2000 Nov 10, 275(45), 35116 - 21
A covariant change of the two highly conserved bases in the GTPase-associated center of 28 S rRNA in silkworms and other moths; Uchiumi T et al.; The GTPase-associated center in 23/28 S rRNA is one of the most conserved functional domains throughout all organisms . We detected a unique sequence of this domain in Bombyx mori species in which the bases at positions 1094 and 1098 (numbering from Escherichia coli 23 S rRNA) are C and G instead of the otherwise universally conserved bases U and A, respectively . These changes were also observed in four other species of moths, but not in organisms other than the moths . Characteristics of the B . mori rRNA domain were investigated by native polyacrylamide gel electrophoresis using RNA fragments containing residues 1030-1128 . Although two bands of protein-free RNA appeared on gel, they shifted to a single band when bound to Bombyx ribosomal proteins Bm-L12 and Bm-P complex, equivalent to E . coli L11 and L8, respectively . Bombyx RNA showed lower binding capacity than rat RNA for the ribosomal proteins and anti-28 S autoantibody, specific for a folded structure of the eukaryotic GTPase-associated domain . When the C(1094)/G(1098) bases in Bombyx RNA were replaced by the conserved U/A bases, the protein-free RNA migrated as a single band, and the complex formation with Bm-L12, Bm-P complex, and anti-28 S autoantibody was comparable to that of rat RNA . The results suggest that the GTPase-associated domain of moth-type insects has a labile structural feature that is caused by an unusual covariant change of the U(1094)/A(1098) bases to C/G.

J Bacteriol, 2000 Sep, 182(18), 5274 - 7
Mutational analysis of genes encoding the early flagellar components of Helicobacter pylori: evidence for transcriptional regulation of flagellin A biosynthesis; Allan E et al.; We investigated the roles of fliF, fliS, flhB, fliQ, fliG, and fliI of Helicobacter pylori, predicted by homology to encode structural components of the flagellar basal body and export apparatus . Mutation of these genes resulted in nonmotile, nonflagellate strains . Western blot analysis showed that all the mutants had considerably reduced levels of both flagellin subunits and of FlgE, the flagellar hook protein . RNA slot blot hybridization showed reduced levels of flaA mRNA, indicating that transcription of the major flagellin gene is inhibited in the absence of the early components of the flagellar-assembly pathway . This is the first demonstration of a checkpoint in H . pylori flagellar assembly.

J Bacteriol, 2000 Sep, 182(18), 5267 - 70
Evidence for multimerization of neu proteins involved in polysialic acid synthesis in Escherichia coli K1 using improved LexA-based vectors; Daines DA et al.; Recently, M . Dmitrova et al . (Mol . Gen . Genet . 257:205-212, 1998) described a LexA-based genetic system to monitor protein-protein interactions in an Escherichia coli background . However, the plasmids used in this system, pMS604 and pDP804, were not readily amenable for general use . In this report, we describe modifications of both plasmids that allow fragments of DNA to be fused to either vector in any reading frame . Homodimerization and heterodimerization of full-length proteins involved in polysialic acid synthesis in E . coli K1, as well as heterodimerization between a full-length protein and a protein fragment, demonstrate the usefulness of the modified plasmids for investigating bacterial protein-protein interactions in vivo.

J Bacteriol, 2000 Sep, 182(18), 5231 - 7
Function of the sigma(E) regulon in dead-cell lysis in stationary-phase Escherichia coli; Nitta T et al.; Elevation of active sigma(E) levels in Escherichia coli by either repressing the expression of rseA encoding an anti-sigma(E) factor or cloning rpoE in a multicopy plasmid, led to a large decrease in the number of dead cells and the accumulation of cellular proteins in the medium in the stationary phase . The numbers of CFU, however, were nearly the same as those of the wild type or cells devoid of the cloned gene . In the wild-type cells, rpoE expression was increased in the stationary phase and a low-level release of intracellular proteins was observed . These results suggest that dead cell lysis in stationary-phase E . coli occurs in a sigma(E)-dependent fashion . We propose there is a novel physiological function of the sigma(E) regulon that may guarantee cell survival in prolonged stationary phase by providing nutrients from dead cells for the next generation.

J Bacteriol, 2000 Sep, 182(18), 5167 - 71
A positive cis-acting DNA element is required for high-level transcription in Chlamydia; Schaumburg CS et al.; The spacer A/T region is a positive cis-acting DNA element that was identified in the Chlamydia trachomatis rRNA promoter region . We have now demonstrated that similar sequences in other chlamydial promoters are important for transcription . Substitution of candidate spacer A/T regions in four chlamydial promoters decreased transcription by partially purified C . trachomatis RNA polymerase in an in vitro transcription assay . Addition of a spacer A/T region to the dnaK promoter, which does not contain an identifiable spacer A/T region, increased transcription 16-fold . Transcription of Escherichia coli promoters by C . trachomatis RNA polymerase also appeared to be dependent on the spacer A/T region . However, the effect of the spacer A/T region on transcription by E . coli RNA polymerase was small . In summary, the spacer A/T region is a novel DNA element that is required for high-level transcription of many promoters by chlamydial RNA polymerase.

J Bacteriol, 2000 Sep, 182(18), 5153 - 66
ZipA-induced bundling of FtsZ polymers mediated by an interaction between C-terminal domains; Hale CA et al.; FtsZ and ZipA are essential components of the septal ring apparatus, which mediates cell division in Escherichia coli . FtsZ is a cytoplasmic tubulin-like GTPase that forms protofilament-like homopolymers in vitro . In the cell, the protein assembles into a ring structure at the prospective division site early in the division cycle, and this marks the first recognized event in the assembly of the septal ring . ZipA is an inner membrane protein which is recruited to the nascent septal ring at a very early stage through a direct interaction with FtsZ . Using affinity blotting and protein localization techniques, we have determined which domain on each protein is both sufficient and required for the interaction between the two proteins in vitro as well as in vivo . The results show that ZipA binds to residues confined to the 20 C-terminal amino acids of FtsZ . The FtsZ binding (FZB) domain of ZipA is significantly larger and encompasses the C-terminal 143 residues of ZipA . Significantly, we find that the FZB domain of ZipA is also required and sufficient to induce dramatic bundling of FtsZ protofilaments in vitro . Consistent with the notion that the ability to bind and bundle FtsZ polymers is essential to the function of ZipA, we find that ZipA derivatives lacking an intact FZB domain fail to support cell division in cells depleted for the native protein . Interestingly, ZipA derivatives which do contain an intact FZB domain but which lack the N-terminal membrane anchor or in which this anchor is replaced with the heterologous anchor of the DjlA protein also fail to rescue ZipA(-) cells . Thus, in addition to the C-terminal FZB domain, the N-terminal domain of ZipA is required for ZipA function . Furthermore, the essential properties of the N domain may be more specific than merely acting as a membrane anchor.

J Bacteriol, 2000 Sep, 182(18), 5076 - 81
Recognition of overlapping nucleotides by AraC and the sigma subunit of RNA polymerase; Dhiman A et al.; The Escherichia coli promoter p(BAD), under the control of the AraC protein, drives the expression of mRNA encoding the AraB, AraA, and AraD gene products of the arabinose operon . The binding site of AraC at p(BAD) overlaps the RNA polymerase -35 recognition region by 4 bases, leaving 2 bases of the region not contacted by AraC . This overlap raises the question of whether AraC substitutes for the sigma subunit of RNA polymerase in recognition of the -35 region or whether both AraC and sigma make important contacts with the DNA in the -35 region . If sigma does not contact DNA near the -35 region, p(BAD) activity should be independent of the identity of the bases in the hexamer region that are not contacted by AraC . We have examined this issue in the p(BAD) promoter and in a second promoter where the AraC binding site overlaps the -35 region by only 2 bases . In both cases promoter activity is sensitive to changes in bases not contacted by AraC, showing that despite the overlap, sigma does read DNA in the -35 region . Since sigma and AraC are thus closely positioned at p(BAD), it is possible that AraC and sigma contact one another during transcription initiation . DNA migration retardation assays, however, showed that there exists only a slight degree of DNA binding cooperativity between AraC and sigma, thus suggesting either that the normal interactions between AraC and sigma are weak or that the presence of the entire RNA polymerase is necessary for significant interaction.

J Biomol NMR, 2000 Jul, 17(3), 195 - 202
{15N,1H}/{13C,1H}-TROSY for simultaneous detection of backbone 15N-1H, aromatic 13C-1H and side-chain 15N-1H2 correlations in large proteins; Pervushin K et al.; This paper describes a {15N,1H}/{13C,1H}-TROSY experiment for the simultaneous acquisition of the heteronuclear chemical shift correlations of backbone amide 15N-1H groups, side chain 15N-1H2 groups and aromatic 13C-1H groups in otherwise highly deuterated proteins . The 15N-1H and 13C-1H correlations are extracted from two subspectra of the same data set, thus preventing possible spectral overlap of aromatic and amide protons in the 1H dimension . The side-chain 15N-1H2 groups, which are suppressed in conventional {15N,1H)-TROSY, are observed with high sensitivity in the 15N-1H subspectrum . {15N,1H}/{13C,1H}-TROSY was used as the heteronuclear correlation block in a 3D {1H,1H}-NOESY-{15N,1H}/{13C,1H}-TROSY experiment with the membrane protein OmpA reconstituted in detergent micelles of molecular weight 80000 Da, which enabled the detection of numerous NOEs between backbone amide protons and both aromatic protons and side chain 15N-1H2 groups.

Adv Exp Med Biol, 2000, 480, 295 - 305
Immunological aspects of pregnancy-associated glycoproteins; Dosogne H et al.; The incidence of severe cases of acute E . coli mastitis in dairy cows is highest during early lactation . This phenomenon has been associated with a decreased function and decreased numbers of circulating polymorphonuclear neutrophil leukocytes (PMN) . The cause of this impaired function and decreased number is poorly understood . Stress, hormonal and metabolic alterations around parturition and the onset of lactation may play a role in this phenomenon . Several molecules, such as cortisol and beta-hydroxybutyrate have been found to alter the oxidative burst activity of circulating PMN around parturition . Pregnancy-Associated Glycoprotein (bPAG) could also be involved . The theory of immunosuppression by bPAG was investigated because analogous glycoproteins produced by the placenta of other species exert local immunosuppression in order to maintain the histoincompatible feto-maternal unit . The production and subsequent release into the maternal circulation of bPAG is ensured by the binucleate cells from the trophoblast and starts already at implantation . However, peak levels are only reached 1 week before parturition . Due to the long half-life time of this molecule, high levels are found in plasma until 2 weeks after calving . The co-occurrence of the impairment of PMN oxidative burst activity in the early postpartum period and a peak in plasma bPAG concentrations might support the hypothesis of an immunosuppressive effect of PAG . Moreover, an inhibitory effect of bPAG on the proliferation of bovine bone marrow progenitor cells has been found recently in our laboratory . bPAG occurs in colostrum, but its effect on milk cells has not been clarified . It is concluded that interaction between the physiology of reproduction and lactation on the one side and immune function on the other side in dairy cattle requires further research.

J Dermatol Sci, 2000 Aug, 23(3), 183 - 90
Production of the entire extracellular domain of BP180 (type XVII collagen) by baculovirus expression; Hata Y et al.; Bullous pemphigoid (BP) is an acquired autoimmune skin disease, and its target antigens are a 230 kDa plaque protein (BP230) and a 180 kDa transmembrane protein with interrupted collagenous domains (BP180, type XVII collagen), which localize at the hemidesmosome . In this study we have attempted to express the entire extracellular domain of BP180 (rBP180EC) as a secreted protein by baculovirus expression . Seventy out of 83 BP sera (84.4%) showed positive reactivity against rBP180EC by immunoblot analysis, and 56 out of 83 BP sera (67.5%) were positive against rBP180EC by ELISA . These figures were comparable with those when a bacterial recombinant protein encoding the NC16a domain of BP180 (rNC16a) was used as an antigen source . Reactivity of BP sera against rBP180EC by ELISA was completely abolished or significantly reduced by immunocompetition with rNC16a in 11 out of 14 BP sera tested, while the reactivity was not altered in the rest of the three sera . These findings indicate that the NC16a domain represents the major epitopes on the extracellular domain of BP180, although there are some other minor epitopes outside of NC16a which are uniquely expressed by rBP180EC . rBP180EC will be useful to develop a diagnostic tool for BP as well as to dissect a molecular role for BP180 in interactions of keratinocytes with epidermal basement membrane.

Virus Res, 2000 Jul, 68(2), 155 - 60
Rubella virus nonstructural protein 2 is a minor immunogen; Hofmann J et al.; The full-length nonstructural protein P90 of rubella virus (RV) was expressed as recombinant protein in Escherichia coli bacteria, as well as in Vero cells . Monoclonal antibodies raised against the protein specifically reacted with the protein in both P90-transfected and RV infected Vero cells . Ninety human sera obtained from reconvalescents, vaccinees and patients with acute RV infection were tested for reactivity against the P90 protein . A weak immune reaction was detected only in a small minority (8%), indicating that P90 is minor immunogen for RV and is not suitable for diagnostic purposes.

Virus Res, 2000 Jul, 68(2), 99 - 107
Construction of a stable and highly infectious intron-containing cDNA clone of plum pox potyvirus and its use to infect plants by particle bombardment; Lopez-Moya JJ et al.; An infectious plum pox potyvirus cDNA clone was constructed placing a copy of the full-length sequence of the virus genome between an enhanced cauliflower mosaic virus 35S promoter and a nopaline synthase termination signal . Stabilization of the clone and faster growth of bacteria, in addition to higher plasmid yield, followed a modification consisting of the insertion of an intron which interrupted the viral open reading frame at the P3 region . This intron-containing clone was infectious when inoculated into plants after undergoing in vivo transcription and splicing . Particle bombardment delivery of the cDNA greatly increased the efficiency of plant infection.

Mol Cell Biol, 2000 Sep, 20(18), 6816 - 25
Human genomic sequences that inhibit splicing; Fairbrother WG et al.; Mammalian genes are characterized by relatively small exons surrounded by variable lengths of intronic sequence . Sequences similar to the splice signals that define the 5' and 3' boundaries of these exons are also present in abundance throughout the surrounding introns . What causes the real sites to be distinguished from the multitude of pseudosites in pre-mRNA is unclear . Much progress has been made in defining additional sequence elements that enhance the use of particular sites . Less work has been done on sequences that repress the use of particular splice sites . To find additional examples of sequences that inhibit splicing, we searched human genomic DNA libraries for sequences that would inhibit the inclusion of a constitutively spliced exon . Genetic selection experiments suggested that such sequences were common, and we subsequently tested randomly chosen restriction fragments of about 100 bp . When inserted into the central exon of a three-exon minigene, about one in three inhibited inclusion, revealing a high frequency of inhibitory elements in human DNA . In contrast, only 1 in 27 Escherichia coli DNA fragments was inhibitory . Several previously identified silencing elements derived from alternatively spliced exons functioned weakly in this constitutively spliced exon . In contrast, a high-affinity site for U2AF65 strongly inhibited exon inclusion . Together, our results suggest that splicing occurs in a background of repression and, since many of our inhibitors contain splice like signals, we suggest that repression of some pseudosites may occur through an inhibitory arrangement of these sites.

Vet Rec, 2000 Jul 29, 147(5), 123 - 8
Clinical and laboratory findings in cases of toxic mastitis in cows in Northern Ireland; Menzies FD et al.; This paper describes the clinical and laboratory findings from 264 cases of toxic mastitis in cows in Northern Ireland between October 1995 and May 1997 . Nearly all the cases occurred during the winter housing period, with 84 per cent occurring between November and March inclusive, and 30 per cent in March . Sixty per cent of the cases occurred within one month of calving, and 29 per cent within four days of calving . The most common clinical signs were lethargy (92 per cent), discoloured milk (90 per cent), anorexia (72 per cent), tachypnoea (23 per cent), diarrhoea (23 per cent), recumbency (18 per cent) and staggering (15 per cent) . Severe pyrexia (18 per cent) and clinical dehydration (44 per cent) were relatively common findings . Pure growths of Escherichia coli were isolated from 50 per cent of the milk samples, but 11 per cent yielded no bacterial growth . In vitro sensitivity tests indicated that enrofloxacin was effective against 98 per cent of the bacteria isolated, and framycetin and amoxycillin/clavulanic acid against 91 per cent . Abnormally high blood urea levels were observed in 31 per cent of cases, high blood creatinine levels in 42 per cent, and severe leucopenia in 56 per cent . Of the cases which were followed up, 14 per cent died, 21 per cent were culled early and a further 22 per cent lost milk production from the affected quarter.

Acta Crystallogr D Biol Crystallogr, 2000 Sep, 56 ( Pt 9), 1194 - 7
Crystallization and preliminary X-ray diffraction studies of the peptide methionine sulfoxide reductase from Escherichia coli; Tete-Favier F et al.; Peptide methionine sulfoxide reductase mediates the reduction of protein sulfoxide methionyl residues back to methionines and could thus be implicated in the antioxidant defence of organisms . Hexagonal crystals of the Escherichia coli enzyme (MsrA) were obtained by the hanging-drop vapour-diffusion technique . They belong to space group P6(5)22, with unit-cell parameters a = b = 102.5, c = 292.3 A, gamma = 120 degrees . A native data set was collected at 1.9 A resolution . Crystals of selenomethionine-substituted MsrA were also grown under the same crystallization conditions . A three-wavelength MAD experiment has led to the elucidation of the positions of the Se atoms and should result in a full structure determination.

Acta Crystallogr D Biol Crystallogr, 2000 Sep, 56 ( Pt 9), 1191 - 3
Purification, crystallization and preliminary crystallographic data for rat cytosolic selenocysteine 498 to cysteine mutant thioredoxin reductase; Zhong L et al.; Mammalian cytosolic thioredoxin reductase is a homodimer of 55 kDa subunit containing an essential penultimate selenocysteine residue . An active analogue of the rat enzyme in which cysteine replaces selenocysteine has been expressed in Escherichia coli cells at high levels and purified to homogeneity . The pure enzyme contains one FAD per subunit and shows spectral properties identical to that of the wild-type thioredoxin reductase . The isolated enzyme in its oxidized and reduced forms or the enzyme complexed with NADP(+) was crystallized by the hanging-drop vapour-diffusion method . The diffraction pattern extends to 3 A resolution . The crystals are monoclinic, space group P2(1), with unit-cell parameters a = 78.9, b = 140.5, c = 170.8 A, alpha = 94.6 degrees . There are three dimeric molecules in the asymmetric unit.

Acta Crystallogr D Biol Crystallogr, 2000 Sep, 56 ( Pt 9), 1185 - 6
Crystallographic study of intein homing endonuclease II encoded in the archaeal DNA polymerase gene; Hashimoto H et al.; Intein homing endonucleases are proteins spliced out from a precursor protein and site-specific enzymes that make double-strand breaks in inteinless alleles . Crystals of intein homing endonuclease II from the hyperthermophilic archaeon Pyrococcus kodakaraensis strain KOD1 (PI-PkoII) have been grown at room temperature using ammonium sulfate as a precipitant . The diffraction pattern of the crystal extends to 3.0 A resolution at room temperature upon exposure to synchrotron X-rays at KEK-PF, Japan . The crystals have symmetry consistent with space group C222(1), with unit-cell parameters a = 107.6, b = 150.5, c = 146.8 A . A full set of X-ray diffraction data were collected to 3.0 A Bragg spacing from a native crystal with an overall R(merge) of 4.8% and a completeness of 96.6%.

Acta Crystallogr D Biol Crystallogr, 2000 Sep, 56 ( Pt 9), 1180 - 2
Crystallization and preliminary X-ray diffraction analysis of a rat biliverdin reductase; Sun D et al.; Biliverdin reductase (BVR) catalyzes the final step of haem degradation and converts biliverdin to bilirubin using NAD(P)H as an electron donor . This paper deals with the first crystallization and preliminary crystallographic study of recombinant rat BVR expressed in Escherichia coli . Crystals of BVR were obtained by the sitting-drop vapour-diffusion method . Using synchrotron radiation at station BL44B2 of SPring-8, Japan, BVR diffraction data were collected to 1.6 A resolution . Crystals belong to the orthorhombic space group P2(1)2(1)2(1), with unit-cell parameters a = 58.89, b = 70.41, c = 87.76 A . The complete determination of the crystallographic structure is currently in progress using MAD (multiwavelength anomalous diffraction) data from an Ir-derivative crystal.

Acta Crystallogr D Biol Crystallogr, 2000 Sep, 56 ( Pt 9), 1170 - 2
Crystallization of the dI component of transhydrogenase, a proton-translocating membrane protein; Sedelnikova SE et al.; Nicotinamide nucleotide transhydrogenase couples the exchange of a hydride-ion equivalent between NAD(H) and NADP(H) to the translocation of protons across an energy-transducing membrane . Peripheral components of 380 and 200 residues bind NAD(H) (dI) and NADP(H) (dIII), respectively, while a third component forms a membrane-spanning region (dII) . The NAD(H)-binding component dI of Rhodospirillum rubrum transhydrogenase has been crystallized in a form which diffracts to beyond 3.0 A resolution and is in space group P2 or P2(1), with unit-cell parameters a = 69.3, b = 117.8, c = 106.6 A, beta = 107.2 degrees and two dimers in the asymmetric unit . The sequence of the dI component is similar to that of alanine dehydrogenase . A full structure determination will lead to important information on the mode of action of this proton pump and will permit the comparison of the structure-function relationships of dI with those of alanine dehydrogenase.

Acta Crystallogr D Biol Crystallogr, 2000 Sep, 56 ( Pt 9), 1166 - 9
Crystallization, preliminary X-ray analysis of a native and selenomethionine D-hydantoinase from Thermus sp; Abendroth J et al.; A D-hydantoinase from Thermus sp . was expressed in Escherichia coli, purified to homogeneity and crystallized both as native and Se-Met labelled protein . The crystals belong to the orthorhombic space group C222(1), with unit-cell parameters a = 125.9, b = 215.8, c = 207.5 A . A three-wavelength MAD data set was collected to 2.5 A resolution and a native data set was collected to 1.7 A resolution . Crystal packing and self-rotation calculations led to the assumption of six protomers per asymmetric unit, corresponding to a V(M) value of 2.28 A(3) Da(-1) and a solvent content of 46% . As each protomer contains nine Se-Met residues, 54 selenium sites per asymmetric unit were present and could be unambigously located in the course of the MAD experiment . This selenium substructure is one of the largest selenium substructures that have been solved to date . The resulting phases obtained at a high-resolution limit of 3.0 A could be extended to 1.7 A and refined by application of density-modification techniques, especially non-crystallographic symmetry.

J Biol Chem, 2000 Nov 10, 275(45), 34867 - 72
The second naphthol reductase of fungal melanin biosynthesis in Magnaporthe grisea: tetrahydroxynaphthalene reductase; Thompson JE et al.; Mutants of Magnaporthe grisea harboring a defective gene for 1,3, 8-trihydroxynaphthalene reductase retain the capability to produce scytalone, thus suggesting the existence of a second naphthol reductase that can catalyze the reduction of 1,3,6, 8-tetrahydroxynaphthalene to scytalone within the fungal melanin biosynthetic pathway . The second naphthol reductase gene was cloned from M . grisea by identification of cDNA fragments with weak homology to the cDNA of trihydroxynaphthalene reductase . The amino acid sequence for the second naphthol reductase is 46% identical to that of trihydroxynaphthalene reductase . The second naphthol reductase was produced in Esherichia coli and purified to homogeneity . Substrate competition experiments indicate that the second reductase prefers tetrahydroxynaphthalene over trihydroxynaphthalene by a factor of 310; trihydroxynaphthalene reductase prefers trihydroxynaphthalene over tetrahydroxynaphthalene by a factor of 4.2 . On the basis of the 1300-fold difference in substrate specificities between the two reductases, the second reductase is designated tetrahydroxynaphthalene reductase . Tetrahydroxynaphthalene reductase has a 200-fold larger K(i) for the fungicide tricyclazole than that of trihydroxynaphthalene reductase, and this accounts for the latter enzyme being the primary physiological target of the fungicide . M . grisea mutants lacking activities for both trihydroxynaphthalene and tetrahydroxynaphthalene reductases do not produce scytalone, indicating that there are no other metabolic routes to scytalone.

J Biol Chem, 2000 Nov 10, 275(45), 35471 - 7
Identification of repair enzymes for 5-formyluracil in DNA . Nth, Nei, and MutM proteins of Escherichia coli; Zhang QM et al.; 5-Formyluracil (5-foU) is a potentially mutagenic lesion of thymine produced in DNA by ionizing radiation and various chemical oxidants . Although 5-foU has been reported to be removed from DNA by Escherichia coli AlkA protein in vitro, its repair mechanisms are not fully understood . In this study, we used the borohydride trapping assay to detect and characterize repair activities for 5-foU in E . coli extracts with site-specifically designed oligonucleotides containing a 5-foU at defined sites . The trapping assay revealed that there are three kinds of proteins that form covalent complexes with the 5-foU-containing oligonucleotides . Extracts from strains defective in the nth, nei, or mutM gene lacked one of the proteins . All of the trapped complexes were completely lost in extracts from the nth nei mutM triple mutant . The introduction of a plasmid carrying the nth, nei, or mutM gene into the E . coli triple mutant restored the formation of the corresponding protein-DNA complex . Purified Nth, Nei, and MutM proteins were trapped by the 5-foU-containing oligonucleotide to form the complex in the presence of NaBH(4) . Furthermore, the purified Nth, Nei, and MutM proteins efficiently cleaved the oligonucleotide at the 5-foU site . In addition, 5-foU was site-specifically incorporated into plasmid pSVK3, and the resulting plasmid was replicated in E . coli . The mutation frequency of the plasmid was significantly increased in the E . coli nth nei mutM alkA mutant, compared with the wild-type and alkA strains . From these results it is concluded that the Nth, Nei, and MutM proteins are involved in the repair pathways for 5-foU that serve to avoid mutations in E . coli.

J Biol Chem, 2000 Nov 17, 275(46), 35825 - 30
Studies with substrate and cofactor analogues provide evidence for a radical mechanism in the chorismate synthase reaction; Osborne A et al.; Chorismate synthase catalyzes the conversion of 5-enolpyruvylshikimate 3-phosphate (EPSP) to chorismate . The strict requirement for a reduced FMN cofactor and a trans-1,4-elimination are unusual . (6R)-6-Fluoro-EPSP was shown to be converted to chorismate stoichiometrically with enzyme-active sites in the presence of dithionite . This conversion was associated with the oxidation of FMN to give a stable flavin semiquinone . The IC(50) of the fluorinated substrate analogue was 0.5 and 250 microm with the Escherichia coli enzyme, depending on whether it was preincubated with the enzyme or not . The lack of dissociation of the flavin semiquinone and chorismate from the enzyme appears to be the basis of the essentially irreversible inhibition by this analogue . A dithionite-dependent transient formation of flavin semiquinone during turnover of (6S)-6-fluoro-EPSP has been observed . These reactions are best rationalized by radical chemistry that is strongly supportive of a radical mechanism occurring during normal turnover . The lack of activity with 5-deaza-FMN provides additional evidence for the role of flavin in catalysis by the E . coli enzyme.

J Biol Chem, 2000 Nov 10, 275(45), 35457 - 70
A pollen-specific novel calmodulin-binding protein with tetratricopeptide repeats; Safadi F et al.; Calcium is essential for pollen germination and pollen tube growth . A large body of information has established a link between elevation of cytosolic Ca(2+) at the pollen tube tip and its growth . Since the action of Ca(2+) is primarily mediated by Ca(2+)-binding proteins such as calmodulin (CaM), identification of CaM-binding proteins in pollen should provide insights into the mechanisms by which Ca(2+) regulates pollen germination and tube growth . In this study, a CaM-binding protein from maize pollen (maize pollen calmodulin-binding protein, MPCBP) was isolated in a protein-protein interaction-based screening using (35)S-labeled CaM as a probe . MPCBP has a molecular mass of about 72 kDa and contains three tetratricopeptide repeats (TPR) suggesting that it is a member of the TPR family of proteins . MPCBP protein shares a high sequence identity with two hypothetical TPR-containing proteins from Arabidopsis . Using gel overlay assays and CaM-Sepharose binding, we show that the bacterially expressed MPCBP binds to bovine CaM and three CaM isoforms from Arabidopsis in a Ca(2+)-dependent manner . To map the CaM-binding domain several truncated versions of the MPCBP were expressed in bacteria and tested for their ability to bind CaM . Based on these studies, the CaM-binding domain was mapped to an 18-amino acid stretch between the first and second TPR regions . Gel and fluorescence shift assays performed with CaM and a CaM-binding synthetic peptide further confirmed MPCBP binding to CaM . Western, Northern, and reverse transcriptase-polymerase chain reaction analysis have shown that MPCBP expression is specific to pollen . MPCBP was detected in both soluble and microsomal proteins . Immunoblots showed the presence of MPCBP in mature and germinating pollen . Pollen-specific expression of MPCBP, its CaM-binding properties, and the presence of TPR motifs suggest a role for this protein in Ca(2+)-regulated events during pollen germination and growth.

J Biol Chem, 2000 Nov 10, 275(45), 34989 - 97
Thyroid hormone regulates carnitine palmitoyltransferase Ialpha gene expression through elements in the promoter and first intron; Jansen MS et al.; Carnitine palmitoyltransferase I (CPT-I) catalyzes the transfer of long chain fatty acyl groups from CoA to carnitine for translocation across the mitochondrial inner membrane . CPT-Ialpha is a key regulatory enzyme in the oxidation of fatty acids in the liver . CPT-Ialpha is expressed in all tissues except skeletal muscle and adipose tissue, which express CPT-Ibeta . Expression of CPT-Ialpha mRNA and enzyme activity are elevated in the liver in hyperthyroidism, fasting, and diabetes . CPT-Ialpha mRNA abundance is increased 40-fold in the liver of hyperthyroid compared with hypothyroid rats . Here, we examine the mechanisms by which thyroid hormone (T3) stimulates CPT-Ialpha gene expression . Four potential T3 response elements (TRE), which contain direct repeats separated by four nucleotides, are located 3000-4000 base pairs 5' to the start site of transcription in the CPT-Ialpha gene . However, only one of these elements functions as a TRE . This TRE binds the T3 receptor as well as other nuclear proteins . Surprisingly, the first intron of the CPT-Ialpha gene is required for the T3 induction of CPT-Ialpha expression, but this region of the gene does not contain a TRE . In addition, we show that CPT-Ialpha is induced by T3 in cell lines of hepatic origin but not in nonhepatic cell lines.

J Med Chem, 2000 Jul 27, 43(15), 2929 - 37
Discovery of novel and potent retinoic acid receptor alpha agonists: syntheses and evaluation of benzofuranyl-pyrrole and benzothiophenyl-pyrrole derivatives; Yoshimura H et al.; In the course of our studies on retinoic acid receptor (RAR) agonists, we have designed and synthesized a series of benzofuran and benzothiophene derivatives . Some of these compounds (1a,b,e,f,j) markedly inhibited LPS-induced B-lymphocyte proliferation and exerted RARalpha selectivity . One of them, 4-{5-(4,7-dimethylbenzofuran-2-yl)pyrrol-2-yl}benzoic acid (1b), when orally administered significantly inhibited mouse antibody production and delayed type hypersensitivity (DTH) responses from a dose of 0.1 mg/kg.

Biochemistry, 2000 Aug 29, 39(34), 10454 - 67
Interactions of Escherichia coli replicative helicase PriA protein with single-stranded DNA; Jezewska MJ et al.; Quantitative analyses of the interactions of the Escherichia coli replicative helicase PriA protein with a single-stranded DNA have been performed, using the thermodynamically rigorous fluorescence titration technique . The analysis of the PriA helicase interactions with nonfluorescent, unmodified nucleic acids has been performed, using the macromolecular competition titration (MCT) method . Thermodynamic studies of the PriA helicase binding to ssDNA oligomers, as well as competition studies, show that independently of the type of nucleic acid base, as well as the salt concentration, the type of salt in solution, and nucleotide cofactors, the PriA helicase binds the ssDNA as a monomer . The enzyme binds the ssDNA with significant affinity in the absence of any nucleotide cofactors . Moreover, the presence of AMP-PNP diminishes the intrinsic affinity of the PriA protein for the ssDNA by a factor approximately 4, while ADP has no detectable effect . Analyses of the PriA interactions with different ssDNA oligomers, over a large range of nucleic acid concentrations, indicates that the enzyme has a single, strong ssDNA-binding site . The intrinsic affinities are salt-dependent . The formation of the helicase-ssDNA complexes is accompanied by a net release of 3-4 ions . The experiments have been performed with ssDNA oligomers encompassing the total site size of the helicase-ssDNA complex and with oligomers long enough to encompass only the ssDNA-binding site of the enzyme . The obtained results indicate that salt dependence of the intrinsic affinity results predominantly, if not exclusively, from the interactions of the ssDNA-binding site of the helicase with the nucleic acid . There is an anion effect on the studied interactions, which suggests that released ions originate from both the protein and the nucleic acid . Contrary to the intrinsic affinities, cooperative interactions between bound PriA molecules are accompanied by a net uptake of approximately 3 ions . The PriA protein shows preferential intrinsic affinity for pyrimidine ssDNA oligomers . In our standard conditions (pH 7.0, 10 degrees C, 100 mM NaCl), the intrinsic binding constant for the pyrimidine oligomers is approximately 1 order of magnitude higher than the intrinsic binding constant for the purine oligomers . The significance of these results for the mechanism of action of the PriA helicase is discussed.

Biochemistry, 2000 Aug 29, 39(34), 10431 - 8
In vitro replication of primer-templates containing benzo{a}pyrene adducts by exonuclease-deficient Escherichia coli DNA polymerase I (Klenow fragment): effect of sequence context on lesion bypass; Alekseyev YO et al.; The presence of benzo{a}pyrene diol epoxide (B{a}PDE) adducts in DNA is known to interfere with DNA replication . Kinetic studies of nucleotide insertion by exonuclease-deficient E . coli DNA polymerase I (Klenow fragment) across from either the (+)-trans- or the (+)-cis-B{a}P-N(2)-dG adduct in the 5'-CGT-3' sequence context indicated that the rate of nucleotide incorporation followed the order: dAMP > dGMP > dTMP > dCMP, which did not correlate with the mutational spectrum observed for these adducts in this sequence in E . coli (mostly G-->A transitions) . Interestingly, a kinetic analysis of extension past the adduct showed that, unlike other sequences studied, the primer-template was extended best when dT was positioned at the 3'-terminus of the primer across from either a (+)-trans- or a (+)-cis-B{a}P-N(2)-dG adduct . In contrast, when the (+)-trans-B{a}P-N(2)-dG adduct was positioned in the 5'-TGC-3' sequence context, which gives predominantly G-->T mutations in E . coli, extension was detectable only when dA was positioned across from the adduct . These data provide the first in vitro evidence that may explain why G-->A transitions, rather than the G-->T transversions found in other sequences, are preferred in the 5'-CGT-3' sequence in vivo.

Biochemistry, 2000 Aug 22, 39(33), 10224 - 35
Escherichia coli double-strand uracil-DNA glycosylase: involvement in uracil-mediated DNA base excision repair and stimulation of activity by endonuclease IV; Sung JS et al.; Escherichia coli double-strand uracil-DNA glycosylase (Dug) was purified to apparent homogeneity as both a native and recombinant protein . The molecular weight of recombinant Dug was 18 670, as determined by matrix-assisted laser desorption-ionization mass spectrometry . Dug was active on duplex oligonucleotides (34-mers) that contained site-specific U.G, U.A, ethenoC.G, and ethenoC.A targets; however, activity was not detected on DNA containing a T.G mispair or single-stranded DNA containing either a site-specific uracil or ethenoC residue . One of the distinctive characteristics of Dug was that the purified enzyme excised a near stoichiometric amount of uracil from U.G-containing oligonucleotide substrate . Electrophoretic mobility shift assays revealed that the lack of turnover was the result of strong binding by Dug to the reaction product apyrimidinic-site (AP) DNA . Addition of E . coli endonuclease IV stimulated Dug activity by enhancing the rate and extent of uracil excision by promoting dissociation of Dug from the AP . G-containing 34-mer . Catalytically active endonuclease IV was apparently required to mediate Dug turnover, since the addition of 5 mM EDTA mitigated the effect . Further support for this interpretation came from the observations that Dug preferentially bound 34-mer containing an AP.G target, while binding was not observed on a substrate incised 5' to the AP-site . We also investigated whether Dug could initiate a uracil-mediated base excision repair pathway in E . coli NR8052 cell extracts using M13mp2op14 DNA (form I) containing a site-specific U.G mispair . Analysis of reaction products revealed a time dependent appearance of repaired form I DNA; addition of purified Dug to the cell extract stimulated the rate of repair.

Biochemistry, 2000 Aug 22, 39(33), 10196 - 206
RecA realigns suboptimally paired frames of DNA repeats through a process that requires ATP hydrolysis; Sen S et al.; Microsatellite repeats such as mono-, di-, and trinucleotides are highly abundant and viable targets for homologous recombination in the genome . However, if recombination ensues in such repetitive regions, they are intrinsically prone to frame misalignments during pairing and might eventually give rise to genetic instabilities . Suboptimally paired frames lead to an abrogation of branch migration at the junctions of mixed sequences and repeats, due to a heterologous register . If so, can recombination machinery rectify such misalignments in order to avoid subsequent arrest in branch migration? We analyzed Escherichia coli RecA, the universal prototype of a recombinase, for its pairing abilities across repeats . We used a complementary pairing assay to test whether RecA can mediate realignments of stochastically paired suboptimal frames to a maximally aligned register . Here, we demonstrate that RecA-single stranded DNA filament indeed facilitates such a realignment, probably by sliding the paired strands across mono- and di- as well as trinucleotide repeats . These realignments apparently have no net directional bias . Such a putative "motor" function of RecA seems to be ATP hydrolysis-dependent.

Biochemistry, 2000 Aug 22, 39(33), 10098 - 109
A single engineered point mutation in the adenine glycosylase MutY confers bifunctional glycosylase/AP lyase activity; Williams SD et al.; The E . coli adenine glycosylase MutY is a member of the base excision repair (BER) superfamily of DNA repair enzymes . MutY plays an important role in preventing mutations caused by 7, 8-dihydro-8-oxo-2'-deoxyguanosine (OG) by removing adenine from OG:A base pairs . Some enzymes of the BER superfamily catalyze a strand scission even concomitant with base removal . These bifunctional glycosylase/AP lyases bear a conserved lysine group in the active site region, which is believed to be the species performing the initial nucleophilic attack at C1' in the catalysis of base removal . Monofunctional glycosylases such as MutY are thought to perform this C1' nucleophilic displacement by a base-activated water molecule, and, indeed, the conservation of amine functionality positioning has not been observed in protein sequence alignments . Bifunctional glycosylase/AP lyase activity was successfully engineered into MutY by replacing serine 120 with lysine . MutY S120K is capable of catalyzing DNA strand scission at a rate equivalent to that of adenine excision for both G:A and OG:A mispair substrates . The extent of DNA backbone cleavage is independent of treating reaction aliquots with 0.1 M NaOH . Importantly, the replacement of the serine with lysine results in a catalytic rate that is compromised by at least 20-fold . The reduced efficiency in the glycosylase activity is also reflected in a reduced ability of S120K MutY to prevent DNA mutations in vivo . These results illustrate that the mechanisms of action of the two classes of these enzymes are quite similar, such that a single amino acid change is sufficient, in the case of MutY, to convert a monofunctional glycosylase to a bifunctional glycosylase/AP lyase.

Biochemistry, 2000 Aug 22, 39(33), 10017 - 22
Unusual catalytic triad of Escherichia coli outer membrane phospholipase A; Kingma RL et al.; Escherichia coli outer membrane phospholipase A (OMPLA) is an integral membrane enzyme . OMPLA is active as a homodimer and requires calcium as a cofactor . The crystal structures of the monomeric and the inhibited dimeric enzymes were recently determined {Snijder, H . J., et al . (1999) Nature 401, 717-721} and revealed that OMPLA monomers are folded into a 12-stranded antiparallel beta-barrel . The active site consists of previously identified essential residues Ser144 and His142 in an arrangement resembling the corresponding residues of a serine hydrolase catalytic triad . However, instead of an Asp or Glu that normally is present in the triad of serine hydrolases, a neutral asparagine (Asn156) was found in OMPLA . In this paper, the importance of the catalytic Asn156 is addressed by site-directed mutagenesis studies . All variants were purified at a 30 mg scale, and were shown to be properly folded using SDS-PAGE and circular dichroism spectroscopy . Using chemical cross-linking, it was shown that all variants were not affected in their calcium-dependent dimerization properties . The Asn156Asp variant exhibited a 2-fold lower activity than wild-type OMPLA at neutral pH . Interestingly, the activity of the variant is 1 order of magnitude higher than that of the wild type at pH >10 . Modest residual activities (5 and 2.5%, respectively) were obtained for the Asn156Ala and Asn156Gln mutants, showing that the active site of OMPLA is more tolerant toward replacements of this third residue of the catalytic triad than other serine hydrolases, and that the serine and histidine residues are minimally required for catalysis . In the X-ray structure of dimeric OMPLA, the cofactor calcium is coordinating the putative oxyanion via two water molecules . We propose that this may lessen the importance for the asparagine in the catalytic triad of OMPLA.

Microbios, 2000, 102(403), 135 - 45
Oral administration of Escherichia coli glutamic acid decarboxylase has immunomodulatory effects in streptozotocin-induced diabetes; Yazdchi-Marandi L et al.; The extent of destruction of insulin-secreting beta cells of the Islets of Langerhans was investigated in an animal model using oral administration of glutamic acid decarboxylase (GAD) isolated from Escherichia coli . The extent of lymphocytic infiltration of the pancreatic Islet cells and the severity of diabetes were significantly reduced by oral administration of GAD to rats 14 days before intraperitoneal injections of streptozotocin (STZ, 40 mg/kg body wt on 5 consecutive days) . In addition, oral administration of GAD to rats 14 days before or 3 days after STZ treatment significantly (p <0.05) reduced the levels of GAD-specific antibodies and improved the in vitro proliferative response of splenocytes to concanavalin A (Con A) . These data demonstrate that oral GAD administration probably generates active cellular mechanisms which suppress the disease and therefore raise the possibility of using E . coli GAD as a new means for the immunomodulation of autoimmune diabetes.

Allergy, 2000 Aug, 55(8), 712 - 7
PCR-based cloning, isolation, and IgE-binding properties of recombinant latex profilin (rHev b 8); Rihs HP et al.; BACKGROUND: Profilin (Hev b 8) in natural rubber latex (NRL) has been assumed to be an important allergen . Since latex profilin has a molecular mass similar to two other latex allergens (Hev b 1 and Hev b 6.03) in the 14-kDa range, it is difficult to obtain sufficient amounts of purified native profilin for investigations and diagnostics . The present study aimed to produce recombinant latex profilin (rHev b 8) and study its IgE-binding reactivity . METHODS: A profilin-specific cDNA encoding the latex profilin from Hevea brasiliensis leaves was synthesized and subcloned, and the rHev b 8 was overexpressed in fusion with the maltose-binding protein (MBP) in E . coli . The IgE-binding reactivity of rHev b 8 was studied by immunoblotting, immunoblot inhibition experiments, and the Pharmacia CAP method, with 25 sera from health-care workers with latex allergy and 17 sera from latex-sensitive spina bifida patients . RESULTS: rHev b 8 was found to have 131 amino acids and a sequence identity of 75% with birch profilin (Bet v 2) . Analysis by the CAP system revealed the presence of rHev b 8-specific IgE antibodies in two out of 17 sera from spina bifida patients and in five out of 25 sera (20%) from health-care workers . Two subjects of the latter group with rHev b 8-specific IgE showed negative results in the skin prick tests with tree-pollen extracts and had no IgE to rBet v 2, indicating the presence of IgE-binding epitopes on the Hev b 8-molecule which do not cross-react with birch profilin . Immunoblot inhibition assays using MBP-rHev b 8 as inhibitor confirmed the presence of latex profilin in the NRL extract . IgE binding to the native latex profilin could be completely inhibited by the MBP-rHev b 8 . CONCLUSIONS: Latex profilin represents a minor allergen in NRL and may have IgE-binding epitopes different from Bet v 2.

Pediatr Surg Int, 2000, 16(5-6), 440 - 2
Indications for laparotomy in infection with verotoxigenic Escherichia coli; Vanamo K; Verotoxigenic types of Escherichia coli have emerged as serious and important human pathogens . The clinical disease most frequently manifests as a painful form of bloody diarrhea, which can progress to life-threatening systemic microangiopathic hemolytic anemia, known as the hemolytic-uremic syndrome (HUS) . Three children with hemorrhagic enteritis due to verotoxigenic E . coli are presented to illustrate the unique diagnostic, therapeutic, and operative management dilemmas associated with this disease . When contemplating surgery, one should seek to determine the anatomic and transmural extent of the disease.

Hum Gene Ther, 2000 Aug 10, 11(12), 1625 - 35
Expression of wild-type and noncleavable Fas ligand by tetracycline-regulated adenoviral vectors to limit intimal hyperplasia in vascular lesions; Mano T et al.; Proliferation of vascular smooth muscle cells (VSMCs) and the infiltration of T cells and macrophages into vessel wall are considered to be important for intimal lesion formation after balloon angioplasty . Previous studies have shown that Fas ligand (FasL) gene transfer to balloon-injured vessels inhibits lesion formation by killing both proliferating VSMCs and infiltrating inflammatory cells . Here, we describe the construction and utility of a binary, tetracycline-regulated adenovirus system that provides controlled transgene expression in vitro and in vivo . In this system, optimal transgene expression required cotransfection with an adenovirus encoding the tetracycline-dependent trans-activator (rtTA) and induction with doxycycline hydrochloride (DOX), an analog of tetracycline . Using this system, adenovirus constructs were designed that allow regulated expression of wild-type FasL and a noncleavable mutant of FasL (FasL-NC) . Transduction of FasL and FasL-NC induced similar extents of apoptosis in proliferating VSMCs in vitro in a manner that was dependent on the doses of the rtTA adenovirus and the presence of DOX in the medium . Furthermore, inhibition of intimal hyperplasia in injured carotid arteries by FasL or FasL-NC transduction was also dependent on cotransfection with the rtTA adenovirus and administration of DOX by subcutaneous injection . In contrast to wild-type FasL, transduction of FasL-NC did not result in the production of soluble (cleaved) FasL in the medium of infected cells in vitro, or in the serum of rats after local gene transfer to carotid arteries . In conclusion, this binary tetracycline-inducible adenovirus system may allow for safer delivery of cytotoxic genes for therapeutic purposes.

Proc Natl Acad Sci U S A, 2000 Aug 29, 97(18), 10144 - 9
Human interleukin-10-related T cell-derived inducible factor: molecular cloning and functional characterization as an hepatocyte-stimulating factor; Dumoutier L et al.; IL-10-related T cell-derived inducible factor (IL-TIF or IL-21) is a new cytokine structurally related to IL-10 and originally identified in the mouse as a gene induced by IL-9 in T cells and mast cells . Here, we report the cloning of the human IL-TIF cDNA, which shares 79% amino acid identity with mouse IL-TIF and 25% identity with human IL-10 . Recombinant human IL-TIF was found to activate signal transducer and activator of transcription factors-1 and -3 in several hepatoma cell lines . IL-TIF stimulation of HepG2 human hepatoma cells up-regulated the production of acute phase reactants such as serum amyloid A, alpha1-antichymotrypsin, and haptoglobin . Although IL-10 and IL-TIF have distinct activities, antibodies directed against the beta chain of the IL-10 receptor blocked the induction of acute phase reactants by IL-TIF, indicating that this chain is a common component of the IL-10 and IL-TIF receptors . Similar acute phase reactant induction was observed in mouse liver upon IL-TIF injection, and IL-TIF expression was found to be rapidly increased after lipopolysaccharide (LPS) injection, suggesting that this cytokine contributes to the inflammatory response in vivo.

J Biol Chem, 2000 Nov 10, 275(45), 35408 - 12
Steady state kinetic model for the binding of substrates and allosteric effectors to Escherichia coli phosphoribosyl-diphosphate synthase; Willemoes M et al.; A steady state kinetic investigation of the P(i) activation of 5-phospho-d-ribosyl alpha-1-diphosphate synthase from Escherichia coli suggests that P(i) can bind randomly to the enzyme either before or after an ordered addition of free Mg(2+) and substrates . Unsaturation with ribose 5-phosphate increased the apparent cooperativity of P(i) activation . At unsaturating P(i) concentrations partial substrate inhibition by ribose 5-phosphate was observed . Together these results suggest that saturation of the enzyme with P(i) directs the subsequent ordered binding of Mg(2+) and substrates via a fast pathway, whereas saturation with ribose 5-phosphate leads to the binding of Mg(2+) and substrates via a slow pathway where P(i) binds to the enzyme last . The random mechanism for P(i) binding was further supported by studies with competitive inhibitors of Mg(2+), MgATP, and ribose 5-phosphate that all appeared noncompetitive when varying P(i) at either saturating or unsaturating ribose 5-phosphate concentrations . Furthermore, none of the inhibitors induced inhibition at increasing P(i) concentrations . Results from ADP inhibition of P(i) activation suggest that these effectors compete for binding to a common regulatory site.

J Biol Chem, 2000 Nov 10, 275(45), 35070 - 6
Mechanism of phosphoanhydride cleavage by baculovirus phosphatase; Martins A et al.; Baculovirus phosphatase (BVP) is a member of the metazoan RNA triphosphatase enzyme family that includes the RNA triphosphatase component of the mRNA capping apparatus . BVP and other metazoan RNA triphosphatases belong to a superfamily of phosphatases that act via the formation and hydrolysis of a covalent cysteinyl-phosphate intermediate . Here we demonstrate the formation of a BVP phosphoenzyme upon reaction with {gamma-(32)P}ATP and identify the linkage as a thiophosphate based on its chemical lability . We surmise that the phosphate is linked to Cys(119) of BVP because replacement of Cys(119) by alanine or serine abrogates phosphoenzyme formation and phosphohydrolase activity . The catalytic cysteine is situated within a conserved phosphate-binding loop ((118)HCTHGINRTGY(128)) . We show that all of the non-aliphatic side chains of the phosphate-binding loop are functionally important, insofar as mutants H118A, H121A, N124A, R125A, T126A, and Y128A were inactive in gamma phosphate hydrolysis and the T120A mutant was 7% as active as wild-type BVP . Structure-activity relationships at the essential positions of the phosphate-binding loop were elucidated by conservative substitutions . A conserved aspartic acid (Asp(60)) invoked as a candidate general acid catalyst was dispensable for phosphohydrolase activity and phosphoenzyme formation by BVP . We propose that the low pK(a) of the bridging oxygen of the beta phosphate leaving group circumvents a requirement for expulsion by a proton donor during attack by cysteine on the gamma phosphorus . In contrast, a conserved aspartic acid is essential for the phosphomonoesterase reactions catalyzed by protein phosphatases, where the serine or tyrosine leaving groups have a much higher pK(a) than does ADP.

Proc Natl Acad Sci U S A, 2000 Aug 29, 97(18), 9919 - 24
A trans-acting RNA as a control switch in Escherichia coli: DsrA modulates function by forming alternative structures; Lease RA et al.; DsrA is an 87-nucleotide regulatory RNA of Escherichia coli that acts in trans by RNA-RNA interactions with two different mRNAs, hns and rpoS . DsrA has opposite effects on these transcriptional regulators . H-NS levels decrease, whereas RpoS (final sigma(s)) levels increase . Here we show that DsrA enhances hns mRNA turnover yet stabilizes rpoS mRNA, either directly or via effects on translation . Computational and RNA footprinting approaches led to a refined structure for DsrA, and a model in which DsrA interacts with the hns mRNA start and stop codon regions to form a coaxial stack . Analogous bipartite interactions exist in eukaryotes, albeit with different regulatory consequences . In contrast, DsrA base pairs in discrete fashion with the rpoS RNA translational operator . Thus, different structural configurations for DsrA lead to opposite regulatory consequences for target RNAs.

Nucleic Acids Res, 2000 Sep 1, 28(17), 3260 - 8
Werner syndrome exonuclease catalyzes structure-dependent degradation of DNA; Shen JC et al.; Werner syndrome (WS) is an autosomal recessive disease characterized by early onset of many features of aging, by an unusual spectrum of cancers, and by genomic instability . The WS protein (WRN) possesses 3'-->5' DNA helicase and associated ATPase activities, as well as 3'-->5' DNA exonuclease activity . Currently, WRN is the only member of the widely distributed RecQ DNA helicase family with documented exonuclease activity . It is not known whether deficiency of the exonuclease or helicase/ATPase activities of WRN, or all of them, is responsible for various elements of the WS phenotype . WRN exonuclease has limited homology to Escherichia coli RNaseD, a tRNA processing enzyme . We show here that WRN preferentially degrades synthetic DNA substrates containing alternate secondary structures, with an exonucleolytic mode of action suggestive of RNaseD . We present evidence that structure-dependent binding of WRN to DNA requires ATP binding, while DNA degradation requires ATP hydrolysis . Apparently, the exonuclease and ATPase act in concert to catalyze structure-dependent DNA degradation . We propose that WRN protein functions as a DNA processing enzyme in resolving aberrant DNA structures via both exonuclease and helicase activities.

Nucleic Acids Res, 2000 Sep 1, 28(17), 3240 - 9
Functional significance of conserved residues in the phosphohydrolase module of Escherichia coli MutT protein; Shimokawa H et al.; Escherichia coli MutT protein hydrolyzes 8-oxo-7,8-dihydro-2'-dGTP (8-oxo-dGTP) to the monophosphate, thus avoiding the incorporation of 8-oxo-7,8-dihydroguanine (8-oxo-G) into nascent DNA . Bacterial and mammalian homologs of MutT protein share the phosphohydrolase module (MutT: Gly37-->Gly59) . By saturation mutagenesis of conserved residues in the MutT module, four of the 10 conserved residues (Gly37, Gly38, Glu53 and Glu57) were revealed to be essential to suppress spontaneous A:T-->C:G transversion mutation in a mutT(-) mutator strain . For the other six residues (Lys39, Glu44, Thr45, Arg52, Glu56 and Gly59), many positive mutants which can suppress the spontaneous mutation were obtained; however, all of the positive mutants for Glu44 and Arg52 either partially or inefficiently suppressed the mutation, indicating that these two residues are also important for MutT function . Several positive mutants for Lys39, Thr45, Glu56 and Gly59 efficiently decreased the elevated spontaneous mutation rate, as seen with the wild-type, hence, these four residues are non-essential for MutT function . As Lys38 and Glu55 in human MTH1, corresponding to the non-essential residues Lys39 and Glu56 in MutT, could not be replaced by any other residue without loss of function, different structural features between the two modules of MTH1 and MutT proteins are evident.

Nucleic Acids Res, 2000 Sep 1, 28(17), 3206 - 15
Purification and characterization of a mammalian homolog of Escherichia coli MutY mismatch repair protein from calf liver mitochondria; Parker A et al.; A protein homologous to the Escherichia coli MutY glycosylase, referred to as mtMYH, has been purified from calf liver mitochondria . SDS-polyacrylamide gel electrophoresis, western blot analysis as well as gel filtration chromatography predicted the molecular mass of the purified calf mtMYH to be 35-40 kDa . Gel mobility shift analysis showed that the purified mtMYH formed specific binding complexes with A/8-oxoG, G/8-oxoG and T/8-oxoG, weakly with C/8-oxoG, but not with A/G and A/C mismatches . The purified mtMYH exhibited DNA glycosylase activity removing adenine mispaired with G, C or 8-oxoG and weakly removing guanine mispaired with 8-oxoG . The mtMYH glycosylase activity was insensitive to high concentrations of NaCl and EDTA . The purified mtMYH cross-reacted with antibodies against both intact MutY and a peptide of human MutY homolog (hMYH) . DNA glycosylase activity of mtMYH was inhibited by anti-MutY antibodies but not by anti-hMYH peptide antibodies . Together with the previously described mitochondrial MutT homolog (MTH1) and 8-oxoG glycosylase (OGG1, a functional MutM homolog), mtMYH can protect mitochondrial DNA from the mutagenic effects of 8-oxoG.

Mol Gen Genet, 2000 Jul, 263(6), 1047 - 52
Efficient transformation of the edible basidiomycete Lentinus edodes with a vector using a glyceraldehyde-3-phosphate dehydrogenase promoter to hygromycin B resistance; Hirano T et al.; To construct a vector for high-level expression of heterologous genes in Lentinus edodes, the L . edodes GPD promoter, which is expressed constitutively and strongly, was fused to a hygromycin B phosphotransferase gene (hph) derived from Escherichia coli as a selective marker . Using the resulting pLG-hph construct, L . edodes was efficiently transformed to hygromycin resistance by restriction enzyme-mediated integration (REMI) . The restriction enzyme concentrations yielding the maximal numbers of transformants by the REMI method were 10 U per transformation in the case of BglII and 25-50 U in the case of HindIII . Southern analysis of the transformants indicated that some 50% of plasmid integrations were REMI-mediated events . These results indicate that pLG is a useful vector for transformation of L . edodes . Deletion analysis of the GPD promoter region suggested that the segment between positions -442 bp and -270 bp relative to the transcription start point may be essential for function.

Mol Gen Genet, 2000 Jul, 263(6), 957 - 65
Analysis of the expression of the Rhodobacter sphaeroides lexA gene; Tapias A et al.; The regulation of the Rhodobacter sphaeroides lexA gene has been analyzed using both gel-mobility experiments and lacZ gene fusions . PCR-mediated mutagenesis demonstrated that the second GAAC motif in the sequence GAACN7GAACN7GAAC located upstream of the R . sphaeroides lexA gene is absolutely necessary for its DNA damage-mediated induction . Moreover, mutagenesis of either the first or the third GAAC motif in this sequence reduced, but did not abolish, the inducibility of the R . sphaeroides lexA gene . A R . sphaeroides lexA-defective (Def) mutant has also been constructed by replacing the active lexA gene with an inactivated gene copy constructed in vitro . Crude extracts of the R . sphaeroides lexA(Def) strain are unable to form any protein-DNA complex when added to the wild-type lexA promoter of R . sphaeroides . Likewise, the R . sphaeroides lexA(Def) cells constitutively express the recA and lexA genes . All these data clearly indicate that the lexA gene product is the negative regulator of the R . sphaeroides SOS response . Furthermore, the morphology, growth and viability of R . sphaeroides lexA(Def) cultures do not show any significant change relative to those of the wild-type strain . Hence, R . sphaeroides is so far the only bacterial species whose viability is known not to be affected by the presence of a lexA(Def) mutation.

Life Sci, 2000, 67(9), 1059 - 72
Disorders in the immune responses of T- and B-cells in mice administered intravenous verotoxin 2; Sugatani J et al.; To obtain a better insight into the pathogenesis of verotoxin-producing Escherichia coli (VTEC)-associated diseases, we explored the effect of verotoxin 2 (VT2) on the immune response in mice . The distribution of lymphocyte phenotypes and the lymphocyte immune response were examined after intravenous administration of VT2 to mice . Among the peripheral lymphocytes and splenocytes of 4-week-old C57BL/6 mice, there was first of all a decrease in T-cells, which began 24 h after intravenous administration of VT2 (50 ng/kg, lethal dose) . The CD4+ cell subpopulations of the peripheral blood and spleen were significantly decreased at 24 h, while the B220+ splenocyte subpopulation was markedly decreased at 45 h after VT2 administration . In the thymus, a decrease in CD4+CD8+ cells was predominantly observed near death . Interestingly, in E . coli lipopolysaccharide (LPS)-responder mouse strains (C57BL/6 and C3H/HeN) cotreated with LPS, the susceptibility to VT2 was enhanced, and the increase in B220+ cells induced by LPS alone was suppressed . Furthermore, splenocytes from C57BL/6 mice treated with VT2 (50 ng/kg) 6-24 h earlier reduced LPS-induced proliferative responses to 50-52% of that in control cells, indicating that the effect of VT2 on the immunoresponse seen in vivo may be negatively exerted on the proliferation of the cells . In addition, the number of splenocytes that produced anti-sheep red blood cell antibody was decreased in mice treated with VT2 . These results suggest that VTEC infection may eliminate CD4+ and CD8+ T-cells and B-cells by affecting their survival and proliferative responses, leading to reduced antibody production.

Anticancer Res, 2000 Jul-Aug, 20(4), 2499 - 503
The recombinant human histones H1 zero and H1.2 cause different toxicity profiles on the human leukemia cell line K562; Pohlmeyer K et al.; The human histones H1 zero and H1.2 were expressed in E . coli and purified to homogenity . Their cytotoxicity on the human leukemia cell line K562 and on PBMC from healthy volunteers was compared with the cytotoxic effect of a bovine histone H1 preparation . In this preparation, histone H1.2 was identified as the main compound . All three histone preparations induced a significant dose-dependent toxicity on the leukemia cell line . Compared with the recombinant histone H1 zero, the bovine preparation and recombinant H1.2 showed stronger cytotoxicities . Cytotoxic effects on K562 cells were observed immediately after addition of the histones, whereas the histone preparations failed to induce significant cytotoxicity on PBMC during the first hour of incubation . However, after 24 hours all three histone preparations induced toxic effects on PBMC which were comparable to those observed on the leukemia cell line.

Anticancer Res, 2000 Jul-Aug, 20(4), 2377 - 81
Expression and purification of recombinant HIV-1 tat protein using HIV-1-tat specific monoclonal antibodies; Demirhan I et al.; We expressed tat protein from HIV-1 coding AA 1-67 (strain HIV-Bru) in the inducible vector pTrc 99A in E . coli . The tat coding region was cloned in the ATG site of the expression vector . A sequence coding for 15 AA was added at the 3'region as a molecular marker . After sonification, the tat protein was routinely detected in Western blots using the Mab developed by us . Following precipitation and centrifugation the resulting pellets were dissolved and purified by three different methods: 1 . immunoaffinity-chromatography using Affi-gel HZ coupled with a Mab recognizing the N-terminal sequence of HIV-1-tat; 2 . ion exchange chromatography using DEAE-52 cellulose, and 3 . isoelectric focusing in free solution . The resulting protein extracts obtained from the three purification protocols were checked in ELISA with the antibody . The peak fraction from all the procedures showed tat activity . No cross reaction in the presence of sera from uninfected persons was observed . The results showed that the purification of tat protein using monoclonal antibodies leads to highly purified preparations.

Mol Pharmacol, 2000 Sep, 58(3), 641 - 8
Viral entry as the primary target for the anti-HIV activity of chicoric acid and its tetra-acetyl esters; Pluymers W et al.; The antiviral activity of L-chicoric acid against HIV-1 has been attributed previously to the inhibition of HIV-1 integration . This conclusion was based on the inhibition of integrase activity in enzymatic assays and the isolation of a resistant HIV strain with a mutation (G140S) in the integrase gene . Here we show that the primary antiviral target of L-CA and its analogs in cell culture is viral entry . L- and D-chicoric acid (L-CA and D-CA) and their respective tetra-acetyl esters inhibit the replication of HIV-1 (III(B) and NL4.3) and HIV-2 (ROD) in MT-4 cells at a 50% effective concentration (EC(50)) ranging from 1.7 to 70.6 microM . In a time-of-addition experiment, L-CA, D-CA, L-CATA, and D-CATA were found to interfere with an early event in the viral replication cycle . Moreover, L-CA, D-CA, and their analogs did not inhibit the replication of virus strains that were resistant toward polyanionic and polycationic compounds at subtoxic concentrations . Furthermore, HIV-1 strains resistant to L-CA and D-CA were selected in the presence of L-CA and D-CA, respectively . Mutations were found in the V2, V3, and V4 loop region of the envelope glycoprotein gp120 of the L-CA and D-CA-resistant NL4.3 strains that were not present in the wild-type NL4.3 strain . Recombination of the gp120 gene of the L-CA and D-CA resistant strain in a NL4.3 wild-type molecular clone fully rescued the phenotypic resistance toward L-CA and D-CA . No significant mutations were detected in the integrase gene of the drug-resistant virus strains . Although inhibition of HIV integrase activity by L-CA and its derivatives was confirmed in an oligonucleotide-driven assay, integrase carrying the G140S mutation was inhibited to the same extent as the wild-type integrase.

J Biol Chem, 2000 Nov 10, 275(45), 35242 - 7
The structure of a CREB bZIP.somatostatin CRE complex reveals the basis for selective dimerization and divalent cation-enhanced DNA binding; Schumacher MA et al.; The cAMP responsive element-binding protein (CREB) is central to second messenger regulated transcription . To elucidate the structural mechanisms of DNA binding and selective dimerization of CREB, we determined to 3.0 A resolution, the structure of the CREB bZIP (residues 283-341) bound to a 21-base pair deoxynucleotide that encompasses the canonical 8-base pair somatostatin cAMP response element (SSCRE) . The CREB dimer is stabilized in part by ionic interactions from Arg(314) to Glu(319') and Glu(328) to Lys(333') as well as a hydrogen bond network that links the carboxamide side chains of Gln(322')-Asn(321)-Asn(321')-Gln(322) . Critical to family selective dimerization are intersubunit hydrogen bonds between basic region residue Tyr(307) and leucine zipper residue Glu(312), which are conserved in all CREB/CREM/ATF-1 family members . Strikingly, the structure reveals a hexahydrated Mg(2+) ion bound in the cavity between the basic region and SSCRE that makes a water-mediated DNA contact . DNA binding studies demonstrate that Mg(2+) ions enhance CREB bZIP:SSCRE binding by more than 25-fold and suggest a possible physiological role for this ion in somatostatin cAMP response element and potentially other CRE-mediated gene expression.

J Biol Chem, 2000 Nov 10, 275(45), 35091 - 7
Osmotic stress regulates the stability of cyclin D1 in a p38SAPK2-dependent manner; Casanovas O et al.; We report here that different cell stresses regulate the stability of cyclin D1 protein . Exposition of Granta 519 cells to osmotic shock, oxidative stress, and arsenite induced the post-transcriptional down-regulation of cyclin D1 . In the case of osmotic shock, this effect was completely reversed by the addition of p38(SAPK2)-specific inhibitors (SB203580 or SB220025), indicating that this effect is dependent on p38(SAPK2) activity . Moreover, the use of proteasome inhibitors prevented this down-regulation . Thus, osmotic shock induces proteasomal degradation of cyclin D1 protein by a p38(SAPK2)-dependent pathway . The effect of p38(SAPK2) on cyclin D1 stability might be mediated by direct phosphorylation at specific sites . We found that p38(SAPK2) phosphorylates cyclin D1 in vitro at Thr(286) and that this phosphorylation triggers the ubiquitination of cyclin D1 . These results link for the first time a stress-induced MAP kinase pathway to cyclin D1 protein stability, and they will help to understand the molecular mechanisms by which stress transduction pathways regulate the cell cycle machinery and take control over cell proliferation.

J Biol Chem, 2000 Nov 10, 275(45), 35361 - 7
Substrate recognition by the ClpA chaperone component of ClpAP protease; Hoskins JR et al.; ClpA, a member of the Clp/Hsp100 ATPase family, is a molecular chaperone and regulatory component of ClpAP protease . We explored the mechanism of protein recognition by ClpA using a high affinity substrate, RepA, which is activated for DNA binding by ClpA and degraded by ClpAP . By characterizing RepA derivatives with N- or C-terminal deletions, we found that the N-terminal portion of RepA is required for recognition . More precisely, RepA derivatives lacking the N-terminal 5 or 10 amino acids are degraded by ClpAP at a rate similar to full-length RepA, whereas RepA derivatives lacking 15 or 20 amino acids are degraded much more slowly . Thus, ClpA recognizes an N-terminal signal in RepA beginning in the vicinity of amino acids 10-15 . Moreover, peptides corresponding to RepA amino acids 4-13 and 1-15 inhibit interactions between ClpA and RepA . We constructed fusions of RepA and green fluorescent protein, a protein not recognized by ClpA, and found that the N-terminal 15 amino acids of RepA are sufficient to target the fusion protein for degradation by ClpAP . However, fusion proteins containing 46 or 70 N-terminal amino acids of RepA are degraded more efficiently in vitro and are noticeably stabilized in vivo in clpADelta and clpPDelta strains compared with wild type.

J Biol Chem, 2000 Nov 10, 275(45), 35063 - 9
The human lysyl-tRNA synthetase gene encodes both the cytoplasmic and mitochondrial enzymes by means of an unusual alternative splicing of the primary transcript; Tolkunova E et al.; Two cDNAs encoding human lysyl-tRNA synthetase have been identified . One encodes the cytoplasmic form of the enzyme identified previously . The second cDNA contains the same sequence but with a 180-bp insertion at the 5'-end of the mRNA . This results in a predicted protein whose carboxyl 576 amino acids are identical to those of the cytoplasmic enzyme but with a different amino terminus of 49 amino acids that contains a putative mitochondrial targeting sequence . Expression of the two lysyl-tRNA synthetase-green fluorescent protein gene fusions in a human cell line confirmed that the cytoplasmic form was targeted to the cytoplasm and the mitochondrial form to mitochondria . The genomic lysyl-tRNA synthetase gene consisted of 15 exons . The two isoforms were created by alternative splicing of the first three exons of the gene . The cytoplasmic form was created by splicing exon 1 to exon 3 . The inclusion of exon 2 between exons 1 and 3 produced an mRNA encoding the mitochondrial isoform with an additional upstream small open reading frame, consisting mainly of a portion of the 5' coding region of the cytoplasmic isoform . This is the first example of mitochondrial targeting sequence being encoded on the second exon of a gene . Ribonuclease protection analysis showed that the mRNA encoding the cytoplasmic isoform makes up approximately 70%, and the mitochondrial isoform approximately 30%, of the mature transcripts from the lysyl-tRNA synthetase gene . The mitochondrial form of the enzyme, purified after expression in Escherichia coli, aminoacylated in vitro transcripts corresponding to both the cytoplasmic and mitochondrial tRNA(Lys), despite the difference in the discriminator base sequence in the acceptor stems of these tRNAs.

J Biol Chem, 2000 Nov 10, 275(45), 35570 - 6
Phosphorylation of geranyl and farnesyl pyrophosphates by Nm23 proteins/nucleoside diphosphate kinases; Wagner PD et al.; The biochemical mechanism(s) by which Nm23 proteins/nucleoside diphosphate kinases suppress tumor metastasis, inhibit cell motility, and affect cellular differentiation are not known . Here we report that Nm23 proteins can phosphorylate geranyl and farnesyl pyrophosphates to give triphosphates . Wild type Nm23-H1 had higher geranyl and farnesyl pyrophosphate kinase activities than did mutants of Nm23-H1 that do not inhibit cell motility . The phosphorylation of farnesyl pyrophosphate appears to occur in vivo as cells with an elevated level of Nm23-H1 contained more farnesyl triphosphate than did control cells . To our knowledge, this is the first report that farnesyl triphosphate exists in cells . The phosphorylation of farnesyl pyrophosphate by Nm23 proteins could alter isoprenoid metabolism, and cells with an elevated level of Nm23 proteins were found to contain more farnesylated 46- and 24-kDa proteins than did control cells . The phosphorylation of geranyl and farnesyl pyrophosphates by Nm23 proteins provides a novel mechanism by which these proteins might exert their biological effects.

J Biol Chem, 2000 Nov 10, 275(45), 35106 - 15
Biological function and site II Ca2+-induced opening of the regulatory domain of skeletal troponin C are impaired by invariant site I or II Glu mutations; Pearlstone JR et al.; To investigate the roles of site I and II invariant Glu residues 41 and 77 in the functional properties and calcium-induced structural opening of skeletal muscle troponin C (TnC) regulatory domain, we have replaced them by Ala in intact F29W TnC and in wild-type and F29W N domains (TnC residues 1-90) . Reconstitution of intact E41A/F29W and E77A/F29W mutants into TnC-depleted muscle skinned fibers showed that Ca(2+)-induced tension is greatly reduced compared with the F29W control . Circular dichroism measurements of wild-type N domain as a function of pCa (= -log{Ca(2+)}) demonstrated that approximately 90% of the total change in molar ellipticity at 222 nm ({theta}(222 nm)) could be assigned to site II Ca(2+) binding . With E41A, E77A, and cardiac TnC N domains this {theta}(222 nm) change attributable to site II was reduced to < or =40% of that seen with wild type, consistent with their structures remaining closed in +Ca(2+) . Furthermore, the Ca(2+)-induced changes in fluorescence, near UV CD, and UV difference spectra observed with intact F29W are largely abolished with E41A/F29W and E77A/F29W TnCs . Taken together, the data indicate that the major structural change in N domain, including the closed to open transition, is triggered by site II Ca(2+) binding, an interpretation relevant to the energetics of the skeletal muscle TnC and cardiac TnC systems.

Nucleic Acids Res . 2000 Sep 1;28(17):E81.
Recovery and potential utility of YACs as circular YACs/BACs; Cocchia M et al.; A method has been established to convert pYAC4-based linear yeast artificial chromosomes (YACs) into circular chromosomes that can also be propagated in Escherichia coli cells as bacterial artificial chromosomes (BACs) . The circularization is based on use of a vector that contains a yeast dominant selectable marker (G418R), a BAC cassette and short targeting sequences adjacent to the edges of the insert in the pYAC4 vector . When it is introduced into yeast, the vector recombines with the YAC target sequences to form a circular molecule, retaining the insert but discarding most of the sequences of the YAC telomeric arms . YACs up to 670 kb can be efficiently circularized using this vector . Re-isolation of megabase-size YAC inserts as a set of overlapping circular YAC/BACs, based on the use of an Alu-containing targeting vector, is also described . We have shown that circular DNA molecules up to 250 kb can be efficiently and accurately transferred into E.coli cells by electroporation . Larger circular DNAs cannot be moved into bacterial cells, but can be purified away from linear yeast chromosomes . We propose that the described system for generation of circular YAC derivatives can facilitate sequencing as well as functional analysis of genomic regions.

Nucleic Acids Res . 2000 Sep 1;28(17):E79.
Insertion of disease-causing mutations in BACs by homologous recombination in Escherichia coli; Nefedov M et al.; We have used GET Recombination, an inducible homologous recombination system for Escherichia coli, to insert one of the most common thalassaemia mutations into the intact beta-globin locus in a second generation BAC vector . We first inserted a PCR fragment carrying the tetracycline resistance gene (TetR) into the beta-globin gene . All recombinant clones examined contained the TetR gene at the correct target site . Next, a PCR fragment with the IVS I-110 G-->A splicing mutation but no selectable marker was used to replace the TetR gene in a second round of GET Recombination . Recombinant clones were selected by plating on medium containing chlorotetracycline and fusaric acid . Although counterselection for the TetR gene is not very efficient, four recombinant colonies with the IVS I-110 mutation were identified among 480 clones screened . Analysis of the recombinant clones did not show any other modifications or rearrangements . Thus the TetR gene can be used in combination with GET Recombination to introduce point mutations and other modifications in BACs without leaving behind any operational sequences, in order to generate accurate cell and transgenic mouse models for various diseases.

J Neuropathol Exp Neurol, 2000 Aug, 59(8), 652 - 63
Intracellular processing and toxicity of the truncated androgen receptor: nuclear congophilia-associated cell death; Feng B et al.; The pathogenesis of the selective motor neuron death in spinal bulbar muscular atrophy (SBMA) is not fully understood . Similar to observations with other mutant polyglutamine (poly Q) expanded proteins, truncated androgen receptor (AR) with expanded poly Q tract cause intracellular aggregates; however, the precise relationship between aggregates and disease pathogenesis is unresolved . In order to have a better understanding of the cellular processing and toxicity of the mutant AR, we focused on a short N-terminal portion of AR containing normal or expanded poly Q repeats, and have carried out biochemical, immunocytochemical, cytochemical and ultrastructural studies of BHK cells at different intervals after transfection . In cells expressing mutant truncated AR, using an anti-AR N-terminal antibody, we observed no immune staining in the nucleus and identified immune negative aggregates surrounded by immunopositive material in the cytoplasm . Congo red staining identified a component of aggregates with a beta-pleated secondary structure in both cytosol and nucleus, while electron microscopy revealed a fibrillary-granular material as the ultrastructural correlate . In addition, acid phosphatase staining and ubiquitin immunocytochemistry demonstrated that in transfected cells, both lysosomal and nonlysosomal degradation systems are actively involved in handling the mutant truncated AR . The temporal relationship of nuclear congophilia to a subsequent massive cell death suggests that entry of proteolytic cleavage products into the nucleus, perhaps the expanded poly Q stretch itself, may play an important role in cell toxicity.

Appl Microbiol Biotechnol, 2000 Jul, 54(1), 78 - 83
A green fluorescent protein fusion strategy for monitoring the expression, cellular location, and separation of biologically active organophosphorus hydrolase; Wu CF et al.; Organophosphorus hydrolase (OPH) is capable of degrading a variety of pesticides and nerve agents . We have developed a versatile monitoring technique for detecting the amount of OPH during the expression and purification steps . This involves fusion of the gene for green fluorescent protein (GFP) to the 5' end of the OPH gene and subsequent expression in Escherichia coli . The synthesized fusion protein was directly visualized due to the optical properties of GFP . Western blot analyses showed that the correct fusion protein was expressed after IPTG-induction . Also, the in vivo GFP fluorescence intensity was proportional to the OPH enzyme activity . Moreover, the OPH, which forms a dimer in its active state, retained activity while fused to GFP . Enterokinase digestion experiments showed that OPH was separated from the GFP reporter after purification via immobilized metal affinity chromatography, which in turn was monitored by fluorescence . The strategy of linking GFP to OPH has enormous potential for improving enzyme production efficiency, as well as enhancing field use, as it can be monitored at low concentrations with inexpensive instrumentation based on detecting green fluorescence.

Appl Microbiol Biotechnol, 2000 Jul, 54(1), 52 - 8
Expression in Escherichia coli, purification and kinetic analysis of the aspartokinase and aspartate semialdehyde dehydrogenase from the rifamycin SV-producing Amycolatopsis mediterranei U32; Zhang WW et al.; The operon encoding aspartokinase and aspartate semialdehyde dehydrogenase was cloned and sequenced from rifamycin-SV-producing Amycolatopsis mediterranei U32 previously . In the present work, these two genes were introduced into the auxotrophic Escherichia coli strain CGSC5074 (ask-) and E . coli X6118 (asd-), respectively . The A . mediterranei U32 asparto-kinase and aspartate semialdehyde dehydrogenase genes can be functionally expressed in E . coli and the gene products are able to substitute for the E . coli enzymes . Histidine-tagged aspartokinase and aspartate semialdehyde dehydrogenase were partially purified from E . coli cellular extracts and their kinetic characteristics were studied . Both aspartokinase and aspartate semialdehyde dehydrogenase showed typical Michaelis-Menten type substrate saturation patterns . Aspartokinase has Km values of 3.4 mM for aspartate and 2.3 mM for ATP, while aspartate semialdehyde dehydrogenase has Km values of 1.25 mM for DL-aspartate semialdehyde and 0.73 mM for NADP, respectively . Aspartokinase was inhibited by L-threonine, L-lysine, and L-methionine, but not by L-isoleucine and diaminopimelate . Aspartate semialdehyde dehydrogenase was not inhibited by any of the end-product amino acids at a concentration of less than 5 mM . Hill plot analysis suggested that asparto-kinase was subject to allosteric control by L-threonine . Repression of both aspartokinase and aspartate semi-aldehyde dehydrogenase gene transcription in A . mediterranei U32 by L-lysine, L-methionine, L-threonine, and L-isoleucine were found . The network of regulation of aspartokinase and aspartate semialdehyde dehydrogenase in rifamycin SV-producing A . mediterranei U32 is presented.

Eur J Biochem, 2000 Sep, 267(17), 5638 - 45
ENDOR spectroscopic studies of stable semiquinone radicals bound to the Escherichia coli cytochrome bo3 quinol oxidase; Hastings SF et al.; The putative oxidation of ubiquinol by the cytochrome bo3 terminal oxidase of Escherichia coli in sequential one-electron steps requires stabilization of the semiquinone . ENDOR spectroscopy has recently been used to study the native ubisemiquinone radical formed in the cytochrome bo3 quinone-binding site {Veselov, A.V., Osborne, J.P., Gennis, R.B . & Scholes, C.P . (2000) Biochemistry 39, 3169-3175} . Comparison of these spectra with those from the decyl-ubisemiquinone radical in vitro indicated that the protein induced large changes in the electronic structure of the ubisemiquinone radical . We have used quinone-substitution experiments to obtain ENDOR spectra of ubisemiquinone, phyllosemiquinone and plastosemiquinone anion radicals bound at the cytochrome bo3 quinone-binding site . Large changes in the electronic structures of these semiquinone anion radicals are induced on binding to the cytochrome bo3 oxidase . The changes in electronic structure are, however, independent of the electronic structures of these semiquinones in vitro . Thus it is shown to be the structure of this binding site in the protein, not the covalent structure of the bound quinone, that determines the electronic structure of the protein-bound semiquinone.

Eur J Biochem, 2000 Sep, 267(17), 5621 - 30
Plant mercaptopyruvate sulfurtransferases: molecular cloning, subcellular localization and enzymatic activities; Nakamura T et al.; Mercaptopyruvate sulfurtransferase (MST, EC 2.8.1.2) and thiosulfate sulfurtransferase (TST, rhodanese, EC 2.8.1.1) are evolutionarily related enzymes that catalyze the transfer of sulfur ions from mercaptopyruvate and thiosulfate, respectively, to cyanide ions . We have isolated and characterized two cDNAs, AtMST1 and AtMST2, that are Arabidopsis homologs of TST and MST from other organisms . Deduced amino-acid sequences showed similarity to each other, although MST1 has a N-terminal extension of 57 amino acids containing a targeting sequence . MST1 and MST2 are located in mitochondria and cytoplasm, respectively, as shown by immunoblot analysis of subcellular fractions and by green fluorescent protein (GFP) analysis . However, some regions of MST1 fused to GFP were found to target not only mitochondria, but also chloroplasts, suggesting that the regions on the targeting sequence recognized by protein import systems of mitochondria and chloroplasts are not identical . Recombinant proteins, expressed in Escherichia coli, exhibited MST/TST activity ratios determined from kcat/Km values of 11 and 26 for MST1 and MST2, respectively . This indicates that the proteins encoded by both AtMST1 and AtMST2 are MST rather than TST type . One of the hypotheses proposed so far for the physiological function of MST and TST concerns iron-sulfur cluster assembly . In order to address this possibility, a T-DNA insertion Arabidopsis mutant, in which the AtMST1 was disrupted, was isolated by PCR screening of T-DNA mutant libraries . However, the mutation had no effect on levels of iron-sulfur enzyme activities, suggesting that MST1 is not directly involved in iron-sulfur cluster assembly.

Eur J Biochem, 2000 Sep, 267(17), 5601 - 7
Human brain GABA transaminase tissue distribution and molecular expression; Jeon SG et al.; Human brain gamma-aminobutyrate transaminase is differentially expressed in a tissue-specific manner . mRNA master dot-blot analysis for 50 different human tissues, including different brain regions and fetal tissues, provided a complete map of the tissue distribution . Genomic Southern analysis revealed that the gamma-aminobutyrate transaminase gene is a single copy, at least 15 kb in size . In addition, human brain gamma-aminobutyrate transaminase cDNA was expressed in Escherichia coli using a pGEX expression vector system . Catalytically active gamma-aminobutyrate transaminase was expressed in large quantities and the purified recombinant enzyme had kinetic parameters that were indistinguishable from those isolated from other mammalian brains . The human enzyme was inactivated by a well-known antiepileptic drug vigabatrin . Values of Ki and kinact were 1 mM and 0.35 min-1, respectively . Results from inactivation kinetics suggested that human gamma-aminobutyrate transaminase is more sensitive to the vigabatrin drug than the enzyme isolated from bovine brain.

Eur J Biochem, 2000 Sep, 267(17), 5571 - 9
Characterization of two sulfurtransferase isozymes from Arabidopsis thaliana; Papenbrock J et al.; Sulfurtransferases transfer a sulfane atom from a donor substrate to a thiophilic acceptor molecule . Recently a sulfurtransferase specific for the substrate 3-mercaptopyruvate was isolated from Arabidopsis thaliana {Papenbrock, J . & Schmidt, A . (2000) Eur . J . Biochem . 267, 145-154} . In this study a second sulfurtransferase from Arabidopsis was characterized and compared to the enzyme described previously . Sequences of the mature proteins had an identity of 77.7% . The plant sulfurtransferases formed a distinct group within the known eukaryotic sulfurtransferases . When Southern blots were hybridized with labelled cDNA fragments from each of the plant sulfurtransferases the same pattern of bands was obtained indicating the existence of only these two closely related sulfurtransferases . The new sulfurtransferase was expressed in Escherichia coli fused with an N-terminal His6-tag, purified and tested for enzyme activity . Like the first enzyme, the newly isolated protein preferred 3-mercaptopyruvate to thiosulfate as substrate . The Km of both enzymes determined for 3-mercaptopyruvate and cyanide were almost identical . As a result of database searches it became obvious that sulfurtransferase proteins from higher plants showed high similarities to small senescence- and stress-induced proteins . To prove the involvement of sulfurtransferases in senescence-associated processes 3-mercaptopyruvate sulfurtransferase activity was determined in crude protein extracts from Arabidopsis plants of different ages . 3-mercaptopyruvate sulfurtransferase activity and steady-state RNA levels of sulfurtransferases increased with increasing age . However, steady-state protein levels as measured by using an antibody against the sulfurtransferase protein expressed previously decreased . Putative roles of sulfurtransferases in senescence-associated processes are discussed.

Eur J Biochem, 2000 Sep, 267(17), 5509 - 19
A bifunctional enzyme with lycopene cyclase and phytoene synthase activities is encoded by the carRP gene of Mucor circinelloides; Velayos A et al.; Using functional analyses in Escherichia coli and Mucor circinelloides, it has been shown that a single M . circinelloides gene (carRP) codes for a protein with two different enzymatic activities, lycopene cyclase and phytoene synthase, which are encoded by independent genes in organisms other than fungi . This gene was identified using complementation tests among different classes of carotenoid mutants of M . circinelloides . The carRP gene product contains two domains: the R domain is located at the N-terminus and determines lycopene cyclase activity; the P domain is located at the C-terminus and displays phytoene synthase activity . The R domain is functional even in the absence of the P domain, while the latter needs the proper R domain conformation to carry out its function . The carRP gene is closely linked to the phytoene dehydrogenase (carB) gene, and the promoter regions of both genes are located within only 446 bp . Northern analyses show a co-ordinated regulation of the expression of both genes by blue light . Several motifs found in this promoter region suggest a bi-directional mode of transcription control.

Eur J Biochem, 2000 Sep, 267(17), 5458 - 65
Comparative analyses of secondary gene products of 3-deoxy-D-manno-oct-2-ulosonic acid transferases from Chlamydiaceae in Escherichia coli K-12; Brabetz W et al.; The waaA gene encoding the essential, lipopolysaccharide (LPS)-specific 3-deoxy-Dmanno-oct-2-ulosonic acid (Kdo) transferase was inactivated in the chromosome of a heptosyltransferase I and II deficient Escherichia coli K-12 strain by insertion of gene expression cassettes encoding the waaA genes of Chlamydia trachomatis, Chlamydophila pneumoniae or Chlamydophila psittaci . The three chlamydial Kdo transferases were able to complement the knockout mutation without changing the growth or multiplication behaviour . The LPS of the mutants were serologically and structurally characterized in comparison to the LPS of the parent strain using compositional analyses, high performance anion exchange chromatography, matrix-assisted laser desorption/ionization time-of-flight mass spectrometry and specific monoclonal antibodies . The data show that chlamydial Kdo transferases can replace in E . coli K-12 the host's Kdo transferase and retain the product specificities described in their natural background . In addition, we unequivocally proved that WaaA from C . psittaci transfers predominantly four Kdo residues to lipid A, forming a branched tetrasaccharide with the structure alpha-Kdo-(2-->8)-{alpha-Kdo-(2-->4)}-alpha-Kdo-(2-->4)-alpha-Kdo.

J Biol Chem, 2000 Aug 25, 275(34), 26233 - 40
Restricted passage of reaction intermediates through the ammonia tunnel of carbamoyl phosphate synthetase; Huang X et al.; The x-ray crystal structure of the heterodimeric carbamoyl phosphate synthetase from Escherichia coli has identified an intermolecular tunnel that connects the glutamine binding site within the small amidotransferase subunit to the two phosphorylation sites within the large synthetase subunit . The tunneling of the ammonia intermediate through the interior of the protein has been proposed as a mechanism for the delivery of the ammonia from the small subunit to the large subunit . A series of mutants created within the ammonia tunnel were prepared by the placement of a constriction via site-directed mutagenesis . The degree of constriction within the ammonia tunnel of these enzymes was found to correlate to the extent of the uncoupling of the partial reactions, the diminution of carbamoyl phosphate formation, and the percentage of the internally derived ammonia that is channeled through the ammonia tunnel . NMR spectroscopy and a radiolabeled probe were used to detect and identify the enzymatic synthesis of N-amino carbamoyl phosphate and N-hydroxy carbamoyl phosphate from hydroxylamine and hydrazine . The kinetic results indicate that hydroxylamine, derived from the hydrolysis of gamma-glutamyl hydroxamate, is channeled through the ammonia tunnel to the large subunit . Discrimination between the passage of ammonia and hydroxylamine was observed among some of these tunnel-impaired enzymes . The overall results provide biochemical evidence for the tunneling of ammonia within the native carbamoyl phosphate synthetase.

J Biol Chem, 2000 Nov 17, 275(46), 36094 - 103
NMR structure of the N-terminal J domain of murine polyomavirus T antigens . Implications for DnaJ-like domains and for mutations of T antigens; Berjanskii MV et al.; The NMR structure of the N-terminal, DnaJ-like domain of murine polyomavirus tumor antigens (PyJ) has been determined to high precision, with root mean square deviations to the mean structure of 0.38 A for backbone atoms and 0.94 A for all heavy atoms of ordered residues 5-41 and 50-69 . PyJ possesses a three-helix fold, in which anti-parallel helices II and III are bridged by helix I, similar to the four-helix fold of the J domains of DnaJ and human DnaJ-1 . PyJ differs significantly in the lengths of N terminus, helix I, and helix III . The universally conserved HPD motif appears to form a His-Pro C-cap of helix II . Helix I features a stabilizing Schellman C-cap that is probably conserved universally among J domains . On the helix II surface where positive charges of other J domains have been implicated in binding of hsp70s, PyJ contains glutamine residues . Nonetheless, chimeras that replace the J domain of DnaJ with PyJ function like wild-type DnaJ in promoting growth of Escherichia coli . This activity can be modulated by mutations of at least one of these glutamines . T antigen mutations reported to impair cellular transformation by the virus, presumably via interactions with PP2A, cluster in the hydrophobic folding core and at the extreme N terminus, remote from the HPD loop.

J Biol Chem, 2000 Nov 10, 275(45), 34881 - 6
Identification of residues important both for primary receptor binding and specificity in fibroblast growth factor-7; Sher I et al.; Fibroblast growth factors (FGFs) mediate a multitude of physiological and pathological processes by activating a family of tyrosine kinase receptors (FGFRs) . Each FGFR binds to a unique subset of FGFs and ligand binding specificity is essential in regulating FGF activity . FGF-7 recognizes one FGFR isoform known as the FGFR2 IIIb isoform or keratinocyte growth factor receptor (KGFR), whereas FGF-2 binds well to FGFR1, FGFR2, and FGFR4 but interacts poorly with KGFR . Previously, mutations in FGF-2 identified a set of residues that are important for high affinity receptor binding, known as the primary receptor-binding site . FGF-7 contains this primary site as well as a region that restricts interaction with FGFR1 . The sequences that confer on FGF-7 its specific binding to KGFR have not been identified . By utilizing domain swapping and site-directed mutagenesis we have found that the loop connecting the beta4-beta5 strands of FGF-7 contributes to high affinity receptor binding and is critical for KGFR recognition . Replacement of this loop with the homologous loop from FGF-2 dramatically reduced both the affinity of FGF-7 for KGFR and its biological potency but did not result in the ability to bind FGFR1 . Point mutations in residues comprising this loop of FGF-7 reduced both binding affinity and biological potency . The reciprocal loop replacement mutant (FGF2-L4/7) retained FGF-2 like affinity for FGFR1 and for KGFR . Our results show that topologically similar regions in these two FGFs have different roles in regulating receptor binding specificity and suggest that specificity may require the concerted action of distinct regions of an FGF.

Genomics, 2000 Aug 15, 68(1), 106 - 10
Single-step conversion of P1 and P1 artificial chromosome clones into yeast artificial chromosomes; Poorkaj P et al.; Large insert genomic clones are useful for generating transgenic animals, particularly when specific mutations are introduced . To facilitate manipulation of large genomic sequences, we developed a method of converting Escherichia coli P1 artificial chromosomes (PACs) into yeast artificial chromosomes (YACs) . A shuttle vector, pMAX-121, was generated that contains elements needed to generate a YAC (cen4, ars, ura3, his, and two telomere segments) along with approximately 1.3 kb of sequence homologous to P1 and PAC vector sequences . Cotransformation of yeast with the target PAC or P1 clone and pMAX-121 results in two homologous recombination events . The first, between the target clone and pMAX-121, results in a circular molecule . The second is an intramolecular recombination event between the two pMAX-121 telomere sequences, resulting in a linear molecule . The resulting YAC is stably maintained in yeast and can be further modified using homologous recombination . The method was used to convert a 201-kb PAC containing the human tau gene into a stable linear YAC . A second vector, pLys2-neo, was developed to retrofit the YAC with the yeast lys2 gene, a selectable marker replacing the yeast ura3 gene, and a Pgk-neo cassette that confers G418 resistance to mammalian cells . The resulting YAC can be used for generating transgenic animals and stably transfected cell lines . Also, the lys2 marker facilitates introduction of mutations by homologous recombination .

J Histochem Cytochem, 2000 Sep, 48(9), 1195 - 202
Calcyclin (S100A6) binding protein (CacyBP) is highly expressed in brain neurons; Jastrzebska B et al.; The expression of a novel calcyclin (S100A6) binding protein (CacyBP) in different rat tissues was determined by Western and Northern blotting . Polyclonal antibodies against recombinant CacyBP purified from E . coli exhibited the highest reaction in the brain and weaker reaction in liver, spleen, and stomach . CacyBP immunoreactivity was also detected in lung and kidney . Densitometric analysis showed that the concentration of CacyBP in the soluble fractions of total brain and cerebellum is approximately 0.17 and 0 . 34 ng/microg protein, respectively . Northern blotting with a specific cDNA probe confirmed the high level of CacyBP expression in the rat brain and lower levels in other tissues examined . Immunohistochemistry and in situ hybridization of rat brain sections revealed strong expression of CacyBP in neurons of the cerebellum, hippocampus, and cortex . The in situ hybridization detected CacyBP in hippocampus as early as P7 (postnatal day 7) and a peak of expression at P21, and the expression signal was preserved until adulthood . In the entorhinal cortex, the peak of expression was observed at P7, whereas in the cerebellum it was seen at P21 . The results presented here show that CacyBP is predominantly a neuronal protein . (J Histochem Cytochem 48:1195-1202)

J Infect Dis, 2000 Sep, 182(3), 873 - 81 Epub 2000 Aug 17.
Localization of endotoxin in the rat intestinal epithelium; Ge Y et al.; High levels of circulating lipopolysaccharide (LPS) cause intestinal inflammation and increased permeability to bacteria and toxins . To better understand the effects of LPS on the gut, confocal microscopy and immunofluorescence staining were used to examine the distribution of LPS in the rat intestine after intravenous or enteral administration . LPS was localized in macrophages in the lamina propria from 1 h to >28 days after intravenous injection . LPS was also detected in the epithelial cells from 8 h to 7 days after injection . In contrast, LPS administered enterally was found in the gut lumen in close proximity to the mucosa but was not detected in enterocytes at any time . The concentration of LPS in enterocytes near the villus tip provides a mechanism for the clearance of endotoxin, by the turnover and shedding of LPS-containing enterocytes into the gut lumen, that has not been previously described.

Appl Biochem Biotechnol, 2000 May, 87(2), 103 - 15
Effect of Escherichia coli chaperonin GroELS on heterologously expressed human immunodeficiency virus type 1 reverse transcriptase in vivo and in vitro; Maier G et al.; The two subunits of human immunodeficiency virus type 1 (HIV-1) reverse transcriptase (HIV-1 RT), p66 and p51, were coexpressed in Escherichia coli along with the E . coli chaperonin system GroEL/GroES . Coexpression increases the yield of heterodimeric HIV-1 RT by a factor of 4 to 5 and improves the nucleic acid binding affinity of HIV-1 RT by a factor of 1.6 . We have analyzed the reasons for the improvements . The total increase in yield of HIV-1 RT can be attributed to an accumulation of RT subunits in the cells (factor of about 2.8) and an increased growth of the E . coli cells (factor of about 1.4) . One reason for the accumulation in the cells is an improved stability of HIV-1 RT subunits toward bacterial proteases . In vitro studies showed that the nucleic acid binding affinity of HIV-1 RT purified from cells that did not coexpress GroELS was stimulated by adding purified GroELS (approx 1.5-fold), whereas HIV-1 RT stemming from cells coexpressing GroELS was stimulated only marginally (approx 1.1-fold) . The in vivo as well as the in vitro studies suggest that the chaperonin interacts with HIV-1 RT and therefore affects the folding process of HIV-1 RT.

J S Afr Vet Assoc, 2000 Mar, 71(1), 47 - 52
Prognostic indicators of post partum viability of kids born to Escherichia coli-vaccinated or unvaccinated does; Munyua SJ et al.; This study was undertaken to determine some blood and other physiological parameters with potential for use as prognostic indicators of viability of newborn goat kids . Of the 143 kids born during the on-farm study, 97 were crosses of Galla x Small East African (SEA) and 46 were pure SEA . The SEA x Galla kids were 46 single males, with a mean body weight at birth of 2.77 +/- 0.22 kg, 43 females with a mean body weight at birth of 2.36 +/- 0.76 kg and 5 and 3 sets of female and male twins (mean body weight at birth of 1.8 +/- 0.19 kg and 2.05 +/- 0.07 kg for the female and male kids, respectively) . The SEA kids comprised 36 single male and female kids (mean body weight at birth of 2.48 +/- 0.04 kg and 10 sets of twins (both male and female) (mean body weight at birth of 1.50 +/- 0.04 kg ) . Pre-suckling sera obtained on-station from kids born of does vaccinated against Escherichia coli (n = 8) and unvaccinated does (n = 7) had a total protein content of <40.0 g/l and no detectable levels of IgG and A or E . coli antibodies . Sera obtained 12 hours post partum) from kids that survived in both groups contained about 19-22 g of Ig g/l, 50-80 g total protein/l, blood glucose of >5 mmol/l and had an E . coli antibody titre of between 1/160 and 1/640 . On the other hand, kids that died within 48 hours of birth (parturient deaths) and had been classified in categories 3 and 4 righting reaction had low (<40 g/l) total protein, low white blood cell count (4,000/ml) and low blood glucose concentration (<4.9 mmol/l) . It is concluded that kids with delayed righting reaction (>45 minutes), low rectal temperature (<36 degrees C), low birth weights (<1.5 kg for singles and <1.0 kg for twins), low white blood cells (<4,000/ml), low (<2 mmol/l) blood glucose levels, low total protein (<40.0 g/l), low (<1:160) E . coli antibody titre and IgG (< or =3,350 mg/l) in sera obtained 12 hours after birth have a poor prognosis for survival.

Plant Mol Biol, 2000 May, 43(1), 113 - 20
Phytobilin biosynthesis: the Synechocystis sp . PCC 6803 heme oxygenase-encoding ho1 gene complements a phytochrome-deficient Arabidopsis thalianna hy1 mutant; Willows RD et al.; The phytobilin chromophores of phycobiliproteins and phytochromes are biosynthesized from heme in a pathway that begins with the opening of the tetrapyrrole macrocycle of protoheme to form biliverdin IXalpha, in a reaction catalyzed by heme oxygenase . An Arabidopsis thaliana hy1 mutant was previously shown to be deficient in phytochrome responses, and these responses were regained when the plants were administered biliverdin IXalpha . A heme oxygenase-encoding gene, ho1, was recently cloned from the cyanobacterium Synechocystis sp . PCC 6803 . When ho1 was expressed in Escherichia coli, the cells produced active ferredoxin-dependent soluble heme oxygenase . The open reading frame of ho1 was fused in frame with a chloroplast transit peptide-encoding sequence from the oli gene of Antirrhinum majus . This construct was placed in a binary plasmid vectorcontaining a kanamycin resistance marker and a cauliflower mosaic virus 35S promoter to control expression of the chimeric oli-ho1 gene and used to transform A . thaliana hy1 plants . Two independent transformed lines were obtained that had the phenotype of the parental Landsberg erecta line and expressed the chimeric gene, as indicated by detection of its mRNA by reverse transcriptase-polymerase chain reaction . The results indicate that Synechocystis sp . PCC 6803 heme oxygenase encoded by ho1 can substitute for the defective HY1 gene product and that the only required enzyme activity of the HY1 gene product is heme oxygenase.

Plant Mol Biol, 2000 May, 43(1), 23 - 32
Electron transport controls transcription of the thioredoxin gene (trxA) in the cyanobacterium Synechocystis sp . PCC 6803; Navarro F et al.; The trxA gene encoding one of the different thioredoxins of the facultative heterotrophic cyanobacterium Synechocystis sp . PCC 6803 is transcribed as a single mRNA of 450 nucleotides . Transcript accumulation is similar in all standard growth conditions but strongly decreases after transferring cell cultures from light to darkness . In steady-state conditions, trxA transcription is reduced at high (150-500 microE m(-2) s(-1)) compared with moderate (10-50 microE m(-2) s(-1)) light intensities . The stability of the trxA transcript was similar at different light intensities, and also in darkness . Photosynthetic electron transport inhibitors, as well as glucose starvation in a mutant strain lacking photosystem II, promote a strong decline in the level of trxA transcript . Primer extension analysis suggests that trxA is transcribed from two proximal promoters containing a -10 TATA box similar to the Escherichia coli consensus promoters . Unlike the trxA mRNA, the amount of thioredoxin protein was not reduced in the dark, neither at high light intensities, indicating that thioredoxin protein is very stable . Our results indicate that the thioredoxin encoded by the trxA gene is likely to be primarily regulated at the transcriptional level, rather than at the protein level, by the electron transport generated photosynthetically or from glucose metabolism.

J Biotechnol, 2000 Jul 14, 80(3), 217 - 30
Hydantoin racemase from Arthrobacter aurescens DSM 3747: heterologous expression, purification and characterization; Wiese A et al.; In Arthrobacter aurescens DSM 3747 three enzymes are involved in the complete conversion of slowly racemizing 5'-monosubstituted D,L-hydantoins to L-amino acids, a stereoselective hydantoinase, a stereospecific L-N-carbamoylase and a hydantoin racemase . The gene encoding the hydantoin racemase, designated hyuA, was identified upstream of the previously described L-N-carbamoylase gene in the plasmid pAW16 containing genomic DNA of A . aurescens . The gene hyuA which encodes a polypeptide of 25.1 kDa, was expressed in Escherichia coli and the recombinant protein purified to homogeneity and further characterized . The optimal condition for racemase activity were pH 8.5 and 55 degrees C with L-5-benzylhydantoin as substrate . The enzyme was completely inhibited by HgCL2 and iodoacetamide and stimulated by addition of dithiothreitol . No effect on enzyme activity was seen with EDTA . The enzyme showed preference for hydantoins with arylalkyl side chains . Kinetic studies revealed substrate inhibition towards the aliphatic substrate L-5-methylthioethylhydantoin . Enzymatic racemization of D-5-indolylmethylenehydantoin in D2O and NMR analysis showed that the hydrogen at the chiral center of the hydantoin is exchanged against solvent deuterium during the racemization.

Electrophoresis, 2000 Jul, 21(13), 2694 - 702
Global analysis of gene expression in cells of the immune system II . Cell-free translation products and high-density filter hybridization data; Frey JR et al.; We have developed an experimental system for linking information on cell-free transcription and translation products from cDNA clones with data obtained from hybridization signals from complex probes . The work described in this paper consists of two distinct processes, one being the construction of a system of clonal addresses and the other the identification of expressed genes involved in the studied processes . We describe the use of this system to identify genes involved in thymus development . Complex probes from fetal thymuses (GD15, 17 and newborn) of Balb/c mice were used to identify genes, which are up- or downregulated during the process of differentiation . The full set of information is available in the Clone-base of the Basel Institute for Immunology and will be retrievable from the website of the collaborating laboratories.

Electrophoresis, 2000 Jul, 21(13), 2606 - 9
In vitro coupled transcription translation: effects of modification in lysate preparation on protein composition and biosynthesis activity; Schindler PT et al.; Cell-free extracts (lysates) from Escherichia coli were used for protein synthesis in vitro . Essential steps of the lysate preparation were modified and analyzed with respect to their impact on in vitro protein synthesis capacity, using the green fluorescent protein (GFP) as a target protein . Variably manufactured lysates of low, medium and higher protein synthesis activity, were examined by high resolution two-dimensional gel electrophoresis to determine whether the modifications result in substantial alterations in protein composition of the final lysate . The total number of proteins calculated from the gel maps did not vary for lysates with different activity and thus cannot serve as an evaluation parameter . Ribosomal proteins RP-S1, RP-L9, and RP-L10 were found in stoichiometric amounts for each of these lysates and in equal concentrations in comparison among the different lysates . Conversely, depending on the activity profiles, up to 7 different isoforms of the elongation factor EF-Ts were detected in the gel maps.

Mol Cell, 2000 Jul, 6(1), 159 - 71
Periodic conformational changes in rRNA: monitoring the dynamics of translating ribosomes; Polacek N et al.; In protein synthesis, a tRNA transits the ribosome via consecutive binding to the A (acceptor), P (peptidyl), and E (exit) site; these tRNA movements are catalyzed by elongation factor G (EF-G) and GTP . Site-specific Pb2+ cleavage was applied to trace tertiary alterations in tRNA and all rRNAs on pre- and posttranslocational ribosomes . The cleavage pattern of deacylated tRNA and AcPhe-tRNA changed individually upon binding to the ribosome; however, these different conformations were unaffected by translocation . On the other hand, translocation affects 23S rRNA structure . Significantly, the Pb2+ cleavage pattern near the peptidyl transferase center was different before and after translocation . This structural rearrangement emerged periodically during elongation, thus providing evidence for a dynamic and mobile role of 23S rRNA in translocation.

Arch Virol, 2000, 145(6), 1117 - 31
Human immunodeficiency virus reverse transcriptase base misincorporations can promote strand transfer; Diaz L et al.; A system to determine if HIV-reverse transcriptase (RT) base misincorporations can promote strand transfer was constructed . A donor RNA, on which RT-directed DNA synthesis was initiated, shared homology over a 119 base internal region with an acceptor RNA, to which DNAs initiated on the donor could transfer . Products completed on the donor in the presence or absence of acceptor were isolated and PCR was used to amplify these DNAs . PCR products were ligated into a vector which had this same region (near the N-terminus of the alpha-lac gene) removed . Transformed E . coli were screened in an alpha-complementation assay by blue-white phenotype analysis with white colonies scored as those with errors in plasmid-derived alpha-lac . The frequence of white colonies +/- standard deviations was 0.031 +/- 0.006 and 0.0037 +/- 0.009, for plasmids with inserts derived from donor-directed products synthesized with 100 microM dNTPs in the presence and absence of acceptor template, respectively . Statistical analysis indicated a lower white colony frequency in the presence of acceptor (p = 0.0025) . The lower frequency with acceptor implies that a portion of the errors made on the donor are transferred to the acceptor suggesting that base misincorporations can induce strand transfer.

Biotechniques, 2000 Aug, 29(2), 338 - 42
Automated purification of His6-tagged proteins allows exhaustive screening of libraries generated by random mutagenesis; Lanio T et al.; In the course of site-directed mutagenesis or directed evolution experiments, large numbers of protein variants are often generated . To characterize functional properties of individual mutant proteins in vitro, a rapid and reliable protein purification system is required . We have developed an automated method for the parallel purification of 96 different protein variants that takes about two hours . Using a 96-well format, the whole process can be performed automatically by a pipetting robot . Coupled with a suitable assay, again using a 96-well format, all variants can be functionally characterized within a few hours . The protein purification procedure described here is based on the interaction between His6-tagged proteins and Ni-NTA-coated microplates . Typical yields are 3-8 pmol purified protein/well, which is sufficient to analyze most enzymatic activities . Using this procedure, we have purified and characterized variants of the restriction endonuclease EcoRV, which were produced in an effort to enhance the selectivity of this enzyme . For this purpose, three amino acid residues were randomized in a region known from the co-crystal structure to be located at the protein-DNA interface . From a library of about 1200 variants, predominantly single and double mutants, more than 1000 variants were purified and characterized in parallel, which corresponds to an almost complete screening of the library.

Plant Cell, 2000 Aug, 12(8), 1295 - 306
Cloning of the SNG1 gene of Arabidopsis reveals a role for a serine carboxypeptidase-like protein as an acyltransferase in secondary metabolism; Lehfeldt C et al.; Serine carboxypeptidases contain a conserved catalytic triad of serine, histidine, and aspartic acid active-site residues . These enzymes cleave the peptide bond between the penultimate and C-terminal amino acid residues of their protein or peptide substrates . The Arabidopsis Genome Initiative has revealed that the Arabidopsis genome encodes numerous proteins with homology to serine carboxypeptidases . Although many of these proteins may be involved in protein turnover or processing, the role of virtually all of these serine carboxypeptidase-like (SCPL) proteins in plant metabolism is unknown . We previously identified an Arabidopsis mutant, sng1 (sinapoylglucose accumulator 1), that is defective in synthesis of sinapoylmalate, one of the major phenylpropanoid secondary metabolites accumulated by Arabidopsis and some other members of the Brassicaceae . We have cloned the gene that is defective in sng1 and have found that it encodes a SCPL protein . Expression of SNG1 in Escherichia coli demonstrates that it encodes sinapoylglucose:malate sinapoyltransferase, an enzyme that catalyzes a transesterification instead of functioning like a hydrolase, as do the other carboxypeptidases . This finding suggests that SCPL proteins have acquired novel functions in plant metabolism and provides an insight into the evolution of secondary metabolic pathways in plants.

J Biol Chem, 2000 Nov 3, 275(44), 34757 - 65
Two distinct triggers for cycling of the lagging strand polymerase at the replication fork; Li X et al.; There are two modes of DNA synthesis at a replication fork . The leading strand is synthesized in a continuous fashion in lengths that in Escherichia coli can be in excess of 2 megabases . On the other hand, the lagging strand is synthesized in relatively short stretches of 2 kilobases . Nevertheless, identical assemblies of the DNA polymerase III core tethered to the beta sliding clamp account for both modes of DNA synthesis . Yet the same lagging strand polymerase accounts for the synthesis of all Okazaki fragments at a replication fork, cycling repeatedly every 1 or 2 s from the 3'-end of the just-completed fragment to the 3'-end of the new primer . Several models have been invoked to account for the rapid cycling of a polymerase complex that can remain bound to the template for upward of 40 min . By using isolated replication protein-DNA template complexes, we have tested these models and show here that cycling of the lagging strand polymerase can be triggered by either the action of primase binding to the replisome and synthesizing a primer or by collision of the lagging strand polymerase with the 5'-end of the previous Okazaki fragment.

Infect Immun, 2000 Sep, 68(9), 5030 - 6
Antigen detection in enteropathogenic Escherichia coli using secretory immunoglobulin A antibodies isolated from human breast milk; Manjarrez-Hernandez HA et al.; Enteropathogenic Escherichia coli (EPEC) produces a characteristic attaching and effacing (A/E) lesion in the small intestines of infected children . The immune response to EPEC infection remains poorly characterized . The molecular targets that elicit protective immunity against EPEC disease are unknown . In this study protein antigens from EPEC were identified using secretory immunoglobulin A (sIgA) antibodies isolated from milk from Mexican women by Western blot analysis . Purified sIgA antibodies, which inhibit the adherence of EPEC to cells, reacted to many EPEC proteins, the most prominent of which were intimin (a 94-kDa outer membrane protein) and two unknown proteins with apparent molecular masses of 80 and 70 kDa . A culture supernatant protein of 110 kDa also reacted strongly with the sIgA antibodies . The molecular size of this protein and its reactivity with specific anti-EspC antiserum suggest that it is EPEC-secreted protein C (EspC) . These EPEC surface protein antigens were consistently recognized by all the different sIgA samples obtained from 15 women . Screening of clinical isolates of various O serogroups from cases of severe infantile diarrhea revealed that all EPEC strains able to produce the A/E lesion showed expression of intimin and the 80- and 70-kDa proteins . Such proteins reacted strongly with the purified sIgA pool . Moreover, nonvirulent E . coli strains were unable to generate a sIgA response . The immunogenic capacities of the 80- and 70-kDa proteins as virulence antigens have not been previously reported . The strong sIgA response to intimin and the 80- and 70-kDa proteins obtained in this study indicates that such antigens stimulate intestinal immune responses and may elicit protective immunity against EPEC disease.

Infect Immun, 2000 Sep, 68(9), 4986 - 91
DNA-Based immunization with Trypanosoma cruzi complement regulatory protein elicits complement lytic antibodies and confers protection against Trypanosoma cruzi infection; Sepulveda P et al.; A complement regulatory protein (CRP) of Trypanosoma cruzi was evaluated as a vaccine candidate in a murine model of experimental T . cruzi infection . Recombinant CRP derived from an Escherichia coli expression system and a plasmid encoding the full-length crp structural gene under the control of a eukaryotic promoter were used to immunize BALB/c mice . Immunization with both protein and DNA vaccines resulted in a Th1-type T-cell response, comparable antibody titers, and similar immunoglobulin G isotype profiles . Only mice immunized with the crp DNA plasmid produced antibodies capable of lysing the parasites in the presence of complement and were protected against a lethal challenge with T . cruzi trypomastigotes . These results demonstrate the superiority of DNA immunization over protein immunization with the recombinant CRP . The work also supports the further investigation of CRP as a component of a multigene, anti-T . cruzi DNA vaccine.

Infect Immun, 2000 Sep, 68(9), 4972 - 9
Molecular cloning, sequencing, expression, and characterization of an immunogenic 43-kilodalton lipoprotein of Bartonella bacilliformis that has homology to NlpD/LppB; Padmalayam I et al.; A recombinant clone expressing an immunoreactive antigen of Bartonella bacilliformis was isolated by screening a genomic DNA library with serum from a patient with the chronic verruga phase of bartonellosis . The clone, pBIPIM-17, contained a partial open reading frame that expressed an immunoreactive fusion protein . Subsequent rescreening of the library by plaque hybridization resulted in the isolation of recombinant clones that contain the entire open reading frame . The open reading frame (ORF-401) is capable of encoding a protein of 401 amino acids with a predicted molecular mass of 43 kDa . The deduced amino acid sequence of the encoded protein was found to be highly homologous to a recently identified bacterial lipoprotein (LppB/NlpD) which has been associated with virulence . Evidence has been provided to show that the 43-kDa antigen of B . bacilliformis is a lipoprotein and that it is likely to use the same biosynthetic pathway as other bacterial lipoproteins . This is the first report to date that characterizes a lipoprotein of B . bacilliformis . The immunogenicity of the B . bacilliformis LppB homologue was demonstrated by Western blot analysis using sera from patients with clinical bartonellosis . Sera from patients who had a high titer for Bartonella henselae, the causative agent of bacillary angiomatosis and cat scratch disease, also recognized the recombinant 43-kDa antigen, suggesting that a homologue of this antigen is present in B . henselae . Using a cocktail of synthetic peptides corresponding to predicted major antigenic sites, polyclonal antiserum specific for the LppB homologue of B . bacilliformis was generated . This antiserum did not recognize the NlpD homologue of Escherichia coli or the 43-kDa antigen of B . henselae.

Infect Immun, 2000 Sep, 68(9), 4923 - 9
Antipeptide antibody responses following intranasal immunization: effectiveness of mucosal adjuvants; Olszewska W et al.; Toxicity is a major factor limiting the development and use of potent adjuvants for human mucosally delivered vaccines . Novel adjuvant formulations have recently become available, and in the present study two have been used for intranasal immunization with a synthetic peptide immunogen (MAP-M2) . This peptide represents a multiple antigenic peptide containing multiple copies of a mimotope M2, a peptide mimic of a conformational epitope of the fusion protein of measles virus . MAP-M2 was administered intranasally to experimental animals together with synthetic oligodeoxynucleotides containing unmethylated CpG motifs with or without a mutant of wild-type enterotoxin of Escherichia coli (LTR72) . The combination of the mutant toxin LTR72 and the CpG repeats, codelivered with a peptide immunogen, induced both local and systemic peptide- and pathogen-specific humoral and cellular immune responses comparable to those obtained after intranasal immunization with the wild-type toxin LT . In addition, this combination of adjuvants induced a predominantly immunoglobulin G2a antibody response . If both the LTR72 and CpG adjuvants are shown to be safe for use in humans, this particular combination would appear to have potential as an adjuvant for mucosally delivered vaccines in humans.

Science, 2000 Aug 18, 289(5482), 1190 - 4
Twists in catalysis: alternating conformations of Escherichia coli thioredoxin reductase; Lennon BW et al.; In thioredoxin reductase (TrxR) from Escherichia coli, cycles of reduction and reoxidation of the flavin adenine dinucleotide (FAD) cofactor depend on rate-limiting rearrangements of the FAD and NADPH (reduced form of nicotinamide adenine dinucleotide phosphate) domains . We describe the structure of the flavin-reducing conformation of E . coli TrxR at a resolution of 3.0 angstroms . The orientation of the two domains permits reduction of FAD by NADPH and oxidation of the enzyme dithiol by the protein substrate, thioredoxin . The alternate conformation, described by Kuriyan and co-workers, permits internal transfer of reducing equivalents from reduced FAD to the active-site disulfide . Comparison of these structures demonstrates that switching between the two conformations involves a "ball-and-socket" motion in which the pyridine nucleotide-binding domain rotates by 67 degrees.

Biochem J, 2000 Sep 1, 350 Pt 2, 579 - 88
A role for serine-175 in modulating the molecular conformation of calponin; Jin JP et al.; Calponin is an actin filament-associated protein found in smooth muscle and non-muscle cells . Calponin inhibits actin-myosin interaction in a manner that is prevented by protein kinase C (PKC)-catalysed phosphorylation of serine-175 . To investigate the molecular basis of serine-175-mediated regulation, we examined the effect of phosphorylation on the conformation of calponin using monoclonal antibody (mAb) epitope analysis . Eight mAbs against different epitopes on chicken gizzard calponin were developed to monitor the conformational changes in calponin induced by PKC-mediated phosphorylation or serine-175-->alanine (S175A) substitution . The relative affinities of the mAbs for calponins immobilized on microtitre plates or bound to actin-tropomyosin thin filaments were determined, and epitope competitions between free and immobilized calponins were carried out . The changes in binding affinity between mAb paratopes and calponin epitopes demonstrate several serine-175 modification-induced conformational effects: (a) structures of calponin are reconfigured by serine-175 modification, supporting the regulatory function of serine-175; (b) there are submolecular structures unaffected by modification of serine-175 in both free and thin filament-associated calponins, suggesting that the serine-175-based conformational modulation is a targeted allosteric effect; (c) significant conformational changes are detected between free and thin filament-associated calponins, indicating two functional states of the molecular conformation; and (d) the different epitope characteristics between thin filament-bound and free calponins suggest that calponin is a flexible molecule, and the modifications of serine-175 may also determine the structural flexibility to increase the epitope accessibility . These results provide novel information concerning the structure-function relationships of calponin and its regulation by phosphorylation.

Biochem J, 2000 Sep 1, 350 Pt 2, 563 - 8
Internally quenched fluorescent peptide substrates disclose the subsite preferences of human caspases 1, 3, 6, 7 and 8; Stennicke HR et al.; Subsite interactions are considered to define the stringent specificity of proteases for their natural substrates . To probe this issue in the proteolytic pathways leading to apoptosis we have examined the P(4), P(1) and P(1)' subsite preferences of human caspases 1, 3, 6, 7 and 8, using internally quenched fluorescent peptide substrates containing o-aminobenzoyl (also known as anthranilic acid) and 3-nitro-tyrosine . Previous work has demonstrated the importance of the S(4) subsite in directing specificity within the caspase family . Here we demonstrate the influence of the S(1) and S(1)' subsites that flank the scissile peptide bond . The S(1) subsite, the major specificity-determining site of the caspases, demonstrates tremendous selectivity, with a 20000-fold preference for cleaving substrates containing aspartic acid over glutamic acid at this position . Thus caspases are among the most selective of known endopeptidases . We find that the caspases show an unexpected degree of discrimination in the P(1)' position, with a general preference for small amino acid residues such as alanine, glycine and serine, with glycine being the preferred substituent . Large aromatic residues are also surprisingly well-tolerated, but charged residues are prohibited . While this describes the general order of P(1)' subsite preferences within the caspase family, there are some differences in individual profiles, with caspase-3 being particularly promiscuous . Overall, the subsite preferences can be used to predict natural substrates, but in certain cases the cleavage site within a presumed natural substrate cannot be predicted by looking for the preferred peptide cleavage sites . In the latter case we conclude that second-site interactions may overcome otherwise sub-optimal cleavage sequences.

Biochem J, 2000 Sep 1, 350 Pt 2, 429 - 41
Human sphingosine kinase: purification, molecular cloning and characterization of the native and recombinant enzymes; Pitson SM et al.; Sphingosine 1-phosphate (S1P) is a novel lipid messenger that has important roles in a wide variety of mammalian cellular processes including growth, differentiation and death . Basal levels of S1P in mammalian cells are generally low, but can increase rapidly and transiently when cells are exposed to mitogenic agents and other stimuli . This increase is largely due to increased activity of sphingosine kinase (SK), the enzyme that catalyses its formation . In the current study we have purified, cloned and characterized the first human SK to obtain a better understanding of its biochemical activity and possible activation mechanisms . The enzyme was purified to homogeneity from human placenta using ammonium sulphate precipitation, anion-exchange chromatography, calmodulin-affinity chromatography and gel-filtration chromatography . This resulted in a purification of over 10(6)-fold from the original placenta extract . The enzyme was cloned and expressed in active form in both HEK-293T cells and Escherichia coli, and the recombinant E . coli-derived SK purified to homogeneity . To establish whether post-translational modifications lead to activation of human SK activity we characterized both the purified placental enzyme and the purified recombinant SK produced in E . coli, where such modifications would not occur . The premise for this study was that post-translational modifications are likely to cause conformational changes in the structure of SK, which may result in detectable changes in the physico-chemical or catalytic properties of the enzyme . Thus the enzymes were characterized with respect to substrate specificity and kinetics, inhibition kinetics and various other physico-chemical properties . In all cases, both the native and recombinant SKs displayed remarkably similar properties, indicating that post-translational modifications are not required for basal activity of human SK.

Biochem J, 2000 Sep 1, 350 Pt 2, 395 - 404
A novel transcriptional factor with Ser/Thr kinase activity involved in the transforming growth factor (TGF)-beta signalling pathway; Ohta S et al.; Transforming growth factor-beta (TGF-beta) shows a variety of biological activities in various organs or cells . Recently some factors such as Smads (Sma and Mad proteins) and TGF-beta activating kinase 1 ('TAK1') have been characterized as signalling molecules downstream of TGF-beta . Several TGF-beta response elements have been identified such as cAMP response element, Smad binding element, and recognition sites for activating protein-1 and stimulating protein-1 in various gene promoters . We also reported a TGF-beta response element in the human C-type natriuretic peptide (CNP) gene promoter . In this paper, we report on a novel factor which regulates the TGF-beta response promoter . This factor, named TSF1 (TGF-beta stimulated factor 1), possessed DNA-binding ability and activated the TGF-beta responsive CNP promoter or vascular endothelial growth factor gene promoter which possesses a sequence element analogous to the TGF-beta responsive GC-rich element of the CNP promoter . TSF1 did not directly activate a Smads-dependent promoter from plasminogen activator inhibitor 1 gene, but it showed enhancement in co-operation with Smad3 and Smad4 . Interestingly, this factor had the structural features of a Ser/Thr kinase and actually exhibited protein kinase activity . TSF1 mRNA as well as its protein level were stimulated by TGF-beta treatment . Thus, TSF1 is an unique factor with two biological functions, transcriptional regulation and protein phosphorylation, that may be involved in TGF-beta signals.

Biochem J, 2000 Sep 1, 350 Pt 2, 369 - 79
Cloning, post-translational modifications, heterologous expression and ligand-binding of boar salivary lipocalin; Loebel D et al.; Boar submaxillary glands produce the sex-specific salivary lipocalin (SAL), which binds steroidal sex pheromones as endogenous ligands . The cDNA encoding SAL was cloned and sequenced . From a single individual, two protein isoforms, differing in three amino acid residues, were purified and structurally characterized by a combined Edman degradation/MS approach . These experiments ascertained that the mature polypeptide is composed of 168 amino acid residues, that one of the three putative glycosylation sites is post-translationally modified and the structure of the bound glycosidic moieties . Two of the cysteine residues are paired together in a disulphide bridge, whereas the remaining two occur as free thiols . SAL bears sequence similarity to other lipocalins; on this basis, a three-dimensional model of the protein has been built . A SAL isoform was expressed in Escherichia coli in good yields . Protein chemistry and CD experiments verified that the recombinant product shows the same redox state at the cysteine residues and that the same conformation is observed as in the natural protein, thus suggesting similar folding . Binding experiments on natural and recombinant SAL were performed with the fluorescent probe 1-aminoanthracene, which was efficiently displaced by the steroidal sex pheromone, as well as by several odorants.

Mol Ther, 2000 Aug, 2(2), 147 - 52
Adeno-associated virus mediates long-term gene transfer and delivery of chondroprotective IL-4 to murine synovium; Watanabe S et al.; Treatments for rheumatoid arthritis and other inflammatory arthropathies are often ineffective at preventing joint destruction . Long-term genetic modification of the cells lining the joint space (synoviocytes) in vivo represents a potential method for the treatment of these chronic conditions . However, a vector capable of efficiently transducing synoviocytes in vivo for a persistent period has not been available . The present report describes the genetic modification of synoviocytes in vivo using recombinant adeno-associated virus . High-titer adeno-associated virus encoding the gene for Escherichia coli beta-galactosidase was injected into the knee joints of mice . Synovial tissues were then examined for beta-galactosidase transgene expression by in situ staining and by fluorometry . High-efficiency, persistent transgene expression was observed in the synovium with no evidence of vector-induced inflammation . Expression was observed for at least 7 months and was higher in arthritic than nonarthritic mice . Gene transfer of murine IL-4 to the joints of mice with collagen-induced arthritis led to detectable levels of IL-4 in the joint and protection from articular cartilage destruction . These data suggest that adeno-associated virus may be a useful vector for gene delivery to the synovium for the treatment of inflammatory arthropathies.

Mol Ther, 2000 Aug, 2(2), 131 - 9
The interactions of peptides with the innate immune system studied with use of T7 phage peptide display; Sokoloff AV et al.; The icosahedral T7 phage (diameter approximately 65 nm) displaying random peptides at the carboxy-terminus of the phage coat proteins was used as a model for drug and gene delivery vehicles containing peptide ligands . We found that displayed peptides were recognized by natural antibodies and induced complement activation . Strikingly, the phage inactivation by complement was peptide-specific that implied the existence of numerous natural antibodies with different peptide specificity . Selection of phage that avoided inactivation by complement allowed the identification of peptides that protected the phage by binding to serum proteins . In rat blood, peptides with carboxy-terminal lysine or arginine residues protected the phage against complement-mediated inactivation by binding C-reactive protein . In human serum, a number of protective peptides with tyrosine residues were selected . The recognition of displayed peptides by natural antibodies appears to represent a universal mechanism for activation of complement at sites that contain identical or homologous proteins with exposed carboxy-termini.

Genes Cells, 2000 Aug, 5(8), 613 - 26
Two types of localization of the DNA-binding proteins within the Escherichia coli nucleoid; Azam TA et al.; BACKGROUND: The genome DNA of Escherichia coli is folded into the nucleosome-like structure, often called a nucleoid, by the binding of several DNA-binding proteins . We previously determined the specificity and affinity of DNA-binding for 12 species of the E . coli DNA-binding protein, and their intracellular concentrations at various growth phases . The intracellular localization of these proteins in E . coli could be predicted from these data, but no attempt has been made thus far to directly observe the intracellular distribution of the DNA-binding proteins . RESULTS: The intracellular localization in Escherichia coli of 10 species of the nucleoid-associated protein, three components of the transcripton apparatus, and three components of the translation machinery was investigated by indirect immuno-fluorescence microscopy . The DNA-binding proteins could be classified into two groups . The group-I proteins, including the major nucleoid-structural proteins, H-NS, HU, IHF, StpA and Dps, are distributed uniformly within the entire nucleoid . In contrast, the group-II proteins, which are presumed to possess regulatory activities of DNA functions accumulate at specific loci within the nucleoid, forming 2 (SeqA), 3-4 (CbpA and CbpB) and 6-10 (Fis and IciA) immuno-stained dots . Each immuno-stained dot may represent either the association of a hundred to one thousand molecules of each DNA-binding protein at a specific locus of the genome DNA or the assembly of protein-associated DNA segments from different domains of the folded genome . Both the RNA polymerase core enzyme and the sigma70 subunit are mainly associated with the nucleoid, but the anti-sigma70 factor (Rsd) appears to be accumulated at the boundary between the nucleoid and the cytosol in the stationary-phase cells . Here we show that the majority of Hfq is present in cytoplasm together with ribosomal proteins L7/L12 and RMF . CONCLUSION: The DNA-binding proteins of E . coli could be classified into two groups . One group proteins was distributed uniformly within the nucleoid, but the other group of proteins showed an irregular distribution, forming immuno-stained spots or clumps.

Genes Cells, 2000 Jul, 5(7), 571 - 81
Phosphorylation of ERM proteins at filopodia induced by Cdc42; Nakamura N et al.; BACKGROUND: ERM (ezrin, radixin, and moesin) proteins function as membrane-cytoskeletal linkers, and are known to be localized at filopodia and microvilli-like structures . We have shown that Rho-associated kinase (Rho-kinase)/ROKalpha/ROCK II phosphorylates moesin at Thr-558 at the lower stream of Rho, and the phosphorylation is crucial to the formation of microvilli-like structures (Oshiro, N., Fukata, Y . & Kaibuchi, K . (1998) Phosphorylation of moesin by Rho-associated kinase (Rho-kinase) plays a crucial role in the formation of microvilli-like structures . J . Biol . Chem . 273, 34663- 34666) . However, the role of ERM proteins in the formation of filopodia is less well characterized . RESULTS: Here we examined the phosphorylation state of ERM during filopodia formation induced by Cdc42 using the antibody recognizing ERM proteins phosphorylated at COOH (C)-terminal threonine . When NIH 3T3 cells were transfected with constitutively active Cdc42 (Cdc42V12), filopodia formation was induced and phosphorylation of ERM at C-terminal threonine was observed at the tip of filopodia, while the phosphorylation levels of ERM were lower and phosphorylated ERM was distributed throughout the cytoplasm in the control cells . We also showed that Myotonic dystrophy kinase-related Cdc42-binding kinase (MRCK) which has been identified as an effector of Cdc42, phosphorylated moesin at C-terminal threonine in a cell-free system . Coexpression of the dominant negative form of MRCK inhibited both the formation of filopodia and accumulation of C-terminal threonine-phosphorylated ERM proteins at filopodia induced by Cdc42V12 . CONCLUSION: The formation of filopodia induced by Cdc42 is accompanied by phosphorylation of ERM proteins, and MRCK is a candidate for the kinase that phosphorylates ERM proteins at filopodia.

Genes Cells, 2000 Jul, 5(7), 555 - 69
Tuning of the porin expression under anaerobic growth conditions by his-to-Asp cross-phosphorelay through both the EnvZ-osmosensor and ArcB-anaerosensor in Escherichia coli; Matsubara M et al.; BACKGROUND: Widespread bacterial signal transduction circuits are generally referred to as 'two-component systems' or 'histidine (His)-to-aspartate (Asp) phosphorelays.' In Escherichia coli, as many as 30 distinct His-to-Asp phosphorelay signalling pathways operate in response to a wide variety of environmental stimuli, such as medium osmolarity and anaerobiosis . In this regard, it is of interest whether or not some of them together constitute a network of signalling pathways through a physiologically relevant mechanism (often referred to as 'cross-regulation') . We have addressed this issue, with special reference to the osmo-responsive EnvZ and anaero-responsive ArcB phosphorelay signalling pathways in E . coli . RESULTS: Under standard aerobic growth conditions, it is well known that the osmoregulatory profile of the outer membrane porins (OmpC and OmpF) is mainly regulated by the EnvZ-OmpR phosphorelay system in response to medium osmolarity . In this study, it was found that, under anaerobic growth conditions, E . coli cells exhibit a markedly altered expression profile of OmpC and OmpF This profile was significantly different from that observed for the cells grown aerobically . Results from extensive genetic studies showed that, under such anaerobic growth conditions, the arcB gene encoding the anaero-sensory His-kinase appears to be an auxiliary genetic determinant that regulates the expression profile of porins . We then provided several lines of in vivo and in vitro evidence, which taken together, supported the following conclusions . CONCLUSIONS: Under anaerobic growth conditions, porin expression is tuned not only by the authentic osmo-resposive EnvZ sensor, but also by the anaero-responsive ArcB sensor, in an OmpR-dependent manner . It is suggested that such ArcB-mediated cross-regulation plays a physiological role by integrating anaerobic respiratory signals into the porin regulation in E . coli anaerobiosis . The proposed model is a clear example of the interplay of two distinct His-to-Asp phosphorelay signalling pathways.

J Immunol, 2000 Sep 1, 165(5), 2651 - 6
Relevance of the tumor antigen in the validation of three vaccination strategies for melanoma; Bellone M et al.; Many preclinical studies of cancer immunotherapy are based on the testing of a single vaccination strategy in several tumor models . Moreover, most of those studies used xenogeneic Ags, which, owing to their high immunogenicity, may not represent realistic models for the validation of cancer immunotherapies . To address these issues, we compared the vaccination efficacy of three well established strategies (i.e., naked DNA; peptide-pulsed dendritic cells (DC), or a mixture of peptide and the Escherichia coli toxin LTR72) using the xenogeneic OVA or the naturally expressed tyrosinase-related protein 2 (TRP-2) tumor Ag in the B16 melanoma model . C57BL/6 mice received one to three s.c . injections of peptide-pulsed DC or DNA, or one to four mucosal administrations of peptide-toxin mixture . One to 2 wk later, the animals were challenged s.c . with B16 or B16 cells expressing OVA (B16-OVA) . Vaccination of mice with OVA induced in all cases melanoma-specific CTL and protection against B16-OVA . When TRP-2 was used, all three vaccines elicited B16-specific CTL, but only DC pulsed with the immunodominant T cell epitope TRP-2181-188 allowed protection against B16 . Even more importantly, a vaccination regimen with TRP-2-pulsed DC, started 24 h after the injection of a lethal number of B16 cells, caused a therapeutic effect in 60% of the challenged animals . Our results strongly emphasize the relevance of the tumor Ag in the definition of immunotherapeutic strategies for cancer, and support the use of peptide-pulsed DC as cancer vaccine in humans.

Mutat Res, 2000 Aug 21, 469(1), 135 - 45
A comparison of the in vitro genotoxicity of tri- and hexavalent chromium; Blasiak J et al.; Chromium can be found in the environment in two main valence states: hexavalent (Cr(VI)) and trivalent (Cr(III)) . Cr(VI) salts are well known human carcinogens, but the results from in vitro studies are often conflicting . Cr(VI) primarily enters the cells and undergoes metabolic reduction; however, the ultimate product of this reduction, Cr(III) predominates within the cell . In the present work, we compared the effects of tri- and hexavalent chromium on the DNA damage and repair in human lymphocytes using the alkaline single cell gel electrophoresis (comet assay) . Potassium dichromate induced DNA damage in the lymphocytes, measured as the increase in comet tail moment . The effect was dose-dependent . Treated cells were able to recover within a 120-min incubation . Cr(III) caused greater DNA migration than Cr(VI) . The lymphocytes did not show measurable DNA repair . Vitamin C at 50 microM reduced the extent of DNA migration . This was either due to a decrease in DNA strand breaks and/or alkali labile sites induced by Cr(VI) or to the formation of DNA crosslinks by Cr(VI) in the presence of vitamin C . Vitamin C, however, did not modify the effects of Cr(III) . Catalase, an enzyme inactivating hydrogen peroxide, decreased the extent of DNA damage induced by Cr(VI) but not the one induced by Cr(III) . Lymphocytes exposed to Cr(VI) and treated with endonuclease III, which recognizes oxidized pyrimidines, displayed greater extent of DNA damage than those not treated with the enzyme . Such an effect was not observed when Cr(III) was tested . The results obtained suggest that reactive oxygen species and hydrogen peroxide may be involved in the formation of DNA lesions by hexavalent chromium . The comet assay did not indicate the involvement of oxidative mechanisms in the DNA-damaging activity of trivalent chromium and we speculate that its binding to cellular ligands may play a role in its genotoxicity.

Mutat Res, 2000 Aug 30, 460(3-4), 277 - 300
The nucleotide excision repair protein UvrB, a helicase-like enzyme with a catch; Theis K et al.; Nucleotide excision repair (NER) is a universal DNA repair mechanism found in all three kingdoms of life . Its ability to repair a broad range of DNA lesions sets NER apart from other repair mechanisms . NER systems recognize the damaged DNA strand and cleave it 3', then 5' to the lesion . After the oligonucleotide containing the lesion is removed, repair synthesis fills the resulting gap . UvrB is the central component of bacterial NER . It is directly involved in distinguishing damaged from undamaged DNA and guides the DNA from recognition to repair synthesis . Recently solved structures of UvrB from different organisms represent the first high-resolution view into bacterial NER . The structures provide detailed insight into the domain architecture of UvrB and, through comparison, suggest possible domain movements . The structure of UvrB consists of five domains . Domains 1a and 3 bind ATP at the inter-domain interface and share high structural similarity to helicases of superfamilies I and II . Not related to helicase structures, domains 2 and 4 are involved in interactions with either UvrA or UvrC, whereas domain 1b was implicated for DNA binding . The structures indicate that ATP binding and hydrolysis is associated with domain motions . UvrB's ATPase activity, however, is not coupled to the separation of long DNA duplexes as in helicases, but rather leads to the formation of the preincision complex with the damaged DNA substrate . The location of conserved residues and structural comparisons with helicase-DNA structures suggest how UvrB might bind to DNA . A model of the UvrB-DNA interaction in which a beta-hairpin of UvrB inserts between the DNA double strand has been proposed recently . This padlock model is developed further to suggest two distinct consequences of domain motion: in the UvrA(2)B-DNA complex, domain motions lead to translocation along the DNA, whereas in the tight UvrB-DNA pre-incision complex, they lead to distortion of the 3' incision site.

Mutat Res, 2000 Aug 30, 460(3-4), 211 - 29
Abasic site recognition by two apurinic/apyrimidinic endonuclease families in DNA base excision repair: the 3' ends justify the means; Mol CD et al.; DNA damage occurs unceasingly in all cells . Spontaneous DNA base loss, as well as the removal of damaged DNA bases by specific enzymes targeted to distinct base lesions, creates non-coding and lethal apurinic/apyrimidinic (AP) sites . AP sites are the central intermediate in DNA base excision repair (BER) and must be processed by 5' AP endonucleases . These pivotal enzymes detect, recognize, and cleave the DNA phosphodiester backbone 5' of, AP sites to create a free 3'-OH end for DNA polymerase repair synthesis . In humans, AP sites are processed by APE1, whereas in yeast the primary AP endonuclease is termed APN1, and these enzymes are the major constitutively expressed AP endonucleases in these organisms and are homologous to the Escherichia coli enzymes Exonuclease III (Exo III) and Endonuclease IV (Endo IV), respectively . These enzymes represent both of the conserved 5' AP endonuclease enzyme families that exist in biology . Crystal structures of APE1 and Endo IV, both bound to AP site-containing DNA reveal how abasic sites are recognized and the DNA phosphodiester backbone cleaved by these two structurally unrelated enzymes with distinct chemical mechanisms . Both enzymes orient the AP-DNA via positively charged complementary surfaces and insert loops into the DNA base stack, bending and kinking the DNA to promote flipping of the AP site into a sequestered enzyme pocket that excludes undamaged nucleotides . Each enzyme-DNA complex exhibits distinctly different DNA conformations, which may impact upon the biological functions of each enzyme within BER signal-transduction pathways.

Mutat Res, 2000 Aug 30, 460(3-4), 165 - 81
Structure and function in the uracil-DNA glycosylase superfamily; Pearl LH; Deamination of cytosine to uracil is one of the major pro-mutagenic events in DNA, causing G:C-->A:T transition mutations if not repaired before replication . Repair of uracil-DNA is achieved in a base-excision pathway initiated by a uracil-DNA glycosylase (UDG) enzyme of which four families have so far been identified . Family-1 enzymes are active against uracil in ssDNA and dsDNA, and recognise uracil explicitly in an extrahelical conformation via a combination of protein and bound-water interactions . Extrahelical recognition requires an efficient process of substrate location by 'base-sampling' probably by hopping or gliding along the DNA . Family-2 enzymes are mismatch specific and explicitly recognise the widowed guanine on the complementary strand rather than the extrahelical scissile pyrimidine . This allows a broader specificity so that some Family-2 enzymes can excise uracil and 3, N(4)-ethenocytosine from mismatches with guanine . Although structures are not yet available for Family-3 (SMUG) and Family-4 enzymes, sequence analysis suggests similar overall folds, and identifies common active site motifs but with a surprising lack of conservation of catalytic residues between members of the super-family.

Mutat Res, 2000 Aug 30, 460(3-4), 143 - 9
DNA photolyases and cryptochromes; Deisenhofer J; This brief review gives an overview of the gene family of photolyases and cryptochromes, followed by a description of the main features of the three-dimensional structures of photolyases known to date . It then discusses recent biophysical studies of photolyase function, and modelling studies on the interaction between the enzyme and its substrate.

Vet Microbiol, 2000 Sep 25, 76(2), 175 - 84
The afa-related gene cluster in necrotoxigenic and other Escherichia coli from animals belongs to the afa-8 variant; Gerardin J et al.; Six hundred and nine necrotoxigenic Escherichia coli type 1 and 2 (NTEC1 and NTEC2) and non-NTEC isolated in Western and Southern Europe, North Africa and Canada from diseased calves, pigs, humans, poultry, and 55 isolated from asymptomatic calves were studied for the identification of afa-related sequences to the recently described afa-7 and afa-8 gene cluster variants from two bovine Escherichia coli (Lalioui et al., 1999) . Colony hybridization and PCR assays for the afaD-7, afaE-7, afaD-8 and afaE-8 identified the afa-related sequences to the afa-8 gene cluster in most (67/79; 85%) of the E . coli positive with the Afa-f family probe and in 14 additional strains negative with the Afa-f probe . No E . coli was positive for the afa-7 gene cluster . The existence of afa-8 positive strains was thus confirmed among bovine E . coli and for the first time among porcine, poultry and human E . coli . Sequencing of the afaE-8 amplicon of nine strains from the different host species showed a high degree of conservation (>95% at the DNA level; >92% at the amino-acid level) . The afa-8 gene cluster was more frequent in E . coli from diseased calves (18%) than from piglets (12%), humans (6%) and poultry (5%) . Bovine NTEC2 (26%) were more frequently positive than NTEC 1 (20%) and non-NTEC (11%) . E . coli isolated from asymptomatic calves were rarely positive: one NTEC2 (3%) and no non-NTEC . The afa-8 gene cluster was located on the Vir plasmid in 11/23 NTEC2, but no plasmid localization was detected in NTEC1 or non-NTEC.

J Biol Chem, 2000 Nov 3, 275(44), 34580 - 5
Heparan/chondroitin sulfate biosynthesis . Structure and mechanism of human glucuronyltransferase I; Pedersen LC et al.; Human beta1,3-glucuronyltransferase I (GlcAT-I) is a central enzyme in the initial steps of proteoglycan synthesis . GlcAT-I transfers a glucuronic acid moiety from the uridine diphosphate-glucuronic acid (UDP-GlcUA) to the common linkage region trisaccharide Gal beta 1-3Gal beta 1-4Xyl covalently bound to a Ser residue at the glycosaminylglycan attachment site of proteoglycans . We have now determined the crystal structure of GlcAT-1 at 2.3 A in the presence of the donor substrate product UDP, the catalytic Mn(2+) ion, and the acceptor substrate analog Gal beta 1-3Gal beta 1-4Xyl . The enzyme is a alpha/beta protein with two subdomains that constitute the donor and acceptor substrate binding site . The active site residues lie in a cleft extending across both subdomains in which the trisaccharide molecule is oriented perpendicular to the UDP . Residues Glu(227), Asp(252), and Glu(281) dictate the binding orientation of the terminal Gal-2 moiety . Residue Glu(281) is in position to function as a catalytic base by deprotonating the incoming 3-hydroxyl group of the acceptor . The conserved DXD motif (Asp(194), Asp(195), Asp(196)) has direct interaction with the ribose of the UDP molecule as well as with the Mn(2+) ion . The key residues involved in substrate binding and catalysis are conserved in the glucuronyltransferase family as well as other glycosyltransferases.

J Surg Res, 2000 Sep, 93(1), 9 - 15
Influence of amrinone on tissue oxygenation of jejunal mucosa during endotoxemia; Schmidt W et al.; BACKGROUND: The intestinal mucosa is the portion of the gut most susceptible to impaired perfusion and oxygen delivery . The phosphodiesterase (PDE) inhibitor amrinone has been proposed to improve oxygen delivery and tissue perfusion during sepsis . The objective of this study was to investigate the effects of amrinone on arterial oxygenation (Pao(2)) and tissue oxygenation (Ptio(2)) of jejunal mucosa during endotoxemia . MATERIALS AND METHODS: Forty anesthetized and ventilated rats were laparotomized and a jejunal portion was exteriorized and fixed on a plexiglass stage . The jejunum was punctured and a Clark-type microcatheter Po(2) probe and a microthermocouple were placed on the mucosa to measure Ptio(2) . The animals were randomly assigned to receive one of the four treatments: infusion of Escherichia coli lipopolysaccharides (LPS, 2 mg/kg/h) without amrinone pretreatment (LPS group); infusion of LPS with amrinone pretreatment (40 microg/kg/min, start 30 min before LPS infusion, amrinone + LPS group); no treatment with either amrinone or LPS (control group); treatment with amrinone without LPS infusion (amrinone group) . Mean arterial pressure (MAP), heart rate (HR), Pao(2), and Ptio(2) were measured 30 min before and 0, 60, and 120 min after induction of endotoxemia . RESULTS: MAP remained stable in the control and LPS groups . In the amrinone + LPS group MAP decreased within the first 30 min of amrinone infusion and decreased further during endotoxemia . Pao(2) remained stable in the control group and decreased in the LPS group . This endotoxin-induced decrease in Pao(2) was attenuated in the amrinone + LPS group . The mucosal Ptio(2) decreased in the LPS group but remained stable in both the control and amrinone + LPS groups . CONCLUSIONS: Pretreatment with amrinone was able to diminish a decrease in Pao(2) during endotoxemia, indicating that pulmonary dysfunction was attenuated . Endotoxin-induced tissue hypoxia of the intestinal mucosa, however, could be fully prevented, indicating that an additional improvement in compromised tissue perfusion had occurred .

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