Escherichia coli

Mol Microbiol, 1998 Apr, 28(2), 395 - 401
High-affinity binding sites for the initiator protein DnaA on the chromosome of Escherichia coli; Roth A et al.; The initiator protein DnaA of Escherichia coli binds with unusually high affinity to five regions on the chromosome, in addition to the replication origin, oriC . Using a solid-phase DNA binding assay, in which the DNA binding C-terminal domain of DnaA is bound via a biotin tag to magnetic beads, we could fish only fragments with these six regions from different chromosomal digests . Except for oriC, these fragments contain only one or two consensus DnaA binding sites, DnaA boxes . The distribution of these high-affinity DnaA boxes on the chromosome is random.

Mol Microbiol, 1998 Apr, 28(2), 383 - 93
Metronidazole resistance in Helicobacter pylori is due to null mutations in a gene (rdxA) that encodes an oxygen-insensitive NADPH nitroreductase; Goodwin A et al.; Metronidazole (Mtz) is a critical component of combination therapies that are used against Helicobacter pylori, the major cause of peptic ulcer disease . Many H . pylori strains are Mtz resistant (MtzR), however, and here we show that MtzR results from loss of oxygen-insensitive NADPH nitroreductase activity . The underlying gene (called 'rdxA') was identified in several steps: transformation of Mtz-susceptible (MtzS) H . pylori with cosmids from a MtzR strain, subcloning, polymerase chain reaction (PCR) and DNA sequencing . We also found that (i) E . coli (normally MtzR) was rendered MtzS by a functional H . pylori rdxA gene; (ii) introduction of rdxA on a shuttle vector plasmid into formerly MtzR H . pylori rendered it MtzS; and (iii) replacement of rdxA in MtzS H . pylori with an rdxA::camR null insertion allele resulted in a MtzR phenotype . The 630 bp rdxA genes of five pairs of H . pylori isolates from infections that were mixed (MtzR/MtzS), but uniform in overall genotype, were sequenced . In each case, the paired rdxA genes differed from one another by one to three base substitutions . Typical rdxA genes from unrelated isolates differ by 5% in DNA sequence . Therefore, the near identity of rdxA genes from paired MtzR and MtzS isolates implicates de novo mutation, rather than horizontal gene transfer in the development of MtzR . Horizontal gene transfer could readily be demonstrated under laboratory conditions with mutant rdxA alleles . RdxA is a homologue of the classical nitroreductases (CNRs) of the enteric bacteria, but differs in cysteine content (6 vs . 1 or 2 in CNRs) and isoelectric point (pI=7.99 vs . 5.4-5.6), which might account for its reduction of low redox drugs such as Mtz . We suggest that many rdxA (MtzR) mutations may have been selected by prior use of Mtz against other infections . H . pylori itself is an early risk factor for gastric cancer; the possibility that its carcinogenic effects are exacerbated by Mtz use, which is frequent in many societies, or the reduction of nitroaromatic compounds to toxic, mutagenic and carcinogenic products, may be of significant concern in public health.

Mol Microbiol, 1998 Apr, 28(2), 371 - 81
Regulation of type 1 fimbrial expression in uropathogenic Escherichia coli: heterogeneity of expression through sequence changes in the fim switch region; Leathart JB et al.; Over 80% of uropathogenic Escherichia coli express type 1 fimbriae . Expression is phase variable, and regulation of phase switching can differ between isolates . Previously, this was explained by differences in the expression of the fim recombinases, FimB and FimE . Our study of 50 uropathogenic E . coli isolates confirms variation in the regulation of type 1 fimbriae but, in many cases, the variation could be accounted for by sequence changes within and adjacent to the fim switch, rather than by differences in recombinase expression . This was demonstrated by moving the switch from the isolates into an isogenic background and comparing the switching behaviour with that of the original isolate . Isolates could be arranged into groups based on fim switch regulation and sequence similarity . In certain cases, the altered regulation was located to specific basepair changes within the fim switch . Sequence changes were found that had a marked effect on the activity of either FimB or FimE switching, while others affected FimB switching in only one direction . These results emphasize the value of using naturally selected sequence variation to further the understanding of gene regulation.

Mol Microbiol, 1998 Apr, 28(2), 333 - 42
Structure of the chromosomal insertion site for pSAM2: functional analysis in Escherichia coli; Raynal A et al.; The element pSAM2 from Streptomyces ambofaciens integrates into the chromosome through site-specific recombination between the element (attP) and the chromosomal (attB) sites . These regions share an identity segment of 58bp extending from the anti-codon loop through the 3' end of a tRNA(Pro) gene . To facilitate the study of the attB site, the int and xis genes, expressed from an inducible promoter, and attP from pSAM2 were cloned on plasmids in Escherichia coil . Compatible plasmids carrying the different attB regions to be tested were introduced in these E . coli strains . Under these conditions, Int alone could promote site-specific integration; Int and Xis were both required for site-specific excision . This experimental system was used to study the sequences required in attB for efficient site-specific recombination . A 26 bp sequence, centred on the anti-codon loop region and not completely included in the identity segment, retained all the functionality of attB; shorter sequences allowed integration with lower efficiencies . By comparing the 26-bp-long attB with attP, according to the Lambda model, we propose that B and B', C and C' core-type Int binding sites consist of 9 bp imperfect inverted repeats separated by a 5 bp overlap region.

Mol Microbiol, 1998 Apr, 28(2), 305 - 13
Three genes encode distinct AP33 proteins involved in Trichomonas vaginalis cytoadherence; Engbring JA et al.; Adherence to host cells is essential for the initiation and maintenance of infection by mucosal pathogens . The protozoan Trichomonas vaginalis colonizes the human urogenital tract via four surface proteins (AP65, AP51, AP33 and AP23) . To characterize AP33 further, six cDNA clones were examined . Restriction mapping indicated that the six clones represented three similar genes . Southern analysis confirmed the existence of three single-copy AP33 genes and suggested a semi-conservative genomic arrangement between T . vaginalis isolates . Analysis of full-length sequences determined that each contained a 930bp open reading frame encoding a protein of approximately 33,000 Da . Sequence comparisons revealed a high degree of identity at both the DNA and the protein levels . N-terminal protein sequencing established the presence of leader peptides . Each of the three full-length recombinant proteins had a predicted pI of approximately 10, which was verified experimentally for the T . vaginalis AP33 adhesin . A database search revealed that AP33 had significant identity to the succinyl-CoA synthetase alpha-subunit of several different organisms and virtually 100% identity to the reported T . vaginalis subunit . Unlike commercially purchased enzyme, the recombinant proteins retained adhesive properties equal to the natural T . vaginalis AP33 . The characteristics of the AP33 protein are similar to those of the other adhesins and emphasize a complex host-parasite relationship.

Mol Microbiol, 1998 Apr, 28(2), 227 - 33
Immunity proteins and their specificity for endonuclease colicins: telling right from wrong in protein-protein recognition; Kleanthous C et al.; Immunity proteins inhibit colicins, protein toxins released by bacteria during times of environmental stress, by binding and inactivating their cytotoxic domains . This protects the producing organism as it attempts to kill off competing bacteria . The cytotoxic domains of related colicins share a high degree of sequence identity, as do their corresponding immunity proteins, yet specificity and affinity are also high, with little non-cognate biological cross-protection evident under physiological conditions . We review recent work on DNase-specific immunity proteins, which shows that, although both cognate and non-cognate proteins can bind a single toxin, their affinities can differ by as much as 12 orders of magnitude . We have termed this mode of binding dual recognition, because the DNase-binding surface of an immunity protein is made up of two components, one conserved and the other variable . The strength of the binding interaction is dominated by the conserved residues, while neighbouring variable residues control specificity . Similar dual recognition systems may exist in other biological contexts, particularly where a protein must discriminate the right binding partner from numerous, structurally homologous alternatives.

RNA, 1998 Jun, 4(6), 658 - 68
New features of 23S ribosomal RNA folding: the long helix 41-42 makes a "U-turn" inside the ribosome; Baranov PV et al.; 23S rRNA from Escherichia coli was cleaved at single internucleotide bonds using ribonuclease H in the presence of appropriate chimeric oligonucleotides; the individual cleavage sites were between residues 384 and 385, 867 and 868, 1045 and 1046, and 2510 and 2511, with an additional fortuitous cleavage at positions 1117 and 1118 . In each case, the 3' terminus of the 5' fragment was ligated to radioactively labeled 4-thiouridine 5'-,3'-biphosphate ("psUp"), and the cleaved 23S rRNA carrying this label was reconstituted into 50S subunits . The 50S subunits were able to associate normally with 30S subunits to form 70S ribosomes . Intra-RNA crosslinks from the 4-thiouridine residues were induced by irradiation at 350 nm, and the crosslink sites within the 23S rRNA were analyzed . The rRNA molecules carrying psUp at positions 867 and 1117 showed crosslinks to nearby positions on the opposite strand of the same double helix where the cleavage was located, and no crosslinking was detected from position 2510 . In contrast, the rRNA carrying psUp at position 384 showed crosslinking to nt 420 (and sometimes also to 416 and 425) in the neighboring helix in 23S rRNA, and the rRNA with psUp at position 1045 gave a crosslink to residue 993 . The latter crosslink demonstrates that the long helix 41-42 of the 23S rRNA (which carries the region associated with GTPase activity) must double back on itself, forming a "U-turn" in the ribosome . This result is discussed in terms of the topography of the GTPase region in the 50S subunit, and its relation to the locations of the 5S rRNA and the peptidyl transferase center.

RNA, 1998 Jun, 4(6), 639 - 46
Recognition of the universally conserved 3'-CCA end of tRNA by elongation factor EF-Tu; Liu JC et al.; Escherichia coli tRNA(Val) with pyrimidine substitutions for the universally conserved 3'-terminal adenine can be readily aminoacylated . It cannot, however, transfer valine into polypeptides . Conversely, despite being a poor substrate for valyl-tRNA synthetase, tRNA(Val) with a 3'-terminal guanine is active in in vitro polypeptide synthesis . To better understand the function of the 3'-CCA sequence of tRNA in protein synthesis, the effects of systematically varying all three bases on formation of the Val-tRNA(Val):EF-Tu:GTP ternary complex were investigated . Substitutions at C74 and C75 have no significant effect, but replacing A76 with pyrimidines decreases the affinity of valyl-tRNA(Val) for EF-Tu:GTP, thus explaining the inability of these tRNA(Val) variants to function in polypeptide synthesis . Valyl-tRNA(Val) terminating in 3'-guanine is readily recognized by EF-TU:GTP . Dissociation constants of the EF-Tu:GTP ternary complexes with valine tRNAs having nucleotide substitutions at the 3' end increase in the order adenine < guanine < uracil; EF-Tu has very little affinity for tRNA terminating in 3' cytosine . Similar observations were made in studies of the interaction of 3' end mutants of E . coli tRNA(Ala) and tRNA(Phe) with EF-Tu:GTP . These results indicate that EF-Tu:GTP preferentially recognizes purines and discriminates against pyrimidines, especially cytosine, at the 3' end of aminoacyl-tRNAs.

Biomed Mater Eng, 1997, 7(6), 391 - 400
Immunogold electron microscopy in situ end-labeling (EM-ISEL): assay for biomaterial DNA damage detection; Assad M et al.; We have evaluated a genotoxicity assay that combines in situ end-labeling, colloidal gold tagging and electron microscopy in order to adapt it to the measurement of in vitro biomaterial-induced genotoxicity . Human lymphocytes were cultured in semi-physiological medium which had been previously exposed to biomaterial extracts of commercially pure titanium following ISO standards . In order to visualize the location of induced DNA strand breaks, cells were then exposed to exonuclease III which partially digests and amplifies lesions by releasing nucleotides at free 3' hydroxyl ends from nicked double-stranded DNA . The resulting single-stranded DNA was allowed to hybridize with short oligonucleotides of random sequences including biotinylated dUTP . After random priming using Escherichia coli DNA polymerase I, incorporation of biotin-dUTP was detected by immunogold binding to the chromatin . Cells exposed to a mutagenic concentration of methyl methanesulfonate, as a positive control, showed a significantly higher and stronger gold staining than both titanium-exposed and unexposed specimens . This assay allows a precise localization and quantification of both in vitro DNA breakage and DNA repair . It could provide a powerful tool for rapid assessment of the genotoxic potential of new biomaterials.

Cancer Gene Ther, 1998 May-Jun, 5(3), 144 - 9
Purine salvage rescue by xanthine-guanine phosphoribosyltransferase (XGPRT) potentiates methotrexate resistance conferred by transfer of a mutated dihydrofolate reductase gene; Mineishi S et al.; We have previously shown that successful gene transfer of a mutated dihydrofolate reductase (DHFR) cDNA confers resistance to methotrexate (MTX) upon infected cells . We constructed a retrovirus vector, DC/SV6S31GPT, which carries both the Escherichia coli xanthine-guanine phosphoribosyltransferase gene and the mutated Serine 31 DHFR gene . Mouse fibroblast NIH3T3 cells infected with DC/SV6S31 GPT are more resistant to MTX than cells infected with DC/SV6S31, which carries the Serine 31 DHFR and the neomycin resistance gene cDNA . The mechanism of this augmented resistance is the increased salvaging of purines due to expression of xanthine-guanine phosphoribosyltransferase, as the augmentation does not occur when dialyzed serum, containing little xanthine or guanine, is used for cytotoxicity assays . These results indicate that coexpression of a metabolically related gene can potentiate the resistance carried by a drug resistance gene . This vector may be useful in clinical gene therapy to protect bone marrow from the toxic effects of MTX.

J Am Vet Med Assoc, 1998 Jun 1, 212(11), 1746 - 50
Randomized controlled trial of effects of Escherichia coli antiserum on serum immunoglobulin G concentrations and morbidity and mortality rates in foals; Chaffin MK et al.; OBJECTIVE: To determine whether administration of commercially available Escherichia coli antiserum to neonatal foals would affect serum IgG concentration or morbidity and mortality rates during the first 60 days of life . DESIGN: Randomized controlled trial . ANIMALS: 271 neonatal foals on 4 well-managed farms . PROCEDURE: Foals were randomly assigned to a treatment or control group . All foals were allowed to suckle colostrum normally . In addition, treatment-group foals were given E coli antiserum (10 micromilligrams) orally between 0 and 8 hours after birth . Serum samples were obtained between 18 and 36 hours after birth, and serum IgG concentration was determined . Foals were monitored for the first 60 days after birth, and causes of disease or death were recorded . RESULTS: Groups did not differ significantly in regard to breed, sex, month of birth, season of birth, age of dams, parity of dams, duration of gestation, or specific gravity of colostrum before suckling . In addition, groups did not differ significantly in regard to mean serum IgG concentration, prevalence of complete or partial failure of passive transfer of immunity, frequency or causes of disease, or frequency of death from infectious causes . CLINICAL IMPLICATIONS: In this group of foals on well-managed farms, administration of E coli antiserum did not alter serum IgG concentrations or morbidity and mortality rates during the first 60 days of life.

J Hum Genet, 1998, 43(2), 135 - 7
A one-base deletion (183delC) and a missense mutation (D276H) in the T-protein gene from a Japanese family with nonketotic hyperglycinemia; Kure S et al.; Two novel mutations in the gene encoding T-protein, a component of the glycine cleavage system, were identified in a Japanese family with nonketotic hyperglycinemia . The proband had two affected sibs, and enzymatic analysis of the liver sample from the proband revealed the T-protein deficiency . The first mutation, 183delC, was found in exon 1 . One of six cytidine residues (base position 183-188) was deleted . The deletion was located in a coding region of the mitochondrial leader peptide and was deduced to create a truncated peptide with 94 amino acids . The second mutation was a base substitution from G to C at position 955 in exon 7 . The G955C substitution caused an amino acid change from aspartate to histidine at position 276 (D276H) . Aspartic acid at position 276 is evolutionarily conserved among human, bovine, chicken, and pea genes, and replaced by glutamic acid in Escherichia coli, suggesting that the presence of an acidic amino acid at 276 may be crucial for the enzymatic function . No base change other than the 183delC and the G955C was observed in the sequencing analysis . Familial analysis revealed that the 183delC and the D276H mutations were inherited from the father and the mother, respectively . This is the first report of T-protein gene mutation in Oriental patients with nonketotic hyperglycinemia.

J Hematother, 1998 Jun, 7(3), 225 - 39
Retroviral-mediated marker gene transfer in hematopoiesis-supportive marrow stromal cells; Bulabois CE et al.; A Moloney-derived retrovirus containing both LacZ and NeoR genes (G1BgSVNa from Genetic Therapy, Inc.), was used to transduce human and murine bone marrow stromal cells . Different kinds of stromal cells that were able to support hematopoiesis were transduced by incubation for 24 h in the presence of virus-containing supernatant . Semiconfluent layers of MRC-5 (human, myofibroblastic, fetal, pulmonary) and MS-5 (murine, myofibroblastic, medullary) cells were successfully transduced after one 24-h incubation, as demonstrated by G418 resistance and Escherichia coli beta-galactosidase staining . In contrast, human stromal cells, purified from primary confluent layers grown for 3-4 weeks, could not be transduced . However, stromal cells generated after 10-12 days in culture from Stro-1+ and 1B10+ stromal precursors were successfully transduced in the presence of basic fibroblast growth factor . Transduced stromal cells maintained a myofibroblastic phenotype, although with a decreased number of alpha-SM actin-positive microfilaments in MS-5 cells . The ability to support the generation of stroma-adherent colony-forming cells from cocultured cord blood CD34+ cells after 4 weeks in culture was similar before and after transduction and G418 selection . In conclusion, human primary stromal precursors can be efficiently transduced, and the stromal cell phenotype and function are not significantly altered after retroviral-mediated transfer of marker genes.

Emerg Infect Dis, 1998 Apr-Jun, 4(2), 251 - 61
Enteroaggregative Escherichia coli; Nataro JP et al.; Enteroaggregative Escherichia coli (EAEC), an increasingly recognized cause of diarrhea in children in developing countries, has been particularly associated with persistent diarrhea (more than 14 days), a major cause of illness and death . Recent outbreaks implicate EAEC as a cause of foodborne illness in industrialized countries . The pathogenesis of EAEC infection is not well understood, but a model can be proposed in which EAEC adhere to the intestinal mucosa and elaborate enterotoxins and cytotoxins, which result in secretory diarrhea and mucosal damage . EAEC's ability to stimulate the release of inflammatory mediators may also play a role in intestinal illness.

Glycobiology, 1998 Jul, 8(7), 719 - 24
Ion-spray mass spectrometry for identification of the nonreducing terminal sugar of glycosaminoglycan; Takagaki K et al.; Various oligosaccharides from hyaluronic acid, which have glucuronic acid or N- acetylglucosamine at the nonreducing terminal, were prepared by digestion with a combination of testicular hyaluronidase and beta-glucuronidase . These oligo saccharides were analyzed by negative-mode ion-spray mass spectrometry (MS) with an atmospheric pressure ion source . Introduction of collisionally activated dissociation tandem mass spectrometry (CAD-MS/MS) produced ions derived from cleavage of the glycosidic bonds, allowing the structure to be analyzed . The CAD-MS/MS spectrum showed an intense and characteristic fragment ion at m/z 193 for oligosaccharides having glucuronic acid at the nonreducing terminal . On the other hand, this ion was not observed in the spectra of oligosaccharides having N- acetylglucosamine at the nonreducing terminal . Therefore, the fragmentation pattern revealed by CAD-MS/MS provides useful information for distinguishing glucuronic acid and N- acetylglucosamine at the nonreducing terminal of oligosaccharides derived from hyaluronic acid and other glycosaminoglycans . This ion-spray CAD-MS/MS technique was also applied successfully to the characterization of glycosaminoglycans reconstructed by glycotechnology.

J Bacteriol, 1998 Jun, 180(12), 3245 - 9
Appropriate expression of filamentous phage f1 DNA replication genes II and X requires RNase E-dependent processing and separate mRNAs; Kokoska RJ et al.; The products of in-frame overlapping genes II and X carried by the filamentous phage f1 genome are proteins with required but opposing functions in phage DNA replication . Their normal relative levels are important for continuous production of phage DNA without killing infected Escherichia coli hosts . Here we identify several factors responsible for determining the relative levels of pII and pX and that, if perturbed, alter the normal distribution of the phage DNA species in infected hosts . Translation of the two proteins is essentially relegated to separate mRNAs . The mRNAs encoding genes II and X are also differentially sensitive to cleavage dependent on rne, the gene encoding the only E . coli endo-RNase known to have a global role in mRNA stability . Whereas pII levels are limited at the level of mRNA stability, normal pX levels require transcription in sufficient amounts from the promoter for the smaller mRNA encoding only pX.

J Bacteriol, 1998 Jun, 180(12), 3237 - 40
An autonomously replicating transforming vector for Sulfolobus solfataricus; Cannio R et al.; A plasmid able to transform and to be stably maintained both in Sulfolobus solfataricus and in Escherichia coli was constructed by insertion into an E . coli plasmid of the autonomously replicating sequence of the virus particle SSV1 and a suitable mutant of the hph (hygromycin phosphotransferase) gene as the transformation marker . The vector suffered no rearrangement and/or chromosome integration, and its copy number in Sulfolobus was increased by exposure of the cells to mitomycin C.

J Bacteriol, 1998 Jun, 180(12), 3218 - 21
The MTCY428.08 gene of Mycobacterium tuberculosis codes for NAD+ synthetase; Cantoni R et al.; The product of the MTCY428.08 gene of Mycobacterium tuberculosis shows sequence homology with several NAD+ synthetases . The MTCY428.08 gene was cloned into the expression vectors pGEX-4T-1 and pET-15b . Expression in Escherichia coli led to overproduction of glutathione S-transferase fused and His6-tagged gene products, which were enzymatically assayed for NAD synthetase activity . Our results demonstrate that the MTCY428.08 gene of M . tuberculosis is the structural gene for NAD+ synthetase.

J Bacteriol, 1998 Jun, 180(12), 3205 - 8
Effects of carbon source on expression of F0 genes and on the stoichiometry of the c subunit in the F1F0 ATPase of Escherichia coli; Schemidt RA et al.; Expression of the genes for the membrane-bound F0 sector of the Escherichia coli F1F0 proton-translocating ATPase can respond to changes in metabolic conditions, and these changes are reflected in alterations in the subunit stoichiometry of the oligomeric F0 proton channel . Transcriptional and translational lacZ fusions to the promoter and to two F0 genes show that, during growth on the nonfermentable carbon source succinate, transcription of the operon and translation of uncB, encoding the a subunit of F0, are higher than during growth on glucose . In contrast, translation of the uncE gene, encoding the c subunit of F0, is higher during growth on glucose than during growth on succinate . Translation rates of both uncB and uncE change as culture density increases, but transcription rates do not . Quantitation of the c stoichiometry shows that more c subunits are assembled into the F1F0 ATPase in cells grown on glucose than in cells grown on succinate . E . coli therefore appears to have a mechanism for regulating the composition and, presumably, the function of the ATPase in response to metabolic circumstances.

J Bacteriol, 1998 Jun, 180(12), 3120 - 30
Folding-based suppression of extracytoplasmic toxicity conferred by processing-defective LamB; Cosma CL et al.; We have utilized processing-defective derivatives of the outer membrane maltoporin, LamB, to study protein trafficking functions in the cell envelope of Escherichia coli . Our model proteins contain amino acid substitutions in the consensus site for cleavage by signal peptidase . As a result, the signal sequence is cleaved with reduced efficiency, effectively tethering the precursor protein to the inner membrane . These mutant porins are toxic when secreted to the cell envelope . Furthermore, strains producing these proteins exhibit altered outer membrane permeability, suggesting that the toxicity stems from some perturbation of the cell envelope (J . H . Carlson and T . J . Silhavy, J . Bacteriol . 175:3327-3334, 1993) . We have characterized a multicopy suppressor of the processing-defective porins that appears to act by a novel mechanism . Using fractionation experiments and conformation-specific antibodies, we found that the presence of this multicopy suppressor allowed the processing-defective LamB precursors to be folded and localized to the outer membrane . Analysis of the suppressor plasmid revealed that these effects are mediated by the presence of a truncated derivative of the polytopic inner membrane protein, TetA . The suppression mediated by TetA' is independent of the CpxA/CpxR regulon and the sigma E regulon, both of which are involved in regulating protein trafficking functions in the cell envelope.

J Bacteriol, 1998 Jun, 180(12), 3114 - 9
Changes in ribosomal activity of Escherichia coli cells during prolonged culture in sea salts medium; Kalpaxis DL et al.; The activity of ribosomes from a clinical isolate of Escherichia coli, exposed to starvation for 7 days in sea salts medium, was investigated by measuring the kinetic parameters of ribosomal peptidyltransferase, by using the puromycin reaction as a model reaction . No alterations in the extent of peptide bond formation were observed during starvation . In contrast, a 50% reduction in the kmax/Ks ratio could be seen after 24 h of starvation; an additional 6 days of starvation resulted in a progressive but less abrupt decline in the kmax/Ks value . (kmax is the apparent catalytic rate constant of peptidyl transferase, and Ks is the dissociation constant of the encounter complex between acetyl (Ac){3H}Phe-tRNA-poly(U)-ribosome and puromycin.) Although the distribution of ribosomal particles remained constant, a substantial decrease in the number of ribosomes per starved cell and a clear decline in the ability of ribosomes to bind AcPhe-tRNA were observed, particularly during the first day of starvation . Further analysis indicated that rRNA in general, but especially 23S rRNA, was rapidly degraded during the starvation period . In addition, the L12/L7 molar ratio decreased from 1.5 to 1 during the initial phase of starvation (up to 24 h) but remained constant during the subsequent starvation period . Ribosomes isolated from 24-h-starved cells, when artificially depleted of L7/L12 protein and reconstituted with L7/L12 protein from mid-logarithmic-phase cells, regenerated an L12/L7 molar ratio of 1.5 and restored the peptidyltransferase activity to a substantial level . An analogous effect of reconstitution on the efficiency of ribosomes in binding AcPhe-tRNA was evident not only during the initial phase but throughout the starvation period.

J Bacteriol, 1998 Jun, 180(12), 3070 - 9
Promoter region of the Escherichia coli O7-specific lipopolysaccharide gene cluster: structural and functional characterization of an upstream untranslated mRNA sequence; Marolda CL et al.; We report the identification of the promoter region of the Escherichia coli O7-specific lipopolysaccharide (LPS) gene cluster (wbEcO7) . Typical -10 and -35 sequences were found to be located in the intervening region between galF and rlmB, the first gene of the wbEcO7 cluster . Data from RNase protection experiments revealed the existence of an untranslated leader mRNA segment of 173 bp, including the JUMPStart and two ops sequences . We characterized the structure of this leader mRNA by using the program Mfold and a combination of nested and internal deletions transcriptionally fused to a promoterless lac operon . Our results indicated that the leader mRNA may fold into a series of complex stem-loop structures, one of which includes the JUMPStart element . We have also found that one of the ops sequences resides on the predicted stem and the other resides on the loop region, and we confirmed that these sequences are essential for the RfaH-mediated regulation of the O polysaccharide cluster . A very similar stem-loop structure could be predicted in the promoter region of the LPS core operon encoding the waaQGPSBIJYZK genes . We observed another predicted stem-loop, located immediately downstream from the wbEcO7 transcription initiation site, which appeared to be involved in premature termination of transcription . This putative stem-loop is common to many other O polysaccharide gene clusters but is not present in core oligosaccharide genes . wbEcO7-lac transcriptional fusions in single copy numbers were also used to determine the effects of various environmental cues in the transcriptional regulation of O polysaccharide synthesis . No effects were detected with temperature, osmolarity, Mg2+ concentration, and drugs inducing changes in DNA supercoiling . We therefore conclude that the wbEcO7 promoter activity may be constitutive and that regulation takes place at the level of elongation of the mRNA in a RfaH-mediated manner.

J Bacteriol, 1998 Jun, 180(12), 3039 - 48
Target choice and orientation preference of the insertion sequence IS903; Hu WY et al.; We have examined the targeting preference of the bacterial insertion element IS903 by determining the sites of insertion of a large number of transposition events into the 55-kb conjugative plasmid pOX38 . Despite the large target size, all the insertions were clustered in four small distinct regions associated with conjugal DNA transfer . Within these regions, many different sites were used for insertion; however, there were a few sites that IS903 inserted into more than once . Alignment of the insertion sites showed that there was no consensus sequence within the 9-bp target duplication but that there were preferred sequences located symmetrically on either side of the target . This is consistent with target recognition by a dimer or multimer of transposase, with either sequence-specific or structure-specific interactions on both sides of the target . We show further that when one of these preferred regions was cloned into a second conjugative plasmid, pUB307, it was still a preferred target, implying that all the sequences necessary for target selection are contained within this DNA segment . Also, we observed a very strong preference for insertion in a single orientation in pUB307 . We examined the possibility that either DNA replication from the origin of vegetative replication, oriV, or the origin of transfer, oriT, might determine this orientation effect . We find that reversing the direction of vegetative replication had no effect on the orientation of transposon insertions; however, reversing the direction of DNA transfer abolished the orientation effect . This supports the idea that conjugal DNA transfer imparts a polarity on the target that is sensed by the transposon.

J Bacteriol, 1998 Jun, 180(12), 3026 - 30
In vivo analysis of sequence requirements for processing and degradation of the colicin A lysis protein signal peptide; Howard SP et al.; The lipid modification and processing of a number of colicin lysis proteins take place exceedingly slowly and result in the release of a stable signal peptide . It is possible that this peptide or the presence of lipid-modified precursors which result from the slow processing plays a role in the release of colicins and in the quasilysis that occurs in induced colicinogenic cultures . We used in vitro mutagenesis and pulse-chase radiolabeling and immunoprecipitation to examine the reasons for the slow processing and signal peptide degradation reactions for the colicin A lysis protein (Cal) . In one mutant, isoleucine 13 was replaced with serine, and in another, alanine 18, the last residue of the signal peptide, was replaced with glycine . In each case, the mutation caused a striking increase in the rate of maturation of the precursor, and in the case of the serine 13 derivative, the mutation also destabilized the signal peptide . A precursor containing both of these mutations was completely matured and its signal sequence degraded within seconds of its synthesis . The release of colicin A and the quasilysis of producing cultures were unchanged for each of these mutants, indicating that neither the stable signal peptide nor lipid-modified processing intermediates of Cal are required for either of these events in wild-type cells.

Biochem J, 1998 Jun 15, 332 ( Pt 3), 651 - 9
Structure and activity of mouse S-adenosylmethionine decarboxylase gene promoters and properties of the encoded proteins; Nishimura K et al.; The promoter regions of two S-adenosylmethionine decarboxylase genes (AMD genes) were isolated from a mouse genomic library . One promoter was that of the bona fide mouse AMD gene (AMD1) whereas the other was that of the intronless AMD gene (AMD2) . There was no sequence identity between the two promoters . The sequence of the AMD1 promoter was highly homologous to the human AMD1 and rat Amd1B promoters . After transient transfection in various cell lines, the AMD1 promoter was one to two orders of magnitude stronger than the AMD2 promoter . Similar results were obtained by using stably transfected mouse FM3A cells . In S-adenosylmethionine decarboxylase (AdoMetDC)-overproducing SAM-1 cells, the AMD1 gene was amplified over 5-fold . AdoMetDC encoded by the intronless AMD2 gene had two amino acid replacements (Met to Ile at codon 70 and Ala to Val at codon 139), compared with the protein encoded by the AMD1 gene, and exhibited decreased catalytic activity (<50%) and decreased processing activity when expressed in AdoMetDC-deficient Escherichia coli cells . When Ile-70 of the protein encoded by AMD2 was converted into Met, both the catalytic and processing activities recovered markedly, indicating that Met-70 adjacent to the proenzyme-processing site is important for both activities . The third AMD locus (AMD3) in FM3A cells contains a pseudogene, in which deletion of two bases generates a premature termination codon at position 57 . Since the AMD2 promoter had only 1-10% of the strength of the bona fide AMD1 gene and AMD2 protein possessed lower specific activity, the relative contribution of the AMD2-encoded enzyme to total AdoMetDC activity is small . Thus AdoMetDC activity in murine cells is thought to be due mainly to the product of the AMD1 gene.

J Biol Chem, 1998 Jun 5, 273(23), 14582 - 7
Molecular cloning and expression of GDP-D-mannose-4,6-dehydratase, a key enzyme for fucose metabolism defective in Lec13 cells; Ohyama C et al.; Subsets of mammalian cell surface oligosaccharides contain specific fucosylated moieties expressed in lineage- and/or temporal-specific patterns . The functional significance of these fucosylated structures is incompletely defined, although there is evidence that subsets of them, represented by the sialyl Lex determinant, are important participants in leukocyte adhesion and trafficking processes . Genetic deletion of these fucosylated structures in the mouse has been a powerful tool to address functional questions about fucosylated glycans . However, successful use of such approaches can be problematic, given the substantial redundancy in the mammalian alpha-1,3-fucosyltransferase and alpha-1,2-fucosyltransferase gene families . To circumvent this problem, we have chosen to clone the genetic locus encoding a mammalian GDP-D-mannose-4,6-dehydratase (GMD) . This enzyme generates GDP-mannose-4-keto-6-D-deoxymannose from GDP-mannose, which is then converted by the FX protein (GDP-4-keto-6-D-deoxymannose epimerase/GDP-4-keto-6-L-galactose reductase) to GDP-L-fucose . GMD is thus imperative for the synthesis of all fucosylated oligosaccharides . An expression cloning approach and the GMD-deficient CHO host cell line Lec13 were used to generate a population of cDNA molecules enriched in GMD cDNAs . This enriched plasmid population was then screened using a human expressed sequence tag (EST AA065072) with sequence similarity to an Arabidopsis thaliana GMD cDNA . This approach, together with 5'-rapid amplification of cDNA ends, yielded a human cDNA that complements the fucosylation defect in the Lec13 cell line . Northern blot analyses indicate that the GMD transcript is absent in Lec13 cells, confirming the genetic deficiency of this locus in these cells . By contrast, the transcript encoding the FX protein, which forms GDP-L-fucose from the ketosugar intermediate produced by GMD, is present in increased amounts in the Lec13 cells . These results suggest that metabolites generated in this pathway may participate in the transcriptional regulation of the FX protein and possibly the GMD protein . The results also suggest that the genomic structure encoding GMD in Lec13 cells likely has a defect different from a point mutation in the coding region.

J Biol Chem, 1998 Jun 5, 273(23), 14269 - 76
The phosphate carrier from yeast mitochondria . Dimerization is a prerequisite for function; Schroers A et al.; Wild type phosphate carrier (PIC) from Saccharomyces cerevisiae and recombinant PIC proteins with different C-terminal extensions were expressed in Escherichia coli as inclusion bodies . From these, PIC was isolated with the detergent sodium lauroyl sarcosinate in a form, partially monomeric and unfolded . This PIC associates to stable dimers after exchanging the detergent to the polyoxyethylene detergent C12E8 and dialysis . Combining two differently tagged monomers of PIC and following this with affinity chromatography yields defined homo- and heterodimeric forms of PIC, which are all fully active after reconstitution . As a member of the mitochondrial carrier family PIC is supposed to function as a homodimer . We investigated its dimeric nature in the functionally active state after reconstitution . When reconstituting PIC monomers a sigmoidal dependence of transport activity on the amount of inserted protein is observed, whereas insertion of PIC dimers leads to a linear dependence . Heterodimeric PIC constructs consisting of both an active and an inactivated subunit do not catalyze phosphate transport . In contrast, reconstitution of a mixture of active and inactive monomeric subunits led to partially active carrier . These experiments prove (i) that PIC does not function in monomeric form, (ii) that PIC dimers are stable both in the solubilized state and after membrane insertion, and (iii) that transport catalyzed by PIC dimers involves functional cross-talk between the two monomers.

J Biol Chem, 1998 Jun 5, 273(23), 14172 - 8
Pressure-induced dissociation of carbamoyl-phosphate synthetase domains . The catalytically active form is dimeric; Guy HI et al.; Carbamoyl-phosphate synthetase consists of an amidotransferase domain or subunit (GLN) that hydrolyzes glutamine and transfers the ammonia to the synthetase component (CPS) where the biosynthetic reaction occurs . The CPS domain is composed of two homologous subdomains, CPS.A and CPS.B, that catalyze different ATP-dependent reactions involved in carbamoyl phosphate synthesis . When the individual CPS.A and CPS.B subdomains were individually cloned and expressed in Escherichia coli (Guy, H . I., and Evans, D . R . (1996) J . Biol . Chem . 271, 13762-13769), they were found to be functionally equivalent and could each independently catalyze carbamoyl phosphate synthesis . The proposal was advanced that, although the monomers could catalyze the individual partial reactions, overall synthesis of carbamoyl phosphate required a homodimer of CPS.A or CPS.B . To test this hypothesis, the GLN-CPS.B dimer was reversibly dissociated at 1500 bar in a high pressure cell . Dissociation was accompanied by a loss of both glutamine- and ammonia-dependent CPSase activity . Activity was recovered once the protein was returned to atmospheric pressure . If the sample was cross-linked before exposure to high pressure, there was no dissociation and no loss of biosynthetic activity . In contrast, the bicarbonate-dependent ATPase and the carbamoyl phosphate-dependent ATP synthetase activities were largely unaffected by pressure-induced dissociation . These experiments confirmed the hypothesis that the synthesis of carbamoyl phosphate requires the concerted action of the two active sites within the homodimer.

Biochem J, 1998 Apr 15, 331 ( Pt 2), 459 - 64
The reaction of halides with pulsed cytochrome bo from Escherichia coli; Moody AJ et al.; Cytochrome bo forms complexes with chloride, bromide and iodide in which haem o remains high-spin and in which the '630 nm' charge-transfer band is red-shifted by 7-8 nm . The chloride and bromide complexes each have a characteristic set of integer-spin EPR signals arising from spin coupling between haem o and CuB . The rate and extent of chloride binding decreases as the pH increases from 5.5 to 8.5 . At pH 5.5 the dissociation constant for chloride is 2 mM and the first-order rate constant for dissociation is 2 x 10(-4) s-1 . The order of rate of binding, and of affinity, at pH 5.5 is chloride (1) > bromide (0.3) >iodide (0.1) . It is suggested that the halides bind in the binuclear site but, unlike fluoride, they are not direct ligands of the iron of haem o . In addition, both the stability of the halide complexes and the rate of halide binding seem to be increased by the co-binding of a proton.

Biochem J, 1998 Apr 15, 331 ( Pt 2), 437 - 45
The dhnA gene of Escherichia coli encodes a class I fructose bisphosphate aldolase; Thomson GJ et al.; The gene encoding the Escherichia coli Class I fructose-1, 6-bisphosphate aldolase (FBP aldolase) has been cloned and the protein overproduced in high amounts . This gene sequence has previously been identified as encoding an E . coli dehydrin in the GenBanktrade mark database {gene dhnA; entry code U73760; Close and Choi (1996) Submission to GenBanktrade mark} . However, the purified protein overproduced from the dhnA gene shares all its properties with those known for the E . coli Class I FBP aldolase . The protein is an 8-10-mer with a native molecular mass of approx . 340 kDa, each subunit consisting of 349 amino acids . The Class I enzyme shows low sequence identity with other known FBP aldolases, both Class I and Class II (in the order of 20%), which may be reflected by some novel properties of this FBP aldolase . The active-site peptide has been isolated and the Schiff-base-forming lysine residue (Lys236) has been identified by a combination of site-directed mutagenesis, kinetics and electrospray-ionization MS . A second lysine residue (Lys238) has been implicated in substrate binding . The cloning of this gene and the high levels of overexpression obtained will facilitate future structure-function studies.

Biochem J, 1998 Apr 15, 331 ( Pt 2), 423 - 30
Functional implications of ribosomal protein L2 in protein biosynthesis as shown by in vivo replacement studies; Uhlein M et al.; The translational apparatus is a highly complex structure containing three to four RNA molecules and more than 50 different proteins . In recent years considerable evidence has accumulated to indicate that the RNA participates intensively in the catalysis of peptide-bond formation, whereas a direct involvement of the ribosomal proteins has yet to be demonstrated . Here we report the functional and structural conservation of a peptidyltransferase centre protein in all three phylogenetic domains . In vivo replacement studies show that the Escherichia coli L2 protein can be replaced by its homologous proteins from human and archaebacterial ribosomes . These hybrid ribosomes are active in protein biosynthesis, as proven by polysome analysis and poly(U)-dependent polyphenylalanine synthesis . Furthermore, we demonstrate that a specific, highly conserved, histidine residue in the C-terminal region of L2 is essential for the function of the translational apparatus . Replacement of this histidine residue in the human and archaebacterial proteins by glycine, arginine or alanine had no effect on ribosome assembly, but strongly reduced the translational activity of ribosomes containing these mutants.

Biochem J, 1998 Apr 15, 331 ( Pt 2), 409 - 15
Functional effects of single amino acid substitutions in the region of Phe113 to Asp138 in the plasminogen activator inhibitor 1 molecule; Sui GC et al.; Thirteen amino acid substitutions have been introduced within the stretch Phe113 to Asp138 in the plasminogen activator inhibitor 1 (PAI-1) molecule by site-directed mutagenesis . The different proteins and wild-type (wt) PAI-1 have been overexpressed in Escherichia coli and purified by chromatography on heparin-Sepharose and on anhydrotrypsin-agarose . The PAI-1 variants have been characterized by their reactivity with tissue plasminogen activator (tPA), interactions with vitronectin or heparin, and stability . Most PAI-1 variants, except for Asp125-->Lys, Phe126-->Ser and Arg133-->Asp, displayed a high spontaneous inhibitory activity towards tPA, which did not change greatly on reactivation with 4 M guanidinium chloride, followed by dialysis at pH 5.5 . The variants Asp125-->Lys and Arg133-->Asp became much more active after reactivation and they were also more rapidly transformed to inactive forms (t12 22-31 min) at physiological pH and temperature than the other variants . However, in the presence of vitronectin they were both almost equally stable (t12 2.3 h) as wtPAI-1 (t12 3.0 h) . The mutant Glu130-->Lys showed an increased stability, both in the absence and in the presence of vitronectin compared with wtPAI-1 . Nevertheless a similar affinity between all the active PAI-1 variants and vitronectin was observed . Further, all mutants, including the three mutants with low activity, were to a large extent adsorbed on anhydrotrypsin-agarose and were eluted in a similar fashion . In accordance with these data, the three variants with a low activity were all to a large extent cleaved as a result of their reaction with tPA, suggesting that they occurred predominantly in the substrate conformation . Our results do not support the presence of a binding site for vitronectin in this part of the molecule, but rather that it might be involved in controlling the active PAI-1 to substrate transition . Partly, this region of the PAI-1 molecule (Arg115 to Arg118) seems also to be involved in the binding of heparin to PAI-1.

Nature, 1998 May 28, 393(6683), 392 - 6
Atomic structure of progesterone complexed with its receptor; Williams SP et al.; The physiological effects of progestins are mediated by the progesterone receptor, a member of the steroid/nuclear receptor superfamily . As progesterone is required for maintenance of pregnancy, its receptor has been a target for pharmaceuticals . Here we report the 1.8 A crystal structure of a progesterone-bound ligand-binding domain of the human progesterone receptor . The nature of this structure explains the receptor's selective affinity for progestins and establishes a common mode of recognition of 3-oxy steroids by the cognate receptors . Although the overall fold of the progesterone receptor is similar to that found in related receptors, the progesterone receptor has a quite different mode of dimerization . A hormone-induced stabilization of the carboxy-terminal secondary structure of the ligand-binding domain of the progesterone receptor accounts for the stereochemistry of this distinctive dimer, explains the receptor's characteristic pattern of ligand-dependent protease resistance and its loss of repression, and indicates how the anti-progestin RU486 might work in birth control . The structure also indicates that the analogous 3-keto-steroid receptors may have a similar mechanism of action.

J Clin Microbiol, 1998 Jun, 36(6), 1795 - 7
Multiplex PCR for enterotoxigenic, attaching and effacing, and Shiga toxin-producing Escherichia coli strains from calves; Franck SM et al.; A multiplex PCR was developed to identify enterotoxigenic, attaching and effacing, and Shiga toxin-producing Escherichia coli strains by amplifying genes encoding K99 and F41 fimbriae, heat-stable enterotoxin a, intimin, and Shiga toxins 1 and 2 . This multiplex PCR was specific and sensitive . It will be useful for identification of E . coli strains which cause diarrhea in calves.

J Clin Microbiol, 1998 Jun, 36(6), 1604 - 7
Virulence properties of Shiga toxin-producing Escherichia coli (STEC) strains of serogroup O118, a major group of STEC pathogens in calves; Wieler LH et al.; Shiga toxin-producing Escherichia coli (STEC) strains of serogroup 0118 are the most prevalent group among STEC strains in diarrheic calves in Germany (L . H . Wieler, Ph.D . thesis, University of Giessen, 1997) . To define their virulence properties, 42 0118 (0118:H16 {n = 38} and 0118:H- {n = 4}) strains were characterized . The strains displayed three different Stx combinations (Stx1 {36 of 42}, Stx1 and Stx2 {2 of 42}, and Stx2 {4 of 42}) . A total of 41 strains (97.6%) harbored a large virulence-associated plasmid containing hlyEHEC (hly from enterohemorrhagic E . coli) . The strains' adhesive properties varied in relation to the eukaryotic cells tested . Only 28 of 42 strains (66.7%) showed localized adhesion (LA) in the human HEp-2 cell line . In contrast, in bovine fetal calf lung (FCL) cells, the number of LA-positive strains was much higher (37 of 42 {88.1%}) . The locus of enterocyte effacement (LEE) was detected in 41 strains (97.6%) . However, not all LEE-positive strains reacted positively in the fluorescence actin-staining (FAS) test, which indicated the attaching and effacing (AE) lesion . In HEp-2 cells, only 22 strains (52.4%) were FAS positive, while in FCL cells, the number of FAS-positive strains was significantly higher (38 of 42 {90.5%; P < 0.001}) . In conclusion, the vast majority of the 0118 STEC strains from calves (41 of 42 {97.6%}) have a high virulence potential (stx, hlyEHEC, and LEE) . This virulence potential and the high prevalence of STEC 0118 strains in calves suggest that these strains could be a major health threat for humans in the future . In addition, the poor association between results of the geno- and phenotypical tests to screen for the AE ability of STEC strains calls the diagnostic value of the FAS test into question.

J Interferon Cytokine Res, 1998 May, 18(5), 287 - 95
The glycosylation heterogeneity of recombinant human IFN-gamma; Hooker A et al.; The cloning of the cDNA for human interferon-gamma (IFN-gamma) has resulted in its expression in Escherichia coli, baculovirus-infected insect cells, Chinese hamster ovary (CHO) cells, and the mammary gland of transgenic mice . Large quantities of highly purified recombinant IFN-gamma have been generated, aided by the use of highly specific neutralizing monoclonal antibodies, with a view to its production as a human therapeutic protein . The primary source of structural heterogeneity for IFN-gamma during its production in mammalian expression systems is glycosylation, which can profoundly affect the three-dimensional structure of a glycoprotein and its biological function . A number of analytical approaches have been developed recently to allow a detailed analysis of the carbohydrate structures associated with IFN-gamma, the principal advances being in the areas of capillary electrophoresis and mass spectrometry . The implementation of these high-resolution analytical tools to determine the glycosylation profile of IFN-gamma makes it one of the best characterized recombinant glycoproteins . Recombinant human IFN-gamma acts as a model secretory glycoprotein, typifying the intrinsic glycosylation processing events associated with production of a potential therapeutic glycoprotein.

Plant Mol Biol, 1998 May, 37(1), 179 - 85
Isolation, characterisation and expression of a cDNA for pea cholinephosphate cytidylyltransferase; Jones PL et al.; In plants, phosphatidylcholine is the major phospholipid in extra-plastid membranes and is synthesised mainly by the CDP-choline pathway . Evidence from studies in animals, as well as in plants, suggests that the intermediate step catalysed by cholinephosphate cytidylyltransferase (CPCT) has a major control in carbon flux to this lipid . We have isolated a full-length CPCT cDNA (designated PCT2) from Pisum sativum cv . Feltham First using an Arabidopsis probe and the polymerase chain reaction (PCR) . The deduced amino acid of PCT2 is 48%, 43% and 76% identical to the rat, yeast and Brassica napus amino acid sequences, respectively . Expression of the CPCT protein in Escherichia coli confirmed the activity of the enzyme . Expression of the PCT2 mRNA in pea roots and stems was increased by treatment with 0.1 microM indole-3-acetic acid.

Plant Mol Biol, 1998 May, 37(1), 131 - 40
Molecular cloning and characterization of an inorganic pyrophosphatase from barley; Visser K et al.; A cDNA clone with sequence homology to soluble inorganic pyrophosphatase (IPPase) was isolated from a library of developing barley grains . The protein encoded by this clone was produced in transgenic Escherichia coli, and showed IPPase activity . In nondormant barley grains, the gene appeared to be expressed in metabolically active tissue such as root, shoot, embryo and aleurone . During inhibition, a continuous increase of the steady state mRNA level of IPPase was observed in embryos of non-dormant grains . In the embryos of dormant grains its production declined, after an initial increase . With isolated dormant and nondormant embryos, addition of recombinant IPPase, produced by E . coli, enhanced the germination rate . On the other hand, addition of pyrophosphate (PPi), substrate for this enzyme, appeared to reduce the germination rate . A role for this IPPase in germination is discussed.

Plant Mol Biol, 1998 May, 37(1), 99 - 108
Characterization of a single soybean cDNA encoding cytosolic and glyoxysomal isozymes of aspartate aminotransferase; Gebhardt JS et al.; A soybean cDNA clone, pSAT1, which encodes both the cytosolic and glyoxysomal isozymes of aspartate aminotransferase (AAT; EC 2.6.1.1) was isolated . Genomic Southern blots and analysis of genomic clones indicated pSAT1 was encoded by a single copy gene . pSAT1 contained an open reading frame with ca . 90% amino acid identity to alfalfa and lupin cytosolic AAT and two in-frame start codons, designated ATG1 and ATG2 . Alignment of this protein with other plant cytosolic AAT isozymes revealed a 37 amino acid N-terminal extension with characteristics of a peroxisomal targeting signal, designated PTS2, including the modified consensus sequence RL-X5-HF . The second start codon ATG2 aligned with previously reported start codons for plant cytosolic AAT cDNAs . Plasmids constructed to express the open reading frame initiated by each of the putative start codons produced proteins with AAT activity in Escherichia coli . Immune serum raised against the pSAT1-encoded protein reacted with three soybean AAT isozymes, AAT1 (glyoxysomal), AAT2 (cytosolic), and AAT3 (subcellular location unknown) . We propose the glyoxysomal isozyme AAT1 is produced by translational initiation from ATG1 and the cytosolic isozyme AAT2 is produced by translational initiation from ATG2 . N-terminal sequencing of purified AAT1 revealed complete identity with the pSAT1-encoded protein and was consistent with the processing of the PTS2 . Analysis of cytosolic AAT genomic sequences from several other plant species revealed conservation of the two in-frame start codons and the PTS2 sequence, suggesting that these other species may utilize a single gene to generate both cytosolic and glyoxysomal or peroxisomal forms of AAT.

Plant Mol Biol, 1998 May, 37(1), 25 - 37
Intraspecific variability of the tandem repeats in Nicotiana putrescine N-methyltransferases; Hashimoto T et al.; The putrescine N-methyltransferase (PMT) cDNA clone previously isolated from tobacco encodes a spermidine synthase-like protein with an 11 amino acid element repeated four times in tandem at the amino terminus . Genomic Southern blot analyses indicated that this N-terminal repeat array is found in tobacco PMTs but absent in Hyoscyamus and Atropa PMTs . A truncated tobacco PMT in which this repeat array was entirely removed still retained full enzymatic activity when expressed in Escherichia coli . Three PMT genes (NsPMT1, NsPMT2, NsPMT3) isolated from Nicotiana sylvestris encode two, five, and nine tandem repeats, respectively, in the first exon, but otherwise encode highly conserved proteins . Analysis of PCR fragments amplified from the genomes of N . tabacum and its two probable progenitors shows that one of the nine repeat elements in NsPMT3 was precisely deleted in the corresponding N . tabacum gene . These results indicate that direct tandem repeats of a 33 bp sequence that encodes 11 amino acids of no obvious function were added to the ancestral Nicotiana PMT gene, and that the tandem repetition was genetically very unstable, contracting or expanding during evolution of the Nicotiana species.

J Cancer Res Clin Oncol, 1997, 123(11-12), 609 - 13
Expression and purification of single-chain anti-HBx antibody in Escherichia coli; Zhou G et al.; Monoclonal antibodies have been widely used in tumor targeting studies with promising results . However, their clinical application has been limited by heterogeneity and macro-molecular movement of murine antibody . In this study, the variable-region (heavy- and light-chain) fragments of anti-HBx monoclonal antibody were enriched by the polymerase chain reaction . The expression vector, which included a 6x histidine sequence in the 3' terminus of the HBx single-chain antibody (sFv) was recombined with a linker sequence (KLGGGGFSGA) between the variable regions . The expression product from Escherichia coli fused with 6xHis was purified by nickel (Ni2+) nitrilotriacetate chelating resin . The results of enzyme-linked immunosorbent assay and Western blotting showed that sFv had binding affinity with HBxAg, suggesting that it could become a novel targeting carrier in clinical trials.

Virus Res, 1998 Feb, 53(2), 141 - 9
Expression of recombinant hepatitis C virus non-structural protein 5B in Escherichia coli; Al RH et al.; The hepatitis C virus (HCV) represents a major public health problem that can produce liver failure and hepatocellular carcinoma in chronically infected patients . Our goal was to express the HCV non-structural protein 5B (NS5B) protein of HCV genotype 1a in Escherichia coli and initiate studies of its role in HCV genomic replication . In this report we demonstrate that a recombinant NS5B protein with an amino terminal sequence of ASMSYSWTG has RNA-dependent RNA polymerase (RDRP) activity . This recombinant enzyme was active in poly(U) polymerase assays and produced template-sized RNA products when globin mRNA was used as a template . The polymerase activity of recombinant NS5B was primer-dependent and was active for at least 60 min of incubation at 30 degrees C . Deletion of the carboxyl terminal region of HCV NS5B resulted in a loss of RDRP activity indicating that the enzymatic activity observed was due to the full-length recombinant enzyme . Recombinant NS5B (RDRP) should assist in understanding the mechanism of HCV replication and the identification of specific enzyme inhibitors.

Genes Cells, 1998 Mar, 3(3), 145 - 56
Rad52 forms ring structures and co-operates with RPA in single-strand DNA annealing; Shinohara A et al.; BACKGROUND: The RAD52 epistasis group in Saccharomyces cerevisiae is involved in various types of homologous recombination including recombinational double-strand break (DSB) repair and meiotic recombination . A RecA homologue, Rad51, plays a pivotal role in homology search and strand exchange . Genetic analysis has shown that among members of its epistasis group, RAD52 alone is required for recombination between direct repeats yielding deletions . Very little has been discovered about the biochemical roles and structure of the Rad52 protein . RESULTS: Purified Rad52 protein binds to both single-stranded DNA (ssDNA) and double-stranded DNA (dsDNA) . Electron microscope observations revealed that Rad52 molecules form multimeric rings . An increase in the intensity of fluorescence when Rad52 is bound to epsilonDNA showed an alteration of the structure of ssDNA . RPA was binding to Rad52 and enhanced the annealing of complementary ssDNA molecules . This enhancement was not observed in Escherichia coli SSB protein or T4 phage gp32 protein . CONCLUSION: Rad52 forms a ring-like structure and binds to ssDNA . Its structure and DNA binding properties are different from those of Rad51 . The interaction of Rad52 with RPA plays an important role in the enhancement of annealing of complementary ssDNAs . We therefore propose that Rad52 mediates the RAD51-independent recombination through an ssDNA annealing, assisted by RPA.

FASEB J, 1998 Jun, 12(9), 653 - 63
Prediction-based threading of the hMSH2 DNA mismatch repair protein; de las Alas MM et al.; Mutations in the genes whose products participate in DNA mismatch repair underlie the increased risk of cancer in families with hereditary nonpolyposis colon carcinoma . Mutations in hMSH2 account for approximately 50% of the mutations found in these families . We sought to predict the 3-dimensional structure of hMSH2 by identifying structural homologues using prediction-based threading and by computer modeling using information from these putative structurally related proteins . Prediction-based threading identified three candidate structural homologues: glycogen phosphorylase (gpb), a 70 kDa soluble lytic transglycosylase, and ribonucleotide reductase protein R1 . An independent approach utilizing a potential-based threading program also identified gpb as a structural homologue . The models based on the structures of these proteins suggest that the ATP binding domain and helix-turn-helix domain are exposed on the outside of the protein . All known bacterial MutS and hMSH2 mutations appear to be clustered in similar vicinities in the theoretical models of hMSH2; the major site is within the ATP binding domain and near the carboxyl-terminal end, whereas a smaller number map to the region coding for exon 5 and the amino-terminal domain . All point mutations also appear to affect amino acids that are exposed on the outside surface of the protein.

Cytokine, 1998 May, 10(5), 390 - 4
Different roles of brain interleukin 1 in the adrenocorticotropin response to central versus peripheral administration of lipopolysaccharide in the rat; Habu S et al.; Although it is well established that peripheral administration of endotoxin activates the hypothalamic-pituitary-adrenal (HPA) axis, information is very limited regarding whether central administration of endotoxin can similarly stimulate the endocrine axis . Moreover, it is also unknown whether a difference exists in the mode of involvement of brain-derived cytokines in determining the HPA response to peripheral vs central administration of endotoxin . In the present study, the authors attempted to gain more knowledge on these issues focusing on interleukin (IL) 1 in the brain, one of key pro-inflammatory cytokines mediating the immuno-endocrine network . In male rats, both intravenous (i.v., 100 micrograms/kg body weight) and intracerebroventricular {i.c.v . (the 3rd ventricle), 10 micrograms} injections of Escherichia coli lipopolysaccharide (LPS) caused a significant elevation of adrenocorticotropin (ACTH) levels in plasma, even though peaked ACTH responses occurred earlier after the i.v . (60 min post-injection) than the i.c.v . (120 min post-injection) LPS . Although the ACTH response to i.c.v . LPS was significantly suppressed by a prior (5 min) i.c.v . administration of IL-1 receptor antagonist (IL-1Ra, 1 microgram), the hormonal response to i.v . LPS was not . That this dose of IL-1Ra was not biologically a small dose was indicated by another experiment that the same dose of i.c.v . IL-1Ra was able to significantly suppress the ACTH response to an i.c.v . injection of recombinant human IL-1 beta (50 ng) . These results suggest that i.c.v . LPS, as i.v . LPS, can stimulate ACTH secretion in the rat, and this hormonal response may, at least in part, be mediated by brain-derived IL-1 . Although there is one previous study reporting an important role of central IL-1 in mediating the HPA response to systemic LPS treatment, our present data suggest that such a mechanism may not operate before and during an early, peak phase of ACTH secretion after i.v . LPS.

Cytokine, 1998 May, 10(5), 319 - 30
Expression of active thrombopoietin and identification of its key residues responsible for receptor binding; Hou J et al.; In this report expression of the biologically active N-terminal half (amino acids 1-153) of thrombopoietin (TPO153) in Escherichia coli is described and the structure-function relationships in TPO are explored . TPO153 was chosen for expression because of its full biological activity . Since natural TPO153 cDNA expressed poorly, synthetic cDNA was constructed with a unique polymerase chain reaction to enhance the expression . In addition, the 5'-end codons of the synthetic cDNA were altered to maximize the expression . The expressed TPO153 was refolded and then purified to homogeneity . The protein is biologically active, and interestingly, the EC50 of this protein is 8-10-fold smaller in a TPO-dependent cell proliferation assay than that of full-length wild-type TPO . In order to identify the amino acid residues that are involved in the interaction between TPO and its receptor, all charged residues and some of the uncharged residues on the four putative helices of TPO were mutated and biological activities of the mutant proteins were examined . The mutagenesis studies suggest that there are at least two clusters of residues that are vital for TPO to be able to interact with its receptor . These residues are centred respectively around arginine 10 on helix 1 and around lysine 138 on helix IV . The successful expression of the protein in E . coli will greatly facilitate biochemical and crystallographic studies of TPO, and the structure-function relationship studies suggest that TPO has two binding sites which may interact with two individual receptors, resulting in dimerization of the receptors.

Parasite Immunol, 1998 Apr, 20(4), 183 - 95
Mapping of B epitopes in GRA4, a dense granule antigen of Toxoplasma gondii and protection studies using recombinant proteins administered by the oral route; Mevelec MN et al.; GRA4, a dense granule protein of Toxoplasma gondii elicits both mucosal and systemic immune responses following oral infection of mice with cysts . We studied the antigenicity and immunogenicity of truncated and soluble forms of GRA4 expressed as glutathione S-transferase fusion proteins in Escherichia coli . Protein C (amino-acids 297-345) was particularly well recognized by serum IgG antibodies, milk IgA antibodies and intestinal IgA antibodies from T . gondii infected mice and by serum IgG antibodies from T . gondii infected humans and T . gondii infected sheep . One major B epitope was localized within the last 11 C-terminal residues of GRA4 . A second epitope, recognized with lower frequency, was mapped within the region 318-334 . In contrast, the N domain of GRA4 (amino acids 25-276) was poorly recognized . Oral immunization of C57BL/6 mice with N, C or NC (amino acids 25-276 fused to 297-345) in association with cholera toxin induced a significant production of serum anti-GRA4 IgG antibodies but a weak and inconsistent intestinal anti-GRA4 IgG antibody response and afforded partial resistance to oral infection with T . gondii . These results provide new molecular and immunological understanding of GRA4 and indicate that it is a potential candidate for oral vaccination against T . gondii.

Parasite Immunol, 1998 Apr, 20(4), 169 - 74
Suppressive effect of Ixodes ricinus salivary gland extract on mechanisms of natural immunity in vitro; Kopecky J et al.; Tick saliva has been shown to contain a variety of pharmacologically active molecules, including those with immunosuppressive activities . There is increasing evidence that the nonspecific suppression of host immunity by tick saliva is exploited by tick-borne pathogens, e.g . the saliva-activated transmission (SAT) of some tick-borne viruses . We have demonstrated the suppressive effect of the salivary gland extract (SGE) derived from partially fed (five days) Ixodes ricinus females on important mechanisms of innate immunity: natural killer (NK) cells, interferon and nitric oxide production . SGE reduced the interferon induction by polyinosinic-polycytidylic acid (poly I:C) or Escherichia coli lipopolysaccharide (LPS) in a culture of Balb/c mouse splenocytes by 94% and 62%, respectively . SGE suppressed the cytotoxicity of nonstimulated and in vivo poly I:C-stimulated mouse NK cells by up to 31% and 26%, respectively . The induction of NK activity in vitro by LPS but not by Concanavalin-A (Con-A) was also downregulated in the presence of SGE . The addition of SGE to cultures of mouse macrophages partially inhibited the production of nitric oxide, induced by LPS . These data suggest that the facilitating effect of SGE on the transmission of some tick-borne pathogens might be associated with the suppression of the host innate resistance mechanisms, represented by interferon, nitric oxide and NK cells.

Curr Genet, 1998 May, 33(5), 368 - 77
Paxilline-negative mutants of Penicillium paxilli generated by heterologous and homologous plasmid integration; Young C et al.; Using a monoclonal antibody based ELISA, 600 pAN7-1 plasmid-tagged mutants of Penicillium paxilli were screened for paxilline accumulation and one paxilline-negative mutant, YI-20, was identified . A molecular analysis of this mutant showed that pAN7-1 was inserted at a single site but was present as 4-6 copies arranged in a head-to-tail tandem array . Rescue of flanking sequences and analysis of the corresponding genomic region revealed that YI-20 has an extensive deletion at the site of pAN7-1 integration . Probing of a CHEF gel with the same sequences showed that associated with the deletion is a rearrangement of chromosome Va . Targeted gene disruption of wild-type sequences adjacent to the site where pAN7-1 inserted, resulted in the generation of two additional paxilline-negative mutants; both were single crossovers with deletions extending outside the region mapped . Neither of these new mutants had a rearrangement of chromosome Va, suggesting that deletion of genes on this chromosome is responsible for the paxilline-negative phenotype . Telomeric fingerprinting of genomic digests of P . paxilli, combined with pulsed-field gel electrophoresis of chromosomal DNA, established that there are a minimum of eight chromosomes in this fungus.

Microbiol Mol Biol Rev, 1998 Jun, 62(2), 309 - 33
Acylation of Escherichia coli hemolysin: a unique protein lipidation mechanism underlying toxin function; Stanley P et al.; The pore-forming hemolysin (HlyA) of Escherichia coli represents a unique class of bacterial toxins that require a posttranslational modification for activity . The inactive protoxin pro-HlyA is activated intracellularly by amide linkage of fatty acids to two internal lysine residues 126 amino acids apart, directed by the cosynthesized HlyC protein with acyl carrier protein as the fatty acid donor . This action distinguishes HlyC from all bacterial acyltransferases such as the lipid A, lux-specific, and nodulation acyltransferases, and from eukaryotic transferases such as N-myristoyl transferases, prenyltransferases, and thioester palmitoyltransferases . Most lipids directly attached to proteins may be classed as N-terminal amide-linked and internal ester-linked acyl groups and C-terminal ether-linked isoprenoid groups . The acylation of HlyA and related toxins does not equate to these but does appear related to a small number of eukaryotic proteins that include inflammatory cytokines and mitogenic and cholinergic receptors . While the location and structure of lipid moieties on proteins vary, there are common effects on membrane affinity and/or protein-protein interactions . Despite being acylated at two residues, HlyA does not possess a "double-anchor" motif and does not have an electrostatic switch, although its dependence on calcium binding for activity suggests that the calcium-myristoyl switch may have relevance . The acyl chains on HlyA may provide anchorage points onto the surface of the host cell lipid bilayer . These could then enhance protein-protein interactions either between HlyA and components of a host signal transduction pathway to influence cytokine production or between HlyA monomers to bring about oligomerization during pore formation.

Biochem Biophys Res Commun, 1998 May 29, 246(3), 797 - 804
Expression systems for study of mycobacterial gene regulation and development of recombinant BCG vaccines; DasGupta SK et al.; Successful genetic engineering of mycobacteria is crucial for developing new approaches to combat tuberculosis as well as for dissecting out the molecular basis of pathogenesis of Mycobacterium tuberculosis . We have constructed a Mycobacterium-Escherichia coli shuttle expression vector pSD5 . It carries a modular expression cassette which provides sites for cloning of promoters, a ribosome binding site (RBS) with an appropriately placed initiation codon and multiple cloning sites for cloning the genes of interest . We also constructed pDK20, an integration proficient derivative of pSD5, by incorporating mycobacteriophage L5 integration signals in lieu of the origin of DNA replication for mycobacteria . This vector permits stable expression of genes in M.bovis BCG, M.smegmatis, and M.tuberculosis under the transcriptional control of a mycobacterial promoter . These vectors enable the expression of a gene to be regulated by several hundred fold depending upon the strength of mycobacterial promoter . We propose that expression of protective antigens using an appropriate promoter derivative of pDK20 should help in development of recombinant BCG vaccines that can induce an optimal immune response from the host . We have further employed the integration proficient expression system for comparing the efficiency and specificity of transcriptional recognition in M.bovis BCG, M.tuberculosis, and M.smegmatis . We show that fast growing M.smegmatis and slow growing M.tuberculosis and M.bovis BCG recognize mycobacterial promoters with comparable efficiency inspite of differences in their growth rates.

Biochem Biophys Res Commun, 1998 May 29, 246(3), 740 - 5
New paramagnetic species formed at the expense of the transient tyrosyl radical in mutant protein R2 F208Y of Escherichia coli ribonucleotide reductase; Liu A et al.; The highly conserved residue F208 in protein R2 of E . coli ribonucleotide reductase is close to the binuclear iron center, and found to be involved in stabilizing the tyrosyl radical Y122 . in wild type R2 . Upon the reconstitution reaction of the mutant R2 F208Y with ferrous iron and molecular oxygen, we observed a new EPR singlet signal (g = 2.003) formed concomitantly with decay of the transient tyrosyl radical Y122 . (g = 2.005) . This new paramagnetic species (denoted Z) was stable for weeks at 4 degrees C and visible by EPR only below 50 K . The EPR singlet could not be saturated by available microwave power, suggesting that Z may be a mainly metal centered species . The maximum amount of the compound Z in the protein purified from cells grown in rich medium was about 0.18 unpaired spin/R2 . An identical EPR signal of Z was found also in the double mutant R2 F208Y/Y122F . In the presence of high concentration of sodium ascorbate, the amounts of both the transient Y122 . and the new species Z increased considerably in the reconstitution reaction . The results suggest that Z is most likely an oxo-ferryl species possibly in equilibrium with a Y208 ligand radical.

Biochem Biophys Res Commun, 1998 May 29, 246(3), 602 - 5
The flavoprotein component of the Escherichia coli sulfite reductase can act as a cytochrome P450c17 reductase; Zeghouf M et al.; The flavoprotein component (SiR-FP) of the E . coli sulfite reductase was found to support 17 alpha-hydroxylation of pregnenolone in the presence of cytochrome P450c17 . Half maximum activity is obtained for a 1:1 ratio of SiR-FP, expressed as monomer concentration, to P450c17 . When compared to bovine NADPH-cytochrome P450 reductase, SiR-FP is about 12-15 times less efficient . P450c17 was demonstrated to interact specifically with the FMN-binding domain of the protein and the N-terminal part of SiR-FP is suspected to play a role in electron transfer . A cluster of negatively charged residues was found in SiR-FP by amino acid sequence comparison with rat cytochrome P450 reductase . These results argue in favour of the flavodoxin origin of the FMN-binding domain of SiR-FP.

Anal Biochem, 1998 Jun 1, 259(2), 258 - 64
A sandwich hybridization assay employing enzyme amplification for termination of specific ribosomal RNA from unpurified cell lysates; Wicks B et al.; We have employed the power of the cyclic NAD-based enzyme amplification system to the determination of 16S rRNA . This generally applicable system employs two oligonucleotide probes, one of which is captured on a microtiter well surface and the other labeled with alkaline phosphatase . The detection of very low levels of hybridization of the capture probe is then achieved by the means of the ultrasensitive enzyme-amplified assay system, resulting in a highly sensitive, convenient, and rapid technology which can be directly employed on unpurified samples . We have been able to demonstrate the detection of 20 amol (10(7) molecules) of pure rRNA, and specific signals from as few as 2000 bacterial cells have also been demonstrated . The total procedural time can be short-5 to 18 h-depending on the dynamic range and sensitivity required . RNA target in the range of 10(12)-10(8) molecules can be assayed within 5 h . Extending the substrate incubation time enables between 10(11) and 10(7) molecules to be determined within 18 h . The system has great potential use with respect to studying the distribution and physiological states of cellular organisms .

EMBO J, 1998 May 15, 17(10), 2877 - 85
Regulation of crp transcription by oscillation between distinct nucleoprotein complexes; Gonzalez-Gil G et al.; FIS belongs to the group of small abundant DNA-binding proteins of Escherichia coli . We recently demonstrated that, in vivo, FIS regulates the expression of several genes needed for catabolism of sugars and nucleic acids, a majority of which are also transcriptionally regulated by cAMP-cAMP-receptor protein (CRP) complex . Here we provide evidence that FIS represses transcription of the crp gene both in vivo and in vitro . Employing crp promoter-lacZ fusions, we demonstrate that both FIS and cAMP-CRP are required to keep the crp promoter in a repressed state . We have identified in the crp promoter other transcription initiation sites which are located 73, 79 and 80 bp downstream from the previously mapped start site . Two CRP- and several FIS-binding sites with different affinities are located in the crp promoter region, one of them overlapping the downstream transcription initiation sites . We show that initiation of transcription at the crp promoter is affected by the composition of nucleoprotein complexes resulting from the outcome of competition between proteins for overlapping binding sites . Our results suggest that the control of crp transcription is achieved by oscillation in the composition of these regulatory nucleoprotein complexes in response to the physiological state of the cell.

Nucleic Acids Res, 1998 May 15, 26(10), 2508 - 10
New positive/negative selectable markers for mammalian cells on the basis of Blasticidin deaminase-thymidine kinase fusions; Karreman C; Two positive and negative selectable markers were created for use in mammalian cells . They are based on two genes for the resistance to Blasticidin S (BlaS) and on the thymidine kinase (Tk) gene of herpes simplex virus (HSV) . The markers can be selected positively by their ability to induce BlaS resistance and negatively on the induced sensitivity towards gancyclovir (GANC) . Both constructs are also expressed in Escherichia coli and transfer BlaS resistance to this organism as well, making these markers very suitable for the construction of shuttle vectors.

Nucleic Acids Res, 1998 May 15, 26(10), 2505 - 7
Direct visualization of site-specific and strand-specific DNA methylation patterns in automated DNA sequencing data; Rao BS et al.; We report here a simple method of directly visualizing in automated DNA sequencing chromatograms DNA methylations of different types including cytosine methylations in Hpa II and dcm sites as well as adenine methylations in dam sites . This is made possible by the observation that the extent of incorporation of fluorescently labeled dideoxynucleotides is influenced by the methylated bases in template DNA . This simple approach involves routine automated DNA sequencing without any prior treatment of DNA specific for detecting DNA methylation.

Nucleic Acids Res, 1998 May 15, 26(10), 2500 - 1
Preparation of active tRNA gene transcripts devoid of 3'-extended products and dimers; Kholod N et al.; Significant amounts (10-30%) of 3'-extended products with one or two extra nucleotides are synthesized in the course of run-off tRNA gene transcription with T7 RNA polymerase . Denaturing polyacrylamide gel electrophoresis appeared to be insufficient to provide preparative amounts of pure correct-size transcripts . Formation of dimers by tRNA gene transcripts as side products in the course of their activation is also another obstacle in preparation of biologically active transcripts . Here, we have shown that EF-Tu affinity chromatography and/or non-denaturing electrophoresis are simple and efficient tools for isolation of highly active correct-size transcripts . Conditions for transcript activation in vitro should be carefully controlled to prevent dimer formation and obtain reliable data on tRNA transcript structure and function.

Nucleic Acids Res, 1998 May 15, 26(10), 2353 - 8
Cloned human FMR1 trinucleotide repeats exhibit a length- and orientation-dependent instability suggestive of in vivo lagging strand secondary structure; Hirst MC et al.; The normal human FMR1 gene contains a genetically stable (CGG) n trinucleotide repeat which usually carries interspersed AGG triplets . An increase in repeat number and the loss of interspersions results in array instability, predominantly expansion, leading to FMR1 gene silencing . Instability is directly related to the length of the uninterrupted (CGG) n repeat and is widely assumed to be related to an increased propensity to form G-rich secondary structures which lead to expansion through replication slippage . In order to investigate this we have cloned human FMR1 arrays with internal structures representing the normal, intermediate and unstable states . In one replicative orientation, arrays show a length-dependent instability, deletions occurring in a polar manner . With longer arrays these extend into the FMR1 5'-flanking DNA, terminating at either of two short CGG triplet arrays . The orientation-dependent instability suggests that secondary structure forms in the G-rich lagging strand template, resolution of which results in intra-array deletion . These data provide direct in vivo evidence for a G-rich lagging strand secondary structure which is believed to be involved in the process of triplet expansion in humans.

Nucleic Acids Res, 1998 May 15, 26(10), 2273 - 8
Selective inhibition of cell-free translation by oligonucleotides targeted to a mRNA hairpin structure; Le Tinevez R et al.; Using an in vitro selection approach we have previously isolated oligodeoxy aptamers that can bind to a DNA hairpin structure without disrupting the double-stranded stem . We report here that these oligomers can bind to the RNA version of this hairpin, mostly through pairing with a designed 6 nt anchor . The part of the aptamer selected against the DNA hairpin did not increase stability of the RNA-aptamer complex . However, it contributed to the binding site for Escherichia coli RNase H, leading to very efficient cleavage of the target RNA . In addition, a 2'- O -methyloligoribonucleotide analogue of one selected sequence selectively blocked in vitro translation of luciferase in wheat germ extract by binding to the hairpin region inserted upstream of the initiation codon of the reporter gene . Therefore, non-complementary oligomers can exhibit antisense properties following hybridization with the target RNA . Our study also suggests that in vitro selection might provide a means to extend the repertoire of sequences that can be targetted by antisense oligonucleotides to structured RNA motifs of biological importance.

Nucleic Acids Res, 1998 May 15, 26(10), 2265 - 72
Characterization of protein-binding to the spinach chloroplast psbA mRNA 5' untranslated region; Alexander C et al.; RNA-binding proteins play a major role in regulating mRNA metabolism in chloroplasts . In this work we characterized two proteins, of 43 and 47 kDa, which bind to the spinach psbA mRNA 5' untranslated region (psbA encoding the D1 protein of photosystem II) . The 43 kDa protein, which is present in the stroma and in membranes, co-sediments with a complex of 68S . It was purified, and the N-terminal sequence was determined . Upon homology search it was identified as the chloroplast homologue of the Escherichia coli ribosomal protein S1 . The 47 kDa protein, which, in contrast with the 43 kDa protein, sediments with a small sedimentation coefficient, is only detected in the stromal fraction . It is soluble in an uncomplexed form . By deletion analysis, an element within the psbA mRNA 5' untranslated region was identified that is necessary but not sufficient for binding of stromal proteins . The 'central protein binding element' ranges from nucleotide -49 to -9 of the psbA mRNA 5' untranslated region . It comprises the Shine-Dalgarno-like GGAG motif and, 7 nucleotides upstream, an endonucleolytic cleavage site involved in psbA mRNA degradation in vitro . The mechanistic impacts of this region in relation to RNA-binding proteins are discussed.

J Mol Biol, 1998 May 1, 278(2), 307 - 16
Combinatorial effects of NusA and NusG on transcription elongation and Rho-dependent termination in Escherichia coli; Burns CM et al.; The transcription factors NusA and NusG from Escherichia coli are modulators of the RNA polymerase elongation reaction and Rho-dependent transcription termination . NusA decreases the elongation rate and termination efficiency while NusG increases both activities . Both Nus factors are able to physically interact with Rho and with RNA polymerase . Experiments with purified components designed to determine whether these factors act independently or competitively showed that the change in elongation rate was a composite of their individual effects, that the combined effect on termination was dependent on the reaction conditions and that the two factors do not compete for their sites of action for either effect . The two factors were also found not to enhance significantly the slight (20%) inhibition of elongation caused by 200 microM guanosine 3',5'-bisdiphosphate (ppGpp) during transcription in vitro . The results also show that the effects of NusA and NusG on RNA polymerase elongation and Rho function are contrary to the inverse relationship between elongation and termination that is expected for a kinetic coupling of Rho action to RNA polymerase elongation . This property suggests that in addition to their known actions on RNA polymerase that influence the length of pausing, these factors act on some other rate-limiting step of the Rho-dependent termination process .

Shock, 1998 May, 9(5), 364 - 8
Etiology of metabolic acidosis during saline resuscitation in endotoxemia; Kellum JA et al.; We sought to understand the mechanism of metabolic acidosis that results in acute resuscitated endotoxic shock . In six pentobarbital-anesthetized dogs, shock was induced by Escherichia coli endotoxin infusion (1 mg/kg) and was treated with saline infusion to maintain mean arterial pressure > 80 mmHg . Blood gases and strong ions were measured during control conditions and at 15, 45, 90, and 180 min after endotoxin infusion . The mean saline requirement was 1833+/-523 mL over a 3 h period . The total acid load from each source was calculated using the standard base deficit . The mean arterial pH decreased from 7.32 to 7.11 (p < .01); pCO2 and lactate were unchanged . Saline accounted for 42% of the total acid load . However, 52% of the total acid load was unexplained . Although serum Na+ did not change, serum Cl-increased (127.7+/-5.1 mmol/L vs . 137.0+/-6.1 mmol/L; p=.016) . We conclude that saline resuscitation alone accounts for more than one-third of the acidosis seen in this canine model of acute endotoxemia, whereas lactate accounts for less than 10% . A large amount of the acid load can be attributed to differential Na+ and Cl- shifts from extravascular to vascular spaces.

Shock, 1998 May, 9(5), 329 - 35
Selective inhibition of the activity of inducible nitric oxide synthase prevents the circulatory failure, but not the organ injury/dysfunction, caused by endotoxin; Wray GM et al.; Inhibitors of nitric oxide synthase (NOS) attenuate the circulatory failure caused by endotoxin, but the role of NO in the development of multiple organ dysfunction and the relative contribution of NO produced by endothelial NOS and inducible NOS (iNOS) to organ injury remains unclear . Here we report for the first time that 1400W, a novel and highly selective inhibitor of iNOS activity, attenuates the delayed hypotension as well as the rise in the plasma levels of nitrite/nitrate caused by endotoxin in the rat . Inhibition of iNOS activity with 1400W administered either before or 2 h after endotoxin injection did not, however, attenuate the hepatocellular injury, renal dysfunction, or pancreatic injury in this model . Similarly, administration of another selective inhibitor of iNOS activity, L-NIL, 2 h after endotoxin injection abolished the rise in nitrite/nitrate and attenuated the delayed hypotension caused by endotoxin, but failed to ameliorate organ injury . Thus, selective inhibition of iNOS activity with 1400W attenuates the circulatory failure induced by endotoxin in the rat, but fails to influence the degree of organ injury/dysfunction.

Plant Mol Biol, 1998 Jun, 37(3), 495 - 504
Biochemical and immunological characterization of rice homologues of the human immunodeficiency virus-1 Tat binding protein and subunit 4 of human 26S proteasome subunits; Suzuka I et al.; Previously, we isolated two cDNA clones, TBPOs-1 and TBPOs-2, encoding putative ATPases that are the rice homologues of human immunodeficiency virus-1 (HIV-1) Tat binding protein-1 and subunit 4 of human 26S proteasome . In order to determine the RNA-dependent ATPase activity of these putative proteins, the subclones from these cDNA clones were expressed in Escherichia coli as fusion proteins with maltose-binding protein . The recombinant proteins stimulated ATP hydrolysis in the presence of poly(U) and rice total RNA . In contrast, single- and double-stranded forms of HindIII-digested lambda phage DNA are less effective at stimulating ATP hydrolysis . Western blot analysis using antisera against the TBPOs proteins showed a widespread appearance of these proteins in rice tissues and cultured cells . The TBPOs proteins were also found around the region where rice proteasomes would sediment . In addition, the TBPOs-1 protein bound to tobacco TATA-binding protein in vitro . Thus, we suggest that the TBPOs proteins are novel RNA-dependent ATPases characteristic of DEAD-box proteins and propose that the TPBOs proteins can exist in rice proteasomes . Further, the TBPOs-1 protein is thought to play a role in transcriptional events.

Plant Mol Biol, 1998 May, 37(2), 337 - 47
Engineering in vivo instability of firefly luciferase and Escherichia coli beta-glucuronidase in higher plants using recognition elements from the ubiquitin pathway; Worley CK et al.; The ubiquitin pathway targets proteins for degradation through the post-translational covalent attachment of the 76 amino acid protein ubiquitin to epsilon-amino lysyl groups on substrate proteins . Two instability determinants recognized by the ubiquitin pathway in Saccharomyces cerevisiae have been identified . One is described by the N-end rule and requires specific destabilizing residues at the substrate protein N-termini along with a proximal lysyl residue for ubiquitin conjugation . The second is a linear uncleavable N-terminal ubiquitin moiety . The ability of these two determinants to function in higher plants was investigated in tobacco protoplast transient transfection assays using DNA encoding variants of well characterized reporter enzymes as substrates: firefly luciferase that is localized to peroxisomes (pxLUC), a cytosolic version of LUC (cLUC), and Escherichia coli beta-glucuronidase (GUS) . cLUC with phenylalanine encoded at its mature N-terminus was 10-fold less abundant than cLUC with methionine at its mature N-terminus . GUS with phenylalanine encoded at its mature N-terminus was 3-fold less abundant than GUS with methionine at its mature N-terminus . The presence of a uncleavable N-terminal ubiquitin fusion resulted in 50-fold lower protein accumulation of cLUC, but had no effect on GUS . Both instability determinants had a much larger effect on cLUC than on pxLUC, suggesting that these degradation signals are either unrecognized or poorly recognized in the peroxisomes.

Plant Mol Biol, 1998 May, 37(2), 205 - 15
Cloning, characterization and expression of carbonic anhydrase from the cyanobacterium Synechocystis PCC6803; So AK et al.; A 3.3 kb HindIII restriction-digest DNA fragment was isolated from a Synechocystis sp . strain PCC6803 subgenomic plasmid library which strongly hybridized to a 349 bp fragment of the icfA (ccaA) gene from Synechococcus sp . strain PCC7942 . DNA sequence analysis of the fragment revealed three open reading frames (ORFs), two of which potentially coded for pantothenate synthetase (ORF275) and cytidylate kinase (ORF230) . The third, ORF274, was 825 bp in length, encoding a deduced polypeptide of 274 aa (Mr, 30747) that bears 55% sequence identity to the Synechococcus icfA (ccaA) translation product, a beta-type carbonic anhydrase (CA) . A 932 bp EcoRI fragment containing ORF274 was subcloned into an expression vector and the construct was transformed into Escherichia coli for overexpression . Electrometric assays for CA activity revealed that whole cell extracts containing the recombinant protein significantly enhanced the rate of conversion of CO2 to HCO3- and that 98% of this catalytic activity was inhibited by ethoxyzolamide, a well-characterized CA inhibitor . Antisera derived against the overexpressed protein recognized a 30.7 kDa protein that was predominantly associated with the isolated carboxysome fraction from Synechocystis . These results provide molecular and physiological evidence for the identification of a ccaA homologue in Synechocystis PCC6803 that encodes a carboxysomal beta-type CA.

Plant Mol Biol, 1998 May, 37(2), 197 - 204
Identification of a 37 kDa plant protein that interacts with the turnip mosaic potyvirus capsid protein using anti-idiotypic-antibodies; McClintock K et al.; Experimental data are provided for the presence of a plant protein that interacts with the capsid protein (CP) of turnip mosaic potyvirus (TuMV) . The receptor-like protein was identified by exploiting the molecular mimicry potential of anti-idiotypic antibodies . A single-chain Fv molecule derived from the monoclonal antibody 7A (Mab-7A), which recognizes the CP of TuMV, was produced in Escherichia coli and the recombinant protein was used to raise rabbit antibodies . The immune serum reacted with Mab-7A but not with a monoclonal antibody of the same isotype, indicating that anti-idiotypic antibodies were produced . These anti-idiotypic antibodies recognized a 37 kDa protein from Lactuca sativa . Complex formation between the anti-idiotypic antibodies and the plant protein was inhibited by the CP of TuMV which indicates that the plant protein interacts with the viral protein . The 37 kDa protein was localized in chloroplasts and was detected in other plant species.

Curr Eye Res, 1998 May, 17(5), 501 - 5
The role of platelet-activating factor in cell infiltration in endotoxin-induced uveitis in guinea pigs; Tsuji F et al.; PURPOSE: We investigated the role of platelet-activating factor (PAF) in cell infiltration in endotoxin-induced uveitis (EIU) in guinea pigs . METHODS: To elicit EIU, lipopolysaccharide (LPS) was injected into the anterior chamber of the eye . Cell numbers in the aqueous humor after LPS injection were determined by flow cytometry . PAF and prostaglandin E2 (PGE2) production after LPS injection were also examined . RESULTS: Intracameral injection of LPS induced cell infiltration into the anterior chamber, and platelet-activating factor (PAF) was detected in the aqueous humor . In addition, topical apafant (PAF antagonist) partially inhibited cell infiltration . Intracameral injection of PAF scarcely induced cell infiltration but the reaction with EIU was accelerated by intracameral injection of a small amount of PAF . CONCLUSIONS: These results suggest that PAF has weak direct activity on cell infiltration in intraocular inflammation but enhances intraocular inflammation.

Respir Med, 1998 Feb, 92(2), 162 - 6
Budesonide but not terbutaline decreases phagocytosis in alveolar macrophages; Zetterlund A et al.; Alveolar macrophages are the most common cells in bronchoalveolar lavage fluid . The macrophages participate in the inflammatory response and defence of the airways by secretion of mediators and by phagocytizing foreign particles such as bacteria and viruses . beta-Agonists and glucocorticosteroids are the most frequently used drugs in asthma . Alveolar macrophages have beta 2-adrenoceptors on their surface but the functional role of these receptors is unknown . Glucocorticosteroids interact with mediator release from macrophages . However, nothing is known about the effects of those drugs on the phagocytic capacity of alveolar macrophages . Therefore, the present study has investigated phagocytosis of alveolar macrophages from nine healthy volunteers after incubation with a beta 2-agonist, terbutaline (10(-8), 10(-6) and 10(-4) M) and a glucocorticosteroid, budesonide (10(-9), 10(-7) and 10(-5) M) . Alveolar macrophages were incubated with FITC-labelled Escherichia coli, and the drugs and phagocytosis was assessed by flow cytometry . Phagocytosis was measured as the proportion of phagocytizing cells and mean fluorescence intensity (MFI) . MFI was highly correlated with phagocytized E . coli per cell assessed by fluorescence microscopy (r = 0.996) . The proportion of phagocytizing macrophages (control) was {median (25th-75th) percentiles} 46% (40-63) and 29% (18-60), and MFI were 174 (154-205) and 122 (90-271) in the terbutaline and budesonide experiments, respectively . Terbutaline did not affect the phagocytosis significantly, while budesonide decreased the phagocytic capacity (percent phagocytizing cells and MFI) in a dose-dependent manner (P < 0.01) . At the highest budesonide concentration (10(-5) M), phagocytosis was approximately half of the control situation . In conclusion, this in vitro study indicate that a glucocorticosteroid decreases phagocytosis in alveolar macrophages in a concentration that may be relevant in the airway lining fluid . Further investigations regarding the effect on other micro-organisms and in vivo effects are necessary to further elucidate these findings.

Gut, 1998 Apr, 42(4), 460 - 1
Viral vectors expressing immunoregulatory cytokines to treat inflammatory bowel disease; Macdonald TT; Inflammatory bowel disease (IBD) is characterised by altered immunoregulation and augmented synthesis of nitric oxide . The purpose of this study was to determine the effects of exogenous IL-4, introduced by a recombinant human type 5 adenovirus (Ad5) vector, on the tissue injury associated with an experimental model of colonic immune activation and inflammation . Colitis was induced in rats by the intrarectal administration of trinitrobenzene sulfonic acid (TNB) dissolved in 50% ethanol, and control rats received saline via the same route . 1 h later, all rats were randomized into two groups . The first group was injected intraperitoneally (i.p.) with 3.0 x 10(6) plaque forming units (PFUs) of Ad5 transfected with murine interleukin-4 (Ad5IL-4) and the second group was injected i.p . with the same amount of Ad5 expressing the Escherichia coli Lac Z gene (Ad5LacZ) . One-half of the colitic and controls rats were injected again with 3.0 x 10(6) PFUs of Ad5IL-4 or Ad5LacZ on day 3 of the 6-d study . When introduced once or twice via the peritoneal route into control rats Ad5LacZ was localised to the serosal lining of the peritoneal cavity, the diaphragm and the liver on day 6 . One or two injections of Ad5IL-4 into rats also produced measurable levels of circulating IL-4 . TNB-colitis in both Ad5LacZ-treated groups was associated with pronounced elevations in serum IFN-gamma, and mucosal ulceration of the distal colon . Myeloperoxidase and inducible nitric oxide synthase II (NOS II) synthetic activity were also increased by 30- and fivefold, respectively, above control levels in the distal colon . However, two injections of AD5IL-4 into colitic rats caused the overexpression of IL-4, and significantly inhibited tissue damage, serum and colon IFN-gamma levels and myeloperoxidase activity in the distal colon . In addition, NOS II gene expression and NOS II nitric oxide synthesis was significantly inhibited . No therapeutic effect was observed in rats injected once with AD5IL-4 . Thus, IL-4, introduced by Ad5, is therapeutic during acute inflammation in the rat colon . The therapeutic effect of IL-4 was associated with an inhibition of inducible nitric oxide expression and a reduction in nitric oxide synthesis.

Vet Parasitol, 1998 Apr 15, 76(3), 189 - 202
Studies with recombinant proteins of Ehrlichia risticii: identification of strain-specific antigen as a protective antigen; Vemulapalli R et al.; Ehrlichia risticii is the causative agent of Potomac horse fever, an acute infectious disease of equines . To study the role of major antigens of E . risticii in protective immune response, we have expressed the genes of the 55 kDa, 51 kDa and 85/50 kDa-strain-specific antigens of the 90-12 (85 kDa antigen) and 25-D (50 kDa antigen) strains in Escherichia coli using pRSET A, B, C system (Invitrogen, San Diego, CA) . Mice immunized with these purified recombinant proteins of E . risticii developed strong and specific humoral immune response . The recombinant 85 kDa antigen of the 90-12 strain protected mice against challenge infection with both E . risticii strains, whereas its homologue from the 25-D strain, the recombinant 50 kDa antigen, protected mice against only the homologous strain challenge, but not against the heterologous 90-12 strain . Sera from mice immunized with the 85- or 50-kDa antigens did not inhibit the replication of cell-free Ehrlichiae in in vitro neutralization assays . Sera from normal mice and mice immunized with other antigens caused non-specific neutralization of E . risticii . Immunoglobulin G from mice immunized with the 51 kDa protein of the 90-12 strain caused partial in vitro neutralization of both strains of E . risticii . These studies demonstrate that the 85/50-kDa-strain-specific antigen of E . risticii is involved in immunoprotection against PHF.

Anticancer Res, 1998 Mar-Apr, 18(2B), 1255 - 60
GM-CSF in chemotherapy-induced febrile neutropenia--a double-blind randomized study; Arnberg H et al.; Modern chemotherapy programmes render patients susceptible to bacterial and fungal infections, and the risk of developing febrile neutropenia after a chemotherapy course is in proportion to the severity and duration of the neutropenia thus caused . This double-blind randomized study presents details of 29 patients who developed febrile neutropenia an average of 10 days after their course of chemotherapy for different types and stages of malignancy . Fourteen received granulocyte/macrophage colony stimulating factor (GM-CSF) and 15 placebo during 7 consecutive days as subcutaneous injections . The GM-CSF group demonstrated significant increases in total white blood cell count (TWBC) and absolute neutrophil count (ANC) from the morning of the third day of the study . The study concludes that GM-CSF has an important therapeutic role in the treatment of febrile neutropenia that arises during intensive chemotherapy programmes but further studies of dosage and therapy duration are required, as is the development of methods of assessing bone marrow vitality.

Genomics, 1998 May 1, 49(3), 411 - 8
An evolutionarily conserved gene on human chromosome 5q33-q34, UBH1, encodes a novel deubiquitinating enzyme; Hansen-Hagge TE et al.; While cloning breakpoint sequences of a leukemia patient exhibiting a t(5; 14) translocation, we identified a pseudogenic variant of a novel multigene family in proximity to the breakpoint . Chromosomal in situ hybridization suggested that the gene family is clustered on human chromosome 5q33-q34 . The gene family is evolutionarily conserved . Northern blot analysis of mouse tissues revealed low-level expression of a functional member of this gene family in almost all samples . Marked levels of transcripts were detected by in situ hybridization in the retina, the olfactory epithelium, the peripheral neuronal ganglia, and distinct areas of the gut . The predicted protein displays striking similarity to a hypothetical protein of Caenorhabditis elegans (R10E11.3.) and to two yeast deubiquitinating enzymes, Ubp9 and Ubp13, albeit to a lesser extent . We expressed the putative coding region of the human gene in Escherichia coli and demonstrated that it indeed bears deubiquitinating activity based on its ability to cleave ubiquitin from a ubiquitin-beta-galactosidase fusion protein . This new deubiquitinating enzyme has been named UBH1, for ubiquitin hydrolyzing enzyme 1.

J Mol Biol, 1998 May 15, 278(4), 815 - 25
Triple helix-directed psoralen crosslinks are recognized by Uvr(A)BC excinuclease; Duval-Valentin G et al.; Pyrimidine oligonucleotides bind to the major groove of an oligopyrimidine-oligopurine DNA sequence by triple helix formation . A 14-mer oligopyrimidine 3'-psoralen-conjugate (P) and a doubly modified 5'-acridine/3'-psoralen-oligonucleotide (PA) were photo-crosslinked to their target site . The crosslinked complexes were tested regarding their sensitivity to Uvr(A)BC excinuclease/DNA complex formation and excision, and compared to free psoralen crosslinked to the same site (M) . An electrophoretic mobility-shift assay showed that the crosslinked triple-helix did not hamper formation of the (A)2B complex under conditions where the third strand was bound to its target . In vitro excision experiments performed on damaged DNA fragments containing crosslinked 5-methoxypsoralen (M-target) confirmed that the psoralen photoadduct was recognized by Uvr(A)BC and that excision occurred at the crosslinked site . The major cleavage reaction took place on the 5'-side of oligopurine strand . The excision was less efficient on the 5'-side of the pyrimidine strand . The 3'-side incision either on the purine or pyrimidine strand was even weaker . With optimal Uvr(A) concentrations, it was observed that the incision reaction on (P)- and (PA)-modified targets was clearly inhibited compared to the (M)-modified target, reflecting an effect of the oligonucleotide on the recognition/excision process . These results demonstrate that a triple helix is efficient in promoting inhibition of Uvr(A)BC excision nuclease activity . These results could account for divergent findings concerning the effects of triple helix-forming oligonucleotides on repair systems and open new perspectives to study DNA repair processes through the use of bi-substituted triple helix-forming oligonucleotides.

J Mol Biol, 1998 May 15, 278(4), 801 - 13
tRNA anticodon recognition and specification within subclass IIb aminoacyl-tRNA synthetases; Commans S et al.; Subclass IIb aminoacyl-tRNA synthetases (Asn-, Asp- and LysRS) recognize the anticodon triplet of their cognate tRNA (GUU, GUC and UUU, respectively) through an OB-folded N-terminal extension . In the present study, the specificity of constitutive lysyl-tRNA synthetase (LysS) from Escherichia coli was analyzed by cross-mutagenesis of the tRNA(Lys) anticodon, on the one hand, and of the amino acid residues composing the anticodon binding site on the other . From this analysis, a tentative model is deduced for both the recognition of the cognate anticodon and the rejection of non-cognate anticodons . In this model, the enzyme offers a rigid scaffold of amino acid residues along the beta-strands of the OB-fold for tRNA binding . Phe85 and Gln96 play a critical role in this spatial organization . This scaffold can recognize directly U35 at the center of the anticodon . Specification of the correct enzyme:tRNA complex is further achieved through the accommodation of U34 and U36 . The binding of these bases triggers the conformationnal change of a flexible seven-residue loop between strands 4 and 5 of the OB-fold (L45) . Additional free energy of binding is recovered from the resulting network of cooperative interactions . Such a mechanism would not depend on the modifications of the anticodon loop of tRNA(Lys) (mnm5s2U34 and t6A37) . In the model, exclusion by the synthetase of non-cognate anticodons can be accounted for by a hindrance to the positioning of the L45 loop . In addition, Glu135 would repulse a cytosine base at position 35 . Sequence comparisons show that the composition and length of the L45 loop are markedly conserved in each of the families composing subclass IIb aminoacyl-tRNA synthetases . The possible role of the loop is discussed for each case, including that of archaebacterial aspartyl-tRNA synthetases.

J Mol Biol, 1998 May 15, 278(4), 787 - 800
A map of the biotin repressor-biotin operator interface: binding of a winged helix-turn-helix protein dimer to a forty base-pair site; Streaker ED et al.; The Escherichia coli biotin repressor is a member of the "winged helix-turn-helix" class of site-specific DNA binding proteins . The protein binds as a dimer to the 40 bp biotin operator sequence . Although the structure of the aporepressor has been solved by X-ray crystallographic techniques, no structure of the holorepressor-DNA complex is yet available . In order to characterize the structural features of the biotin repressor-biotin operator interface we have applied a number of solution techniques including DNase I, hydroxyl radical and dimethyl sulfate footprinting and the circular permutation or "bending" assay . Results of these combined studies indicate that each repressor monomer forms a bipartite interface with each half-site of the biotin operator sequence . The results imply that, in addition to the helix-turn-helix module of each monomer, a second structural element participates in the protein-DNA interface . The two bipartite protein-DNA interfaces appear, moreover, to primarily involve the two 12 bp termini of the operator site . Results of combined DNase I footprinting and circular permutation analysis indicate, furthermore, that the central 16 bp region that links the two termini becomes distorted concomitant with binding of holoBirA.

J Mol Biol, 1998 May 15, 278(4), 741 - 55
Phi 29 DNA polymerase requires the N-terminal domain to bind terminal protein and DNA primer substrates; Truniger V et al.; A 44 kDa C-terminal fragment of phi 29 DNA polymerase has been separately expressed and purified from Escherichia coli cells . As expected, the truncated protein lacked the 3'-5' exonuclease activity and strand-displacement capacity, previously mapped in the N-terminal domain of phi 29 DNA polymerase . On the other hand, the 44 kDa C-terminal fragment retained polymerase activity when using Mn2+ as metal activator, although the catalytic efficiency was greatly reduced with respect to that of the complete enzyme . Moreover, and in contrast to the high processivity exhibited by phi 29 DNA polymerase (> 70 kb), polymerization by its C-terminal domain was completely distributive . All these polymerization defects were related to a strong impairment of DNA binding, suggesting that additional contacts present in the N-terminal domain are important for an optimal stabilization and translocation of the DNA during polymerization . Moreover, the C-terminal domain showed a very reduced capacity to initiate terminal protein (TP)-primed DNA replication, as a consequence of a weakened interaction with the TP primer, and a lack of activation by protein p6, the initiator of phi 29 DNA replication . We conclude that the C-terminal portion of phi 29 DNA polymerase (residues 188 to 575), although having a structural entity as the domain responsible for the synthetic activities, requires the N-terminal domain to provide important contacts for the two different substrates, DNA and TP, that prime DNA synthesis . These results support the hypothesis of a modular organization of enzymatic activities in DNA-dependent DNA polymerases, but emphasize the functional coordination required for coupling DNA synthesis and proofreading, and for the more specific functions (TP-priming, high processivity and strand-displacement) inherent to phi 29 DNA polymerase.

J Mol Biol, 1998 May 15, 278(4), 713 - 23
Different conformations of nascent peptides on ribosomes; Tsalkova T et al.; The length at which the N terminus of nascent proteins becomes available to antibodies during their synthesis on ribosomes was determined . Three different proteins, bovine rhodanese, bacterial chloramphenicol acetyltransferase and MS2 coat protein, were synthesized with coumarin at their N terminus in a cell-free system derived from Escherichia coli . A derivative of coumarin was cotranslationally incorporated as N-coumarin-methionine at the N terminus of polypeptides . The interaction of specific anti-coumarin antibodies with this N-terminal coumarin of ribosome-bound nascent peptides was examined . The results indicate that short nascent peptides of each of the three proteins are unreactive, that the length at which they become accessible to the antibodies is different for the three proteins, and that longer peptides differ in their reactivity . It is suggested that these differences are due to differences in the conformation acquired by the peptides as they are synthesized on the ribosomes.

Biosci Biotechnol Biochem, 1998 Apr, 62(4), 798 - 800
Need for aromatic residue at position 115 for proteolytic activity found by site-directed mutagenesis of tryptophan 115 in thermolysin; Inouye K et al.; In thermolysin, tryptophan 115 seems to be at the S2 subsite . Trp-115 was replaced with tyrosine, phenylalanine, leucine, and valine during site-directed mutagenesis in order to evaluate the role of Trp-115 in the proteolytic activity of thermolysin . The mutant enzymes with Tyr-115 or Phe-115 had as much proteolytic activity as the wild-type enzyme, but the other two mutant enzymes had no activity . We found earlier that the substitution of Trp-115 with alanine, glutamic acid, lysine, and glutamine causes the enzyme to lose all activity, so an aromatic amino acid at position 115 seems to be essential for thermolysin.

Methods, 1998 May, 15(1), 51 - 62
Purification of native and recombinant double-stranded RNA-specific adenosine deaminases; O'Connell MA et al.; ADAR1 and ADAR2 are members of a family of enzymes that catalyze the conversion of adenosine to inosine in double-stranded RNA . Unlike the other types of RNA editing that involve multiprotein editing complexes, the site-specific deamination of an adenosine to inosine is catalyzed by single enzymes . ADAR1 and ADAR2 have been purified and the genes cloned from various sources . Each gene encodes multiple splice variants . As it is crucial to have an adequate supply of pure protein to investigate this type of RNA editing, we describe in this article methods for both the purification and the overexpression of either full-length or partial ADAR1 and ADAR2 isoforms.

Gene Ther, 1998 Apr, 5(4), 542 - 51
Lipofection of cultured mouse muscle cells: a direct comparison of Lipofectamine and DOSPER; Dodds E et al.; Cationic lipid-DNA complexes (lipoplexes) have been widely used as gene transfer vectors which avoid the adverse immunogenicity and potential for viraemia of viral vectors . With the long-term aim of gene transfer into skeletal muscle in vivo, we describe a direct in vitro comparison of two commercially available cationic lipid formulations, Lipofectamine and DOSPER . Optimisation of transfection was performed in the C2C12 mouse muscle cell line, before further studies in primary mouse myoblasts and C2C12 myotubes . Reporter gene constructs expressing either E . coli beta-galactosidase or green fluorescent protein (GFP) were used in order to evaluate transfection efficiency by histochemical staining or FACS analysis, respectively . Both lipid formulations were able to promote efficient, reproducible gene transfer in C2C12 cells, and to transfect primary mouse myoblast cultures successfully . However, DOSPER exhibited the important advantage of being able to transfect cells in the presence of serum of both bovine and murine origin . This feature allowed increased cell survival during in vitro transfections, and may be advantageous for direct in vivo gene transfer efficacy.

Gene Ther, 1998 Apr, 5(4), 459 - 64
Persistent gene expression in rat liver in vivo by repetitive transfections using HVJ-liposome; Hirano T et al.; Most viral vectors are highly immunogenic and are of limited use for somatic gene therapy that requires repetitive administrations . We have developed a highly efficient gene transduction procedure useful for repetitive transfections using liposome containing hemagglutinating virus of Japan (HVJ-liposome) . The Escherichia coli beta-galactosidase (beta-gal) gene was embodied in HVJ-liposome, and introduced directly into the caudal lobe of rat liver that was transiently isolated from a systemic circulation . A 116 kDa beta-gal protein was detected in transfected rat liver tissues by Western blot analysis and it was expressed in more than two-thirds of the liver by histological staining . It was found that the transfection efficiency was not affected by repetitive transfections . In support of these findings, antibody response to HVJ-liposome detected in the rat sera was weak and transient . Furthermore, cytotoxic T lymphocytes were not elicited against autologous rat hepatocytes that were transfected in vivo using HVJ-liposome . Thus, our results demonstrate that the isolation of a target liver from systemic circulation and the direct administration of foreign genes using HVJ-liposomes are useful for high gene transduction and persistent gene expression in the liver.

Gene Ther, 1998 Apr, 5(4), 440 - 50
Selective astrocytic transgene expression in vitro and in vivo from the GFAP promoter in a HSV RL1 null mutant vector--potential glioblastoma targeting; McKie EA et al.; Due to the lack of any effective therapy, novel approaches are currently being explored for the treatment of primary brain tumours . It has previously been demonstrated that variants of HSV-1 which are deleted in the RL1 gene and fail to produce the virulence factor ICP34.5 are potential candidates for tumour therapy . The RL1 variant 1716 replicates selectively within tumour cells and has the potential to deliver a therapeutic or tumour killing gene directly to the site of tumour growth . As many intracerebral tumours are glial and predominantly astrocytic in origin, we have evaluated the ability of 1716 to deliver a reporter gene specifically to astrocytes in vivo and in vitro using a 2.2 kb fragment which controls expression of the glial fibrillary acidic protein (GFAP), an astrocyte specific protein . Two 1716 variants, 1774 and 1775, were constructed which contain the GFAP-promoter element linked to the E . coli beta-galactosidase gene, inserted into the HSV-1 UL43 and US5 loci, respectively . In primary cultures, human primary tumour cell lines and established tumour cell lines in vitro, 1774 and 1775 gave high levels of expression of beta-galactosidase specifically in astrocytes . In vivo following intracerebral inoculation, both viruses demonstrated high levels of beta-galactosidase expression predominantly in astrocytes . These results indicate that the GFAP promoter element could be used for efficient and selective transgene delivery to human gliomas.

Bioinformatics, 1998, 14(3), 271 - 8
Systematic genomic screening and analysis of mRNA in untranslated regions and mRNA precursors: combining experimental and computational approaches; Dandekar T et al.; MOTIVATION: The untranslated regions (UTRs) of mRNA upstream (5'UTR) and downstream (3'UTR) of the open reading frame, as well as the mRNA precursor, carry important regulatory sequences . To reveal unidentified regulatory signals, we combine information from experiments with computational approaches . Depending on available knowledge, three different strategies are employed . RESULTS: Searching with a consensus template, new RNAs with regulatory RNA elements can be identified in genomic screens . By this approach, we identify new candidate regulatory motifs resembling iron-responsive elements in the 5'UTRs of HemA, FepB and FrdB mRNA from Escherichia coli . If an RNA element is not yet defined, it may be analyzed by combining results from SELEX (selective enrichment of ligands by exponential amplification) and a search of databases from RNA or genomic sequences . A cleavage stimulating factor (CstF) binding element 3 of the polyadenylation site in the mRNA precursor serves as a test example . Alternatively, the regulatory RNA element may be found by studying different RNA foldings and their correlation with simple experimental tests . We delineate a novel instability element in the 3'UTR of the estrogen receptor mRNA in this way . AVAILABILITY: Strategy, methods and programs are available on request from T.Dandekar . CONTACT: dandekar@embl-heidelberg.de

J Bacteriol, 1998 Jun, 180(11), 2999 - 3002
In vitro synthesis of multicopy single-stranded DNA, using separate primer and template RNAs, by Escherichia coli reverse transcriptase; Shimamoto T et al.; A minor population of wild strains of Escherichia coli contains a retron, a retroelement responsible for the synthesis of multicopy single-stranded DNA (msDNA) . The retron is a genetic element consisting of the gene for reverse transcriptase (RT) and the msr-msd region under a single promoter . A single RNA transcript from the msr-msd region serves not only as a template but also as a primer for msDNA synthesis . Here, using a cell-free system with purified RT from retron Ec73, we examined whether the reaction can occur in a bimolecular reaction with use of separately expressed msr and msd transcripts . DNA sequencing of the cell-free product revealed that the sequence of the 5'-end region was identical to that of msDNA-Ec73, indicating that the cDNA synthesis was primed from the 2'-OH group of the specific internal G residue of the primer RNA, identical to the branching G residue in the RNA molecule of msDNA-Ec73 . The present results raise an intriguing possibility for a role of bacterial retrons in vivo, the possibility that cellular mRNAs can be converted into cDNAs in retron-harboring cells if the mRNAs contain a sequence complementary to the sequence directly upstream of the branching G residue of the msr RNA transcript.

J Bacteriol, 1998 Jun, 180(11), 2931 - 5
Novel temperature-sensitive mutants of Escherichia coli that are unable to grow in the absence of wild-type tRNA6Leu; Nakayashiki T et al.; Escherichia coli has only a single copy of a gene for tRNA6Leu (Y . Komine et al., J . Mol . Biol . 212:579-598, 1990) . The anticodon of this tRNA is CAA (the wobble position C is modified to O2-methylcytidine), and it recognizes the codon UUG . Since UUG is also recognized by tRNA4Leu, which has UAA (the wobble position U is modified to 5-carboxymethylaminomethyl-O2-methyluridine) as its anticodon, tRNA6Leu is not essential for protein synthesis . The BT63 strain has a mutation in the anticodon of tRNA6Leu with a change from CAA to CUA, which results in the amber suppressor activity of this strain (supP, Su+6) . We isolated 18 temperature-sensitive (ts) mutants of the BT63 strain whose temperature sensitivity was complemented by introduction of the wild-type gene for tRNA6Leu . These tRNA6Leu-requiring mutants were classified into two groups . The 10 group I mutants had a mutation in the miaA gene, whose product is involved in a modification of tRNAs that stabilizes codon-anticodon interactions . Overexpression of the gene for tRNA4Leu restored the growth of group I mutants at 42 degrees C . Replacement of the CUG codon with UUG reduced the efficiency of translation in group I mutants . These results suggest that unmodified tRNA4Leu poorly recognizes the UUG codon at 42 degreesC and that the wild-type tRNA6Leu is required for translation in order to maintain cell viability . The mutations in the six group II mutants were complemented by introduction of the gidA gene, which may be involved in cell division . The reduced efficiency of translation caused by replacement of the CUG codon with UUG was also observed in group II mutants . The mechanism of requirement for tRNA6Leu remains to be investigated.

J Bacteriol, 1998 Jun, 180(11), 2906 - 10
DNA synthesis and viability of a mutT derivative of Escherichia coli WP2 under conditions of amino acid starvation and relation to stationary-phase (adaptive) mutation; Bridges BA et al.; Escherichia coli WP2 bacteria with an ochre amino acid auxotrophy show no evidence of growth during the first few days after plating at densities above 10(8) on plates lacking the required amino acid . They lose viability for some days, and then a subpopulation recovers and there is cell turnover . At very low plating densities (around 10(2) per plate), almost every cell will eventually form a small but visible colony . At intermediate plating densities (10(6) to 10(7) per plate), there is an immediate increase in the number of viable bacteria . The results are consistent with a model that assumes that growth is dependent on trace amounts of tryptophan or a tryptophan-complementing substance and that death is due to extracellular toxic species in the medium, including active oxygen species . Mutations in mutT bacteria under these conditions result from incorporation of 7,8-dihydro-8-oxo-dGTP into DNA and thus largely reflect DNA synthesis associated with the increase in the number of viable cells at the initial density used (10(7) per plate) . We show that the increase in cell number and much of this DNA synthesis can be eliminated by the presence of 10(8) scavenger bacteria and by removal of early-arising mutant colonies that release the required amino acid . The synthesis that remains is equivalent to less than a quarter of a genome per day and is marginally reduced, if at all, in a polA derivative . We cannot exclude the possibility that this residual DNA synthesis is peculiar to mutT bacteria due to transcriptional leakiness, although there is no evidence that this is a major problem in this strain . If such DNA synthesis also occurs in wild-type bacteria, it may well be important for adaptive mutation since use of a more refined agar in selective plates both eliminated the initial increase in cell number seen at low density (10(7) per plate) and reduced the rate of appearance of mutants at plating densities above 10(8) per plate.

J Bacteriol, 1998 Jun, 180(11), 2842 - 8
Purification and characterization of thin pili of IncI1 plasmids ColIb-P9 and R64: formation of PilV-specific cell aggregates by type IV pili; Yoshida T et al.; Thin pili of the closely related IncI1 plasmids ColIb-P9 and R64 are required only for liquid mating and belong to the type IV family of pili . They were sedimented by ultracentrifugation from culture medium in which Escherichia coli cells harboring ColIb-P9- or R64-derived plasmids had been grown, and then the pili were purified by CsCl density gradient centrifugation . In negatively stained thin pilus samples, long rods with a diameter of 6 nm, characteristic of type IV pili, were observed under an electron microscope . Gel electrophoretic analysis of purified ColIb-P9 thin pili indicated that thin pili consist of two kinds of proteins, pilin and the PilV protein . Pilin was demonstrated to be the product of the pilS gene . Pilin was first synthesized as a 22-kDa prepilin from the pilS gene and subsequently processed to a 19-kDa protein by the function of the pilU product . The N-terminal amino group of the processed protein was shown to be modified . The C-terminal segments of the pilV products vary among six or seven different types, as a result of shufflon DNA rearrangements of the pilV gene . These PilV proteins were revealed to comprise a minor component of thin pili . Formation of PilV-specific cell aggregates by ColIb-P9 and R64 thin pili was demonstrated and may play an important role in liquid mating.

Biochemistry, 1998 May 12, 37(19), 7030 - 8
Mutagenesis by peroxy radical is dominated by transversions at deoxyguanosine: evidence for the lack of involvement of 8-oxo-dG1 and/or abasic site formation; Valentine MR et al.; Oxidative damage of DNA by endogenously generated oxygen radicals contributes to the mutagenic process . Hydroxy, alkoxy, and peroxy radicals all have the potential to react with DNA, giving rise to strand breaks and potentially mutagenic oxidative base damage . Although reactions of the hydroxy radical with DNA have been well studied, far less is known about the reactivities of these other radicals with DNA and their mutation-inducing potential . Frequencies of DNA base modifications and strand break densities caused by peroxy radical (ROO*) oxidation were measured by glyoxal gel electrophoretic analysis . We report the spectrum of mutations induced in Escherichia coli upon transfection with peroxy radical treated DNA carrying the lacZ alpha gene as a reporter . Transfection of DNA exposed to micromolar amounts of peroxy radical resulted in a 30-fold increase in mutation frequency in non-SOS-inducible cells . Sequencing analysis of DNA isolated from mutants showed that among base substitution mutants 88% consisted of transversions at G, with a nearly equal number of G --> C and G --> T mutants . Transition mutations were rarely detected, in contrast to control experiments . Electrophoretic analysis of peroxy radical treated DNA exposed to NaOH, Nth, and Fpg proteins demonstrated that abasic sites are not formed to any detectable degree . The oxidative G lesions are sensitive to digestion by the Fpg protein . We were unable to detect the formation of 8-oxo-dG by HPLC/electrochemical analysis of peroxy radical oxidation of dG, suggesting that the G --> T transversions were not caused by this base lesion.

Biochemistry, 1998 May 12, 37(19), 6911 - 23
Delta subunit of rat liver mitochondrial ATP synthase: molecular description and novel insights into the nature of its association with the F1-moiety; Pan W et al.; The F1 moiety of ATP synthase complexes consists of five subunit types in the stoichiometric ratio alpha 3, beta 3, gamma, delta epsilon . Of these, the delta subunit has received very little attention in the study of F1 preparations from eukaryotic cells . Although recently shown to associate tightly with the beta subunit {Pedersen, P . L., Hullihen, J., Bianchet, M., Amzel, L . M., and Lebowitz, M . S . (1995) J . Biol . Chem . 270, 1775-1784}, the delta subunit is not resolved in the X-ray structure of either the rat liver or bovine heart enzyme . For these reasons, the novel studies reported here were designed both to provide a molecular description of the rat liver delta subunit and to gain insight into the nature of its interaction with F1 . The rat liver delta subunit was cloned from a lambda gt11 library, sequenced, overexpressed in Escherichia coli (E . coli) in fusion with the maltose binding protein, and, after cleavage of the latter protein, purified to homogeneity . The purified delta subunit (MW = 14.7 kDa) was shown by circular dichroism spectroscopy to be highly structured and to exhibit about 25% sequence identity to the chloroplast and E . coli epsilon subunits, frequently regarded as homologues of higher eukaryotic delta subunits . Significantly, and in contrast to the chloroplast and E . coli epsilon subunits, which are readily removed from their parent F1 moieties after treatment respectively with ethanol and lauryldimethylamine oxide, the rat liver delta subunit remained tightly bound to F1 under these relatively mild conditions . For the above reasons, four types of experiments were carried out on rat liver F1 in order to (1) determine the accessibility of the delta subunit to both specific antibodies and to proteases, (2) establish the effect of nucleotides on this subunit's accessibility, (3) identify in cross-linking studies with disuccinimidyl glutarate this subunit's most reactive neighbor, and (4) determine whether this subunit can be dissociated from F1 by using ionic detergents while leaving the remaining complex intact . The data derived from this detailed set of studies (a) supports the view that the rat liver F1-delta subunit is in very close proximity to the gamma subunit near the bottom of the F1 molecule but does not penetrate deeply into the central core, (b) shows that within F1 the delta subunit's N-terminus is exposed while its C-terminus is masked, (c) indicates that access to the delta subunit is shielded in part by the alpha, beta, and gamma subunits and changes during the catalytic cycle of F1, and (d) implicates the delta subunit as important for the structural stability of the F1 unit . These novel findings on a higher eukaryotic F1-delta subunit are discussed in relationship to earlier studies on the related epsilon subunits from both chloroplasts and E . coli.

Biochemistry, 1998 May 12, 37(19), 6905 - 10
Human protoporphyrinogen oxidase: relation between the herbicide binding site and the flavin cofactor; Birchfield NB et al.; Protoporphyrinogen IX oxidase (protox) catalyzes the oxidation of protoporphyrinogen IX to protoporphyrin IX in the penultimate step of heme and chlorophyll biosynthesis in animals and plants . Protox is the target of light-dependent peroxidizing herbicides and is inhibited at nanomolar levels by several chemical classes including tetrahydrophthalimides (discussed below) and diphenyl ethers (e.g., acifluorfen) usually with little selectivity between the mammalian and plant enzymes . The herbicide binding site is examined here with a photoaffinity radioligand optimized on the basis of structure-activity relationships . A radiosynthetic procedure is described for this new herbicidal probe, N-(5-azido-4-chloro-2-fluorophenyl)-3,4,5, 6-{3H}tetrahydrophthalimide ({3H}AzTHP), resulting in high specific activity (2.6 TBq/mmol) . Human protox expressed in Escherichia coli and purified by affinity chromatography is used with {3H}AzTHP to characterize the herbicide/substrate binding site . Specific binding of {3H}AzTHP to human protox is rapid, completely reversible in the absence of light with a Kd of 93 nM, and competitively inhibited by the 5-propargyloxy analogue and by acifluorfen, which are known to bind at the substrate (protoporphyrinogen) site . The Bmax establishes one {3H}AzTHP binding site per FAD . Diphenyleneiodonium, proposed to inhibit protox by interaction with the FAD cofactor, inhibits enzyme activity by 48% at 100 micro M without affecting {3H}AzTHP binding in the presence or absence of substrate, suggesting that the herbicide binding site may not be proximal to FAD . The first step has been taken in photoaffinity labeling the herbicide/substrate site with {3H}AzTHP resulting in apparent covalent derivatization of 13% of the herbicide binding site.

Biochemistry, 1998 May 12, 37(19), 6801 - 9
Kinetic and calcium-binding properties of three calcium-dependent protein kinase isoenzymes from soybean; Lee JY et al.; Calmodulin-like domain protein kinases (CDPKs) are a family of calcium- but not calmodulin-dependent protein kinases found in a wide variety of plants and in protists . CDPKs are encoded by large multigene families, and to assess whether family members play distinct or redundant roles in vivo, we characterized soybean CDPK isoforms alpha, beta, and gamma, which share 60-80% identity in amino acid sequence . RNA blot analysis showed that the three CDPKs were expressed in most plant tissues examined and in suspension-cultured soybean cells . Recombinant CDPKalpha, -beta, and -gamma phosphorylated peptide substrates containing the four-residue motif R/K-X-X-S/T, but CDPKalpha was the most selective for residues outside of the motif . The CDPKs were inhibited by the general protein kinase inhibitors K252a and staurosporine and by calphostin C, which is an inhibitor of protein kinase C . The calcium-binding properties of each CDPK were distinct . The Kd's for Ca2+ determined by flow dialysis in the absence of substrates were 51, 1.4, and 1.6 micro M for CDPKalpha, -beta, and -gamma, respectively . In the presence of the peptide substrate syntide-2 the Kd of CDPKalpha decreased to 0.6 microM . Also, the sensitivity of this isoenzyme's activity to calcium varied with protein substrate . The concentrations of Ca2+ required for half-maximal activity (K0.5) for each CDPK with syntide-2 as substrate were 0.06, 0.4, and 1 micro M, respectively . These results show that members of the CDPK family differ in biochemical properties and support the hypothesis that each isoform may have a distinct role in calcium signal transduction.

Biochemistry, 1998 May 12, 37(19), 6781 - 90
Expression and characterization of four recombinant human dihydrodiol dehydrogenase isoforms: oxidation of trans-7, 8-dihydroxy-7,8-dihydrobenzo{a}pyrene to the activated o-quinone metabolite benzo{a}pyrene-7,8-dione; Burczynski ME et al.; The bioactivation of polycyclic aromatic hydrocarbons (PAHs) to their ultimate carcinogenic forms proceeds via the formation of proximate carcinogen trans-dihydrodiols . Previous studies demonstrated that rat liver 3 alpha-hydroxysteroid dehydrogenase/dihydrodiol dehydrogenase (3 alpha-HSD/DD), a member of the aldo-keto reductase (AKR) superfamily, oxidizes PAH trans-dihydrodiols to redox-cycling o-quinones . Multiple closely related AKRs exist in human liver; however, it is unclear which, if any, participate in PAH activation by catalyzing the NADP+ -dependent oxidation of PAH trans-dihydrodiols . In this study, cDNAs encoding four human DD isoforms were isolated from HepG2 cells using isoform-selective RT-PCR . The recombinant proteins were overexpressed in Escherichia coli, purified to homogeneity, and kinetically characterized . Calculation of KM and kcat values of each isoform for model substrates revealed that they possessed enzymatic activities assigned to native human liver DD1, DD2, DD4, and type 2 3alpha-HSD (DDX) proteins . The ability of human DDs to oxidize the potent proximate carcinogen (+/-)-trans-7,8-dihydroxy-7, 8-dihydrobenzo{a}pyrene (BP-diol) was then examined . A reverse phase HPLC radiochemical assay demonstrated that all four isoforms oxidize (+/-)-BP-diol in the following rank order: DD2 > DD1 > DD4 > DDX . Each DD consumed the entire racemic BP-diol mixture, indicating that both the minor (+)-S,S- and major (-)-R,R-stereoisomers formed in vivo are substrates . First-order decay plots showed that DD1 and DD2 displayed preferences for one of the stereoisomers, and circular dichroism spectroscopy indicated that this isomer was the (+)-7S, 8S-enantiomer . The products of these reactions were trapped as either glycine or thiol ether conjugates of benzo{a}pyrene-7,8-dione (BPQ), indicating that the initial oxidation product was the reactive BPQ . Thus, human liver possesses multiple AKRs which contribute to PAH activation by catalyzing the NADP+-dependent oxidation of PAH trans-dihydrodiols to redox-active o-quinones.

Biochemistry, 1998 May 12, 37(19), 6718 - 26
Phosphorylation destabilizes the amino-terminal domain of enzyme I of the Escherichia coli phosphoenolpyruvate:sugar phosphotransferase system; Nosworthy NJ et al.; Thermal stabilities of enzyme I (63 562 M(r) subunit, in the Escherichia coli phosphoenolpyruvate (PEP):sugar phosphotransferase system (PTS), and a cloned amino-terminal domain of enzyme I (EIN; 28 346 Mr) were investigated by differential scanning calorimetry (DSC) and far-UV circular dichroism (CD) at pH 7.5 . EIN expressed in a delta pts E . coli strain showed a single, reversible, two-state transition with Tm = 57 degrees C and an unfolding enthalpy of approximately 140 kcal/mol . In contrast, monomeric EIN expressed in a wild-type strain (pts+) had two endotherms with Tm congruent with 50 and 57 degrees C and overall delta H = 140 kcal/mol and was converted completely to the more stable form after five DSC scans from 10 to 75 degrees C (without changes in CD: approximately 58% alpha-helices) . Thermal conversion to a more stable form was correlated with dephosphorylation of EIN by mass spectral analysis . Dephospho-enzyme I (monomer right arrow over left arrow dimer) exhibited endotherms for C- and N-terminal domain unfolding with Tm = 41 and 54 degrees C, respectively . Thermal unfolding of the C-terminal domain occurred over a broad temperature range ( approximately 30-50 degrees C), was scan rate- and concentration-dependent, coincident with a light scattering decrease and Trp residue exposure, and independent of phosphorylation . Reversible thermal unfolding of the nonphosphorylated N-terminal domain was more cooperative, occurring from 50 to 60 degrees C . DSC of partially phosphorylated enzyme I indicated that the amino-terminal domain was destabilized by phosphorylation (from Tm = 54 to approximately 48 degrees C) . A decrease in conformational stability of the amino-terminal domain of enzyme I produced by phosphorylation of the active-site His 189 has the physiological consequence of promoting phosphotransfer to the phosphocarrier protein, HP(r).

Biochemistry, 1998 May 12, 37(19), 6679 - 88
Probing the active site of cytochrome P450 2B1: metabolism of 7-alkoxycoumarins by the wild type and five site-directed mutants; Kobayashi Y et al.; A series of 7-alkoxycoumarins (chain length of 1-7 carbon atoms) was utilized as active site probes of purified Escherichia coli-expressed cytochrome P450 2B1 wild type and five site-directed mutants (I114V, F206L, V363A, V363L, and G478S) . The production of 7-hydroxycoumarin, the O-dealkylation product, by the wild-type enzyme exhibited a rank order of C2 > C4 > C3 > C1 > C5 > C6 = C7 . The pattern observed for the P450 I114V mutant was similar to that of the wild-type enzyme, whereas with F206L and G478S mutants, the rate of O-dealkylation was low with all the compounds . In contrast, with V363A, the highest rate of product formation was observed with 7-butoxycoumarin . The V363L mutant preferentially catalyzed the O-dealkylation of 7-methoxy- and 7-ethoxycoumarin, and a further increase in the length of the alkyl chain led to a marked decrease in product formation . The stoichiometry of 7-butoxycoumarin oxidation by V363L suggested that products other than 7-hydroxycoumarin were also formed . HPLC and GC-EIMS analyses revealed that P450 2B1 V363L produced 7-(3-hydroxybutoxy)coumarin and 7-(4-hydroxybutoxy)coumarin as major oxidation products, while the V363A mutant mainly catalyzed the O-dealkylation of 7-butoxycoumarin . Docking of alkoxycoumarins into the active site of a P450 2B1 homology model confirmed the importance of the studied residues in substrate dealkylation and explained the formation of novel 7-butoxycoumarin products by the V363L mutant.

J Bacteriol, 1998 May, 180(10), 2782 - 7
Menaquinone (vitamin K2) biosynthesis: localization and characterization of the menA gene from Escherichia coli; Suvarna K et al.; A key reaction in the biosynthesis of menaquinone involves the conversion of the soluble bicyclic naphthalenoid compound 1, 4-dihydroxy-2-naphthoic acid (DHNA) to the membrane-bound demethylmenaquinone . The enzyme catalyzing this reaction, DHNA-octaprenyltransferase, attaches a 40-carbon side chain to DHNA . The menA gene encoding this enzyme has been cloned and localized to a 2.0-kb region of the Escherichia coli genome between cytR and glpK . DNA sequence analysis of the cloned insert revealed a 308-codon open reading frame (ORF), which by deletion analyses was shown to restore anaerobic growth of a menA mutant . Reverse-phase high-performance liquid chromatography analysis of quinones extracted from the orf-complemented cells independently confirmed the restoration of menaquinone biosynthesis, and similarly, analyses of isolated cell membranes for DHNA octaprenyltransferase activity confirmed the introduction of the menA product into the orf-complemented menA mutant . The validity of an ORF-associated putative promoter sequence was confirmed by primer extension analyses.

J Bacteriol, 1998 May, 180(10), 2779 - 81
Oligoribonuclease is encoded by a highly conserved gene in the 3'-5' exonuclease superfamily; Zhang X et al.; Oligoribonuclease, a 3'-to-5' exoribonuclease specific for small oligoribonucleotides, was purified to homogeneity from extracts of Escherichia coli . The purified protein is an alpha2 dimer of 40 kDa . NH2-terminal sequence analysis of the protein identified the gene encoding oligoribonuclease as yjeR (o204a), a previously reported open reading frame located at 94 min on the E . coli chromosome . However, as a consequence of the sequence information, the translation start site of this open reading frame has been revised . Cloning of yjeR led to overexpression of oligoribonuclease activity, and interruption of the cloned gene with a kanamycin resistance cassette eliminated the overexpression . On the basis of these data, we propose that yjeR be renamed orn . Orthologs of oligoribonuclease are present in a wide range of organisms, extending up to humans.

J Bacteriol, 1998 May, 180(10), 2756 - 8
Suppressor scanning at positions 177 and 236 in the Escherichia coli lactose/H+ cotransporter and stereotypical effects of acidic substituents that suggest a favored orientation of transmembrane segments relative to the lipid bilayer; King SC et al.; Acidic substituents for Ala-177 (helix 6) or Tyr-236 (helix 7) in LacY cause effects on sugar recognition and cosubstrate coupling that are stereotypical of neutral substituents . Thus, helices 6 and 7 are probably oriented to produce little side-chain contact with the low dielectric lipid bilayer at positions 177 and 236.

J Bacteriol, 1998 May, 180(10), 2744 - 8
An rRNA fragment and its antisense can alter decoding of genetic information; Arkov AL et al.; rRNA plays a central role in protein synthesis and is intimately involved in the initiation, elongation, and termination stages of translation . However, the mode of its participation in these reactions, particularly as to the decoding of genetic information, remains elusive . In this paper, we describe a new approach that allowed us to identify an rRNA segment whose function is likely to be related to translation termination . By screening an expression library of random rRNA fragments, we identified a fragment of the Escherichia coli 23S rRNA (nucleotides 74 to 136) whose expression caused readthrough of UGA nonsense mutations in certain codon contexts in vivo . The antisense RNA fragment produced a similar effect, but in neither case was readthrough of UAA or UAG observed . Since termination at UGA in E . coli specifically requires release factor 2 (RF2), our data suggest that the fragments interfere with RF2-dependent termination.

J Bacteriol, 1998 May, 180(10), 2729 - 35
Function of protonatable residues in the flagellar motor of Escherichia coli: a critical role for Asp 32 of MotB; Zhou J et al.; Rotation of the bacterial flagellar motor is powered by a transmembrane gradient of protons or, in some species, sodium ions . The molecular mechanism of coupling between ion flow and motor rotation is not understood . The proteins most closely involved in motor rotation are MotA, MotB, and FliG . MotA and MotB are transmembrane proteins that function in transmembrane proton conduction and that are believed to form the stator . FliG is a soluble protein located on the cytoplasmic face of the rotor . Two other proteins, FliM and FliN, are known to bind to FliG and have also been suggested to be involved to some extent in torque generation . Proton (or sodium)-binding sites in the motor are likely to be important to its function and might be formed from the side chains of acidic residues . To investigate the role of acidic residues in the function of the flagellar motor, we mutated each of the conserved acidic residues in the five proteins that have been suggested to be involved in torque generation and measured the effects on motility . None of the conserved acidic residues of MotA, FliG, FliM, or FliN proved essential for torque generation . An acidic residue at position 32 of MotB did prove essential . Of 15 different substitutions studied at this position, only the conservative-replacement D32E mutant retained any function . Previous studies, together with additional data presented here, indicate that the proteins involved in motor rotation do not contain any conserved basic residues that are critical for motor rotation per se . We propose that Asp 32 of MotB functions as a proton-binding site in the bacterial flagellar motor and that no other conserved, protonatable residues function in this capacity.

J Bacteriol, 1998 May, 180(10), 2682 - 8
Negative regulation of IS2 transposition by the cyclic AMP (cAMP)-cAMP receptor protein complex; Hu ST et al.; Three sequences similar to that of the consensus binding sequence of the cyclic AMP (cAMP)-cAMP receptor protein (CRP) complex were found in the major IS2 promoter region . Experiments were performed to determine whether the cAMP-CRP complex plays a role in the regulation of IS2 transposition . In the gel retardation assay, the cAMP-CRP complex was found to be able to bind the major IS2 promoter . A DNA footprinting assay confirmed that the cAMP-CRP complex binds to the sequences mentioned above . With an IS2 promoter-luciferase gene fusion construct, the cAMP-CRP complex was shown to inhibit transcription from the major IS2 promoter . IS2 was found to transpose at a frequency approximately 200-fold higher in an Escherichia coli host defective for CRP or adenyl cyclase than in a wild-type host . These results suggest that the cAMP-CRP complex is a negative regulator of IS2 transposition.

J Bacteriol, 1998 May, 180(10), 2623 - 9
The L-isoaspartyl protein repair methyltransferase enhances survival of aging Escherichia coli subjected to secondary environmental stresses; Visick JE et al.; Like its homologs throughout the biological world, the L-isoaspartyl protein repair methyltransferase of Escherichia coli, encoded by the pcm gene, can convert abnormal L-isoaspartyl residues in proteins (which form spontaneously from asparaginyl or aspartyl residues) to normal aspartyl residues . Mutations in pcm were reported to greatly reduce survival in stationary phase and when cells were subjected to heat or osmotic stresses (C . Li and S . Clarke, Proc . Natl . Acad . Sci . USA 89:9885-9889, 1992) . However, we subsequently demonstrated that those strains had a secondary mutation in rpoS, which encodes a stationary-phase-specific sigma factor (J . E . Visick and S . Clarke, J . Bacteriol . 179:4158-4163, 1997) . We now show that the rpoS mutation, resulting in a 90% decrease in HPII catalase activity, can account for the previously observed phenotypes . We further demonstrate that a new pcm mutant lacks these phenotypes . Interestingly, the newly constructed pcm mutant, when maintained in stationary phase for extended periods, is susceptible to environmental stresses, including exposure to methanol, oxygen radical generation by paraquat, high salt concentrations, and repeated heating to 42 degrees C . The pcm mutation also results in a competitive disadvantage in stationary-phase cells . All of these phenotypes can be complemented by a functional pcm gene integrated elsewhere in the chromosome . These data suggest that protein denaturation and isoaspartyl formation may act synergistically to the detriment of aging E . coli and that the repair methyltransferase can play a role in limiting the accumulation of the potentially disruptive isoaspartyl residues in vivo.

J Bacteriol, 1998 May, 180(10), 2616 - 22
Molecular and genetic analysis of two closely linked genes that encode, respectively, a protein phosphatase 1/2A/2B homolog and a protein kinase homolog in the cyanobacterium Anabaena sp . strain PCC 7120; Zhang CC et al.; Reversible protein phosphorylation plays important roles in signal transduction . One gene, prpA, encoding a protein similar to eukaryotic types of phosphoprotein phosphatases PP1, PP2A, and PP2B, was cloned from the nitrogen-fixing cyanobacterium Anabaena sp . strain PCC 7120 . Interestingly, a eukaryotic-type protein kinase gene, pknE, was found 301 bp downstream of prpA . This unusual genetic arrangement provides the opportunity for study about how the balance between protein phosphorylation and dephosphorylation can regulate cellular activities . Both proteins were overproduced in Escherichia coli and used to raise polyclonal antibodies . Immunodetection and RNA/DNA hybridization experiments suggest that these two genes are unlikely to be coexpressed, despite their close genetic linkage . PrpA is expressed constitutively under different nitrogen conditions, while PknE expression varies according to the nature of the nitrogen source . Inactivation analysis in vivo suggests that PrpA and PknE function to ensure a correct level of phosphorylation of the targets in order to regulate similar biological processes such as heterocyst structure formation and nitrogen fixation.

J Bacteriol, 1998 May, 180(10), 2599 - 608
Regulation of the carnitine pathway in Escherichia coli: investigation of the cai-fix divergent promoter region; Buchet A et al.; The divergent structural operons caiTABCDE and fixABCX of Escherichia coli are required for anaerobic carnitine metabolism . Transcriptional monocopy lacZ fusion studies showed that both operons are coexpressed during anaerobic growth in the presence of carnitine, respond to common environmental stimuli (like glucose and nitrate), and are modulated positively by the same general regulators, CRP and FNR, and negatively by H-NS . Overproduction of the CaiF specific regulatory protein mediating the carnitine signal restored induction in an fnr mutant, corresponding to its role as the primary target for anaerobiosis . Transcript analysis identified two divergent transcription start points initiating 289 bp apart . DNase I footprinting revealed three sites with various affinities for the binding of the cAMP-CRP complex inside this regulatory region . Site-directed mutagenesis experiments indicated that previously reported perfect CRP motif 1, centered at -41.5 of the cai transcriptional start site, plays a direct role in the sole cai activation . In contrast, mutation in CRP site 2, positioned at -69.5 of the fix promoter, caused only a threefold reduction in fix expression . Thus, the role of the third CRP site, located at -126.5 of fix, might be to reinforce the action of site 2 . A critical 50-bp cis-acting sequence overlapping the fix mRNA start site was found, by deletion analysis, to be necessary for cai transcription . This region is thought to be involved in transduction of the signal mediated by the CaiF regulator.

Genes Dev, 1998 May 1, 12(9), 1348 - 55
Degradation of carboxy-terminal-tagged cytoplasmic proteins by the Escherichia coli protease HflB (FtsH); Herman C et al.; Proteins with short nonpolar carboxyl termini are unstable in Escherichia coli . This proteolytic pathway is used to dispose of polypeptides synthesized from truncated mRNA molecules . Such proteins are tagged with an 11-amino-acid nonpolar destabilizing tail via a mechanism involving the 10Sa (SsrA) stable RNA and then degraded . We show here that the ATP-dependent zinc protease HflB (FtsH) is involved in the degradation of four unstable derivatives of the amino-terminal domain of the lambdacI repressor: three with nonpolar pentapeptide tails (cI104, cI105, cI108) and one with the SsrA tag (cI-SsrA) . cI105 and cI-SsrA are also degraded by the ClpP-dependent proteases . Loss of ClpP can be compensated for by overproducing HflB . In an in vitro system, cI108 and cI-SsrA are degraded by HflB in an energy-dependent reaction, indicating that HflB itself recognizes the carboxyl terminus . These results establish a tail-specific pathway for removing abnormal cytoplasmic proteins via the HflB and Clp proteases.

Genes Dev, 1998 May 1, 12(9), 1338 - 47
The ClpXP and ClpAP proteases degrade proteins with carboxy-terminal peptide tails added by the SsrA-tagging system; Gottesman S et al.; Interruption of translation in Escherichia coli can lead to the addition of an 11-residue carboxy-terminal peptide tail to the nascent chain . This modification is mediated by SsrA RNA (also called 10Sa RNA and tmRNA) and marks the tagged polypeptide for proteolysis . Degradation in vivo of lambda repressor amino-terminal domain variants bearing this carboxy-terminal SsrA peptide tag is shown here to depend on the cytoplasmic proteases ClpXP and ClpAP . Degradation in vitro of SsrA-tagged substrates was reproduced with purified components and required a substrate with a wild-type SsrA tail, the presence of both ClpP and either ClpA or ClpX, and ATP . Clp-dependent proteolysis accounts for most degradation of SsrA-tagged amino-domain substrates at 32 degrees C, but additional proteases contribute to the degradation of some of these SsrA-tagged substrates at 39 degrees C . The existence of multiple cytoplasmic proteases that function in SsrA quality-control surveillance suggests that the SsrA tag is designed to serve as a relatively promiscuous signal for proteolysis . Having diverse degradation systems able to recognize this tag may increase degradation capacity, permit degradation of a wide variety of different tagged proteins, or allow SsrA-tagged proteins to be degraded under different growth conditions.

Genes Dev, 1998 May 1, 12(9), 1327 - 37
A ribosomal function is necessary for efficient splicing of the T4 phage thymidylate synthase intron in vivo; Semrad K et al.; Splicing of the group I intron of the T4 thymidylate synthase (td) gene was uncoupled from translation by introducing stop codons in the upstream exon . This resulted in severe splicing deficiency in vivo . Overexpression of a UGA suppressor tRNA partially rescued splicing, suggesting that this in vitro self-splicing intron requires translation for splicing in vivo . Inhibition of translation by the antibiotics chloramphenicol and spectinomycin also resulted in splicing deficiency . Ribosomal protein S12, a protein with RNA chaperone activity, and CYT-18, a protein that stabilizes the three-dimensional structure of group I introns, efficiently rescued the stop codon mutants . We identified a region in the upstream exon that interferes with splicing . Point mutations in this region efficiently alleviate the effect of a nonsense codon . We infer from these results that the ribosome acts as an RNA chaperone to facilitate proper folding of the intron.

Genes Dev, 1998 May 1, 12(9), 1248 - 53
RadA protein is an archaeal RecA protein homolog that catalyzes DNA strand exchange; Seitz EM et al.; With the discovery that the Saccharomyces cerevisiae Rad51 protein is both structurally and functionally similar to the Escherichia coli RecA protein, the RecA paradigm for homologous recombination was extended to the Eucarya . The ubiquitous presence of RecA and Rad51 protein homologs raises the question of whether this archetypal protein exists within the third domain of life, the Archaea . Here we present the isolation of a Rad51/RecA protein homolog from the archaeon Sulfolobus solfataricus, and show that this protein, RadA, possesses the characteristics of a DNA strand exchange protein: The RadA protein is a DNA-dependent ATPase, forms a nucleoprotein filament on DNA, and catalyzes DNA pairing and strand exchange.

J Appl Physiol, 1998 May, 84(5), 1610 - 4
Effect of platelet-activating factor-receptor antagonism on endotoxin-induced lung dysfunction in sheep; Snapper JR et al.; To further define the role of platelet-activating factor (PAF) in endotoxin-induced lung dysfunction, we examined the effect of ABT-299, a specific and potent PAF-receptor antagonist, on the response to endotoxemia in six chronically instrumented awake sheep . We administered Escherichia coli endotoxin (0.5 microg/kg) intravenously with or without pretreatment with ABT-299 while monitoring mean pulmonary arterial pressure (Ppa), mean systemic arterial pressure (Psa), dynamic compliance of the lungs (Cdyn), and functional residual capacity (FRC) . Endotoxin administration caused pulmonary hypertension, reduced Cdyn, leukopenia, and hypoxemia while having no significant effect on Psa or FRC . Administration of ABT-299 did not affect any of the measured variables at baseline . Pretreatment with ABT-299 attenuated the peak Ppa seen after endotoxin administration but had minimal effects on endotoxin-induced changes in Cdyn, white blood cell count, or alveolar-to-arterial oxygen difference . ABT-299 was shown to completely block the pulmonary hypertension and reduction in Cdyn seen after intravenous administration of exogenous PAF . We conclude that PAF does not play an essential role in the sheep's response to endotoxin.

Proc Natl Acad Sci U S A, 1998 Apr 28, 95(9), 5407 - 12
Compartmentalized expression of two structurally and functionally distinct 4-coumarate:CoA ligase genes in aspen (Populus tremuloides); Hu WJ et al.; 4-Coumarate:CoA ligases (4CLs, EC 6.2.1.12) are a group of enzymes necessary for maintaining a continuous metabolic flux for the biosynthesis of plant phenylpropanoids, such as lignin and flavonoids, that are essential to the survival of plants . So far, various biochemical and molecular studies of plant 4CLs seem to suggest that 4CL isoforms in plants are functionally indistinguishable in mediating the biosynthesis of these phenolics . However, we have discovered two functionally and structurally distinct 4CL genes, Pt4CL1 and Pt4CL2 (63% protein sequence identity), that are differentially expressed in aspen (Populus tremuloides) . The Escherichia coli-expressed and purified Pt4CL1 and Pt4CL2 proteins exhibited highly divergent substrate preference as well as specificity that reveal the association of Pt4CL1 with the biosynthesis of guaiacyl-syringyl lignin and the involvement of Pt4CL2 with other phenylpropanoid formation . Northern hybridization analysis demonstrated that Pt4CL1 mRNA is specifically expressed in lignifying xylem tissues and Pt4CL2 mRNA is specifically expressed in epidermal layers in the stem and the leaf, consistent with the promoter activities of Pt4CL1 and Pt4CL2 genes based on the heterologous promoter-beta-glucouronidase fusion analysis . Thus, the expression of Pt4CL1 and Pt4CL2 genes is compartmentalized to regulate the differential formation of phenylpropanoids that confer different physiological functions in aspen; Pt4CL1 is devoted to lignin biosynthesis in developing xylem tissues, whereas Pt4CL2 is involved in the biosynthesis of other phenolics, such as flavonoids, in epidermal cells.

Proc Natl Acad Sci U S A, 1998 Apr 28, 95(9), 4959 - 63
Evidence that HetR protein is an unusual serine-type protease; Zhou R et al.; The hetR gene plays a very important role in cell differentiation of heterocystous cyanobacteria . To understand the mechanism of the hetR gene product in regulation of heterocyst differentiation, the recombinant HetR protein (rHetR) was overproduced in Escherichia coli . Purified rHetR was unstable and degraded easily in solution . Phenylmethanesulfonyl fluoride, a serine-type protease inhibitor, prevented the degradation and was shown to modify covalently rHetR . Dansyl fluoride (DnsF), another serine-type protease inhibitor, also covalently modifies rHetR as shown by electrophoresis and electroblotting of the labeled rHetR and by MS . The labeling of rHetR with phenylmethanesulfonyl fluoride and DnsF was at the same site of rHetR and required Ca2+ . S179N-rHetR, a mutant protein from strain 216 of Anabaena PCC 7120, which cannot differentiate heterocysts because of the mutation, was also overproduced and characterized . Although S170N-rHetR still can be labeled with DnsF, no proteolysis was observed, suggesting that Ser179 is involved in proteolytic activity . DnsF-labeled rHetR was digested with trypsin, and the labeled peptide was isolated and sequenced . The labeled peptide matches a sequence from HetR . These results show that HetR is a protease.

Proc Natl Acad Sci U S A, 1998 Apr 28, 95(9), 4953 - 8
A stationary phase protein in Escherichia coli with binding activity to the major sigma subunit of RNA polymerase; Jishage M et al.; Switching of the transcription pattern in Escherichia coli during the growth transition from exponential to stationary phase is accompanied by the replacement of the RNA polymerase-associated sigma70 subunit (sigmaD) with sigma38 (sigmaS) . A fraction of the sigma70 subunit in stationary phase cell extracts was found to exist as a complex with a novel protein, designated Rsd (Regulator of sigma D) . The intracellular level of Rsd starts to increase during the transition from growing to stationary phase . The rsd gene was identified at 90 min on the E . coli chromosome . Overexpressed and purified Rsd protein formed complexes in vitro with sigma70 but not with other sigma subunits, sigmaN, sigmaS, sigmaH, sigmaF, and sigmaE . Analysis of proteolytic fragments of sigma70 indicated that Rsd binds at or downstream of region 4, the promoter -35 recognition domain . The isolated Rsd inhibited transcription in vitro to various extents depending on the promoters used . We propose that Rsd is a stationary phase E . coli protein with regulatory activity of the sigma70 function.

Proc Natl Acad Sci U S A, 1998 Apr 28, 95(9), 4918 - 23
Acetylation at Lys-92 enhances signaling by the chemotaxis response regulator protein CheY; Ramakrishnan R et al.; When Escherichia coli cells lacking all chemotaxis proteins except the response regulator CheY are exposed to acetate, clockwise flagellar rotation results, indicating the acetate stimulus has activated signaling by CheY . Acetate can be converted to acetyl-CoA by either of two different metabolic pathways, which proceed through acetyl phosphate or acetyl-AMP intermediates . In turn, CheY can be covalently modified by either intermediate in vitro, leading to phosphorylation or acetylation, respectively . Either pathway is sufficient to support the CheY-mediated response to acetate in vivo . Whereas phosphorylation of Asp-57 is a recognized mechanism for activation of CheY to stimulate clockwise flagellar rotation, acetylation of CheY is less well characterized . We found evidence for multiple CheY acetylation sites by mass spectrometry and directly identified Lys-92 and Lys-109 as acetylation sites by Edman degradation of peptides from {14C}acetate-labeled CheY . Replacement of CheY Lys-92, the preferred acetylation site, with Arg has little effect on chemotaxis but completely prevents the response to acetate via the acetyl-AMP pathway . Thus acetylation of Lys-92 activates clockwise signaling by CheY in vivo . The mechanism by which acetylation activates CheY apparently is not simple charge neutralization, nor does it involve enhanced binding to the FliM flagellar switch protein . Thus acetylation probably affects signal generation by CheY at a step after switch binding.

J Biol Chem, 1998 May 1, 273(18), 11362 - 9
Acceleration of myosin light chain dephosphorylation and relaxation of smooth muscle by telokin . Synergism with cyclic nucleotide-activated kinase; Wu X et al.; Incorporation of 32P into telokin, a smooth muscle-specific, 17-18-kDa, acidic (pI 4.2-4.4) protein, was increased by forskolin (20 microM) in intact rabbit ileum smooth muscle (ileum) and by 8-bromo-cyclic GMP (100 microM) in alpha-toxin-permeabilized ileum . Native telokin (5-20 microM), purified from turkey gizzard, and recombinant rabbit telokin, expressed in Escherichia coli and purified to >90% purity, induced dose-dependent relaxation, associated with a significant decrease in regulatory myosin light chain phosphorylation, without affecting the rate of thiophosphorylation of regulatory myosin light chain of ileum permeabilized with 0.1% Triton X-100 . Endogenous telokin was lost from ileum during prolonged permeabilization (>20 min) with 0.1% Triton X-100, and the time course of loss was correlated with the loss of 8-bromo-cyclic GMP-induced calcium desensitization . Recombinant and native gizzard telokins were phosphorylated, in vitro, by the catalytic subunit of cAMP-dependent protein kinase, cGMP-dependent protein kinase, and p42/44 mitogen-activated protein kinase; the recombinant protein was also phosphorylated by calmodulin-dependent protein kinase II . Exogenous cGMP-dependent protein kinase (0.5 microM) activated by 8-bromo-cyclic GMP (50 microM) phosphorylated recombinant telokin (10 microM) when added concurrently to ileum depleted of its endogenous telokin, and their relaxant effects were mutually potentiated . Forskolin (20 microM) also increased phosphorylation of telokin in intact ileum . We conclude that telokin induces calcium desensitization in smooth muscle by enhancing myosin light chain phosphatase activity, and cGMP- and/or cAMP-dependent phosphorylation of telokin up-regulates its relaxant effect.

J Biol Chem, 1998 May 1, 273(18), 11302 - 12
Membrane association of active plasmid partitioning protein A in Escherichia coli; Lin Z et al.; QsopA and SopA, proteins essential for stable maintenance of low copy number plasmids and encoded on plasmid QpH1 of Coxiella burnetii and the F plasmid of Escherichia coli, respectively, are shown to be membrane associated using three independent approaches: isolation of hybrid protein A-PhoA proteins that display PhoA (bacterial alkaline phosphatase) activity indicating a periplasmic location, biochemical fractionation by flotation gradient centrifugation, and subcellular localization by immunoelectron microscopy . These data provide insight into the mechanism by which partitioning protein A spatially directs plasmids into daughter cells at bacterial division.

J Biol Chem, 1998 May 1, 273(18), 11257 - 66
Activation of gene expression by a ligand-induced conformational change of a protein-DNA complex; Rhee KY et al.; IlvY protein binds cooperatively to tandem operator sites in the divergent, overlapping, promoter-regulatory region of the ilvYC operon of Escherichia coli . IlvY positively regulates the expression of the ilvC gene in an inducer-dependent manner and negatively regulates the transcription of its own divergently transcribed structural gene in an inducer-independent manner . Although binding of IlvY protein to the tandem operators is sufficient to repress ilvY promoter-specific transcription, it is not sufficient to activate transcription from the ilvC promoter . Activation of ilvC promoter-specific transcription requires the additional binding of a small molecule inducer to the IlvY protein-DNA complex . The binding of inducer to IlvY protein does not affect the affinity of IlvY protein for the tandem operator sites . It does, however, cause a conformational change of the IlvY protein-DNA complex, which is correlated with the partial relief of an IlvY protein-induced bend of the DNA helix in the ilvC promoter region . This structural change in the IlvY protein-DNA complex results in a 100-fold increase in the affinity of RNA polymerase binding at the ilvC promoter site . The ability of a protein to regulate gene expression by ligand-responsive modulation of a protein-DNA structure is an emerging theme in gene regulation.

J Biol Chem, 1998 May 1, 273(18), 11121 - 6
Functional analysis of the amino-terminal 8-kDa domain of DNA polymerase beta as revealed by site-directed mutagenesis . DNA binding and 5'-deoxyribose phosphate lyase activities; Prasad R et al.; The amino-terminal 8-kDa domain of DNA polymerase beta functions in binding single-stranded DNA (ssDNA), recognition of a 5'-phosphate in gapped DNA structures, and as a 5'-deoxyribose phosphate (dRP) lyase . NMR and x-ray crystal structures of this domain have suggested several residues that may interact with ssDNA or play a role in the dRP lyase reaction . Nine of these residues were altered by site-directed mutagenesis . Each mutant was expressed in Escherichia coli, and the recombinant protein was purified to near homogeneity . CD spectra of these mutant proteins indicated that the alteration did not adversely affect the global protein structure . Single-stranded DNA binding was probed by photochemical cross-linking to oligo(dT)16 . Several mutants (F25W, K35A, K60A, and K68A) were impaired in ssDNA binding activity, whereas other mutants (H34G, E71Q, K72A, E75A, and K84A) retained near wild-type binding activity . The 5'-phosphate recognition activity of these mutants was examined by UV cross-linking to a 5-nucleotide gap DNA where the 5' terminus in the gap was either phosphorylated or unphosphorylated . The results indicate that Lys35 is involved in 5'-phosphate recognition of DNA polymerase beta . Finally, the dRP lyase activity of these mutants was evaluated using a preincised apurinic/apyrimidinic DNA . Alanine mutants of Lys35 and Lys60 are significantly reduced in dRP lyase activity, consistent with the lower ssDNA binding activity . More importantly, alanine substitution for Lys72 resulted in a greater than 90% loss of dRP lyase activity, without affecting DNA binding . Alanine mutants of Lys68 and Lys84 had wild-type dRP lyase activity . The triple alanine mutant, K35A/K68A/K72A, was devoid of dRP lyase activity, suggesting that the effects of the alanine substitution at Lys72 and Lys35 were additive . The results suggest that Lys72 is directly involved in formation of a covalent imino intermediate and are consistent with Lys72 as the predominant Schiff base nucleophile in the dRP lyase beta-elimination catalytic reaction.

J Biol Chem, 1998 May 1, 273(18), 11115 - 20
The alpha-helical domain near the amino terminus is essential for dimerization of vascular endothelial growth factor; Siemeister G et al.; Vascular endothelial growth factor (VEGF) is an endothelial cell-specific mitogen and a key mediator of aberrant endothelial cell proliferation and vascular permeability in a variety of human pathological situations such as tumor angiogenesis, diabetic retinopathy, or psoriasis . By amino-terminal deletion analysis and by site-directed mutagenesis we have identified a new domain within the amino-terminal alpha-helix that is essential for dimerization of VEGF . VEGF121 variants containing amino acids 8 to 121 or 14 to 121, respectively, either expressed in Escherichia coli and refolded in vitro, or expressed in Chinese hamster ovary cells, were in a dimeric conformation and showed full binding activity to VEGF receptors and stimulation of endothelial cell proliferation as compared with wild-type VEGF . In contrast, a VEGF121 variant covering amino acids 18 to 121, as well as a variant in which the hydrophobic amino acids Val14, Val15, Phe17, and Met18 within the amphipathic alpha-helix near the amino terminus were replaced by serine, failed to form biological active VEGF dimers . From these data we conclude that a domain between amino acids His12 and Asp19 within the amino-terminal alpha-helix is essential for formation of VEGF dimers, and we propose hydrophobic interactions between VEGF monomers to stabilize or favor dimerization.

J Biol Chem, 1998 May 1, 273(18), 11069 - 74
Induction of chromosomal gene mutations in Escherichia coli by direct incorporation of oxidatively damaged nucleotides . New evaluation method for mutagenesis by damaged DNA precursors in vivo; Inoue M et al.; We have developed a new strategy for the evaluation of the mutagenicity of a damaged DNA precursor (deoxyribonucleoside 5'-triphosphate) in Escherichia coli . 8-Hydroxydeoxyguanosine triphosphate (8-OH-dGTP) and 2-hydroxydeoxyadenosine triphosphate (2-OH-dATP) were chosen for this study because they appear to be formed abundantly by reactive oxygen species in cells . We introduced the oxidatively damaged nucleotides into competent E . coli and selected mutants of the chromosomal lacI gene . Both damaged nucleotides induced lacI gene mutations in a dose-dependent manner, whereas unmodified dATP and dGTP did not appear to elicit the mutations . The addition of 50 nmol of 8-OH-dGTP and 2-OH-dATP into an E . coli suspension induced 12- and 9-fold more substitution mutations than the spontaneous event, respectively . The 8-OH-dGTP induced A.T --> C.G transversions, and the 2-OH-dATP elicited G.C --> T.A transversions . These results indicate that the two oxidatively damaged nucleotides are mutagenic in vivo and suggest that 8-OH-dGTP and 2-OH-dATP were incorporated opposite A and G residues, respectively, in the E . coli DNA . This new method enables the evaluation and comparison of the mutagenic potentials of damaged DNA precursors in vivo.

J Biol Chem, 1998 May 1, 273(18), 11032 - 7
The small heat-shock protein IbpB from Escherichia coli stabilizes stress-denatured proteins for subsequent refolding by a multichaperone network; Veinger L et al.; The role of small heat-shock proteins in Escherichia coli is still enigmatic . We show here that the small heat-shock protein IbpB is a molecular chaperone that assists the refolding of denatured proteins in the presence of other chaperones . IbpB oligomers bind and stabilize heat-denatured malate dehydrogenase (MDH) and urea-denatured lactate dehydrogenase and thus prevent the irreversible aggregation of these proteins during stress . While IbpB-stabilized proteins alone do not refold spontaneously, they are specifically delivered to the DnaK/DnaJ/GrpE (KJE) chaperone system where they refold in a strict ATPase-dependent manner . Although GroEL/GroES (LS) chaperonins do not interact directly with IbpB-released proteins, LS accelerate the rate of KJE-mediated refolding of IbpB-released MDH, and to a lesser extent lactate dehydrogenase, by rapidly processing KJE-released early intermediates . Kinetic and gel-filtration analysis showed that denatured MDH preferentially transfers from IbpB to KJE, then from KJE to LS, and then forms a active enzyme . IbpB thus stabilizes aggregation-prone folding intermediates during stress and, as an integral part of a cooperative multichaperone network, is involved in the active refolding of stress-denatured proteins.

J Biol Chem, 1998 May 1, 273(18), 10863 - 7
Reaction of O6-benzylguanine-resistant mutants of human O6-alkylguanine-DNA alkyltransferase with O6-benzylguanine in oligodeoxyribonucleotides; Pegg AE et al.; Inactivation of the human DNA repair protein, O6-alkylguanine-DNA alkyltransferase (AGT), by O6-benzylguanine renders tumor cells susceptible to killing by alkylating agents . AGT mutants resistant to O6-benzylguanine can be made by converting Pro140 to an alanine (P140A) or Gly156 to an alanine (G156A) . These mutations had a much smaller effect on the reaction with O6-benzylguanine when it was incorporated into a short single-stranded oligodeoxyribonucleotide . Such oligodeoxyribonucleotides could form the basis for the design of improved AGT inhibitors . AGT and mutants P140A and G156A preferentially reacted with O6-benzylguanine when incubated with a mixture of two 16-mer oligodeoxyribonucleotides, one containing O6-benzylguanine and the other, O6-methylguanine . When the 6 amino acids located in positions 159-164 in AGT were replaced by the equivalent sequence from the Escherichia coli Ada-C protein (mutant AGT/6ada) the preference for benzyl repair was eliminated . Further mutation incorporating the P140A change into AGT/6ada giving mutant P140A/6ada led to a protein that resembled Ada-C in preference for the repair of methyl groups, but P140A/6ada did not differ from P140A in reaction with the free base O6-benzylguanine . Changes in the AGT active site pocket can therefore affect the preference for repair of O6-benzyl or -methyl groups when present in an oligodeoxyribonucleotide without altering the reaction with free O6-benzylguanine.

J Biol Chem, 1998 May 1, 273(18), 10851 - 6
Inhibitor binding within the NarI subunit (cytochrome bnr) of Escherichia coli nitrate reductase A; Magalon A et al.; We have used inhibitors and site-directed mutants to investigate quinol binding to the cytochrome bnr (NarI) of Escherichia coli nitrate reductase (NarGHI) . Both stigmatellin and 2-n-heptyl-4-hydroxyquinoline-N-oxide (HOQNO) inhibit menadiol:nitrate oxidoreductase activity with I50 values of 0.25 and 6 microM, respectively, and prevent the generation of a NarGHI-dependent proton electrochemical potential across the cytoplasmic membrane . These inhibitors have little effect on the rate of reduction of the two hemes of NarI (bL and bH), but have an inhibitory effect on the extent of nitrate-dependent heme reoxidation . No quinol-dependent heme bH reduction is detected in a mutant lacking heme bL (NarI-H66Y), whereas a slow but complete heme bL reduction is detected in a mutant lacking heme bH (NarI-H56R) . This is consistent with physiological quinol binding and oxidation occurring at a site (QP) associated with heme bL which is located toward the periplasmic side of NarI . Optical and EPR spectroscopies performed in the presence of stigmatellin or HOQNO provide further evidence that these inhibitors bind at a heme bL-associated QP site . These results suggest a model for electron transfer through NarGHI that involves quinol binding and oxidation in the vicinity of heme bL and electron transfer through heme bH to the cytoplasmically localized membrane-extrinsic catalytic NarGH dimer.

J Mol Evol, 1998 May, 46(5), 508 - 20
Evolution of the cytochrome c oxidase proton pump; Musser SM et al.; The superfamily of quinol and cytochrome c terminal oxidase complexes is related by a homologous subunit containing six positionally conserved histidines that ligate a low-spin heme and a heme-copper dioxygen activating and reduction center . On the basis of the structural similarities of these enzymes, it has been postulated that all members of this superfamily catalyze proton translocation by similar mechanisms and that the CuA center found in most cytochrome c oxidase complexes serves merely as an electron conduit shuttling electrons from ferrocytochrome c into the hydrophobic core of the enzyme . The recent characterization of cytochrome c oxidase complexes and structurally similar cytochrome c:nitric oxide oxidoreductase complexes without CuA centers has strengthened this view . However, recent experimental evidence has shown that there are two ubiquinone(ol) binding sites on the Escherichia coli cytochrome bo3 complex in dynamic equilibrium with the ubiquinone(ol) pool, thereby strengthening the argument for a Q(H2)-loop mechanism of proton translocation {Musser SM et al . (1997) Biochemistry 36:894-902} . In addition, a number of reports suggest that a Q(H2)-loop or another alternate proton translocation mechanism distinct from the mitochondrial aa3-type proton pump functions in Sulfolobus acidocaldarius terminal oxidase complexes . The possibility that a primitive quinol oxidase complex evolved to yield two separate complexes, the cytochrome bc1 and cytochrome c oxidase complexes, is explored here . This idea is the basis for an evolutionary tree constructed using the notion that respiratory complexity and efficiency progressively increased throughout the evolutionary process . The analysis suggests that oxygenic respiration is quite an old process and, in fact, predates nitrogenic respiration as well as reaction-center photosynthesis.

Arch Microbiol, 1998 Apr, 169(4), 339 - 45
Structure, organization, and expression of genes coding for envelope components in the archaeon Methanosarcina mazei S-6; Mayerhofer LE et al.; The antigenic mosaics of archaeal species are complex and lead to the distinction of different immunotypes . We began the dissection of the antigenic mosaic of the methanogen Methanosarcina mazei S-6 by gene cloning and sequencing . The analysis of the sequence, organization, and in vitro (heterologous) and in vivo expression of two three-gene clusters that encode proteins localized to the cell envelope and that are recognized by antibodies for surface structures is presented in this report . The amino acid sequences and compositions share characteristics with S-layer proteins and, most notably, have repeats of conserved sequences and secondary structures . Expressed genes produced proteins with a tendency to oligomerize, and one of these proteins was susceptible to breakdown at regular intervals . Altogether, the data reveal a modular system (clusters of homologous genes, proteins of similar sequences with conserved repeats) seemingly suitable for assembling an enormous variety of final molecular structures by rearranging and combining genes, proteins, and repeats, and thus generate the observed wide spectrum of antigenic diversity.

Nucleic Acids Res, 1998 Apr 15, 26(8), 2024 - 30
Cr(III)-mediated crosslinks of glutathione or amino acids to the DNA phosphate backbone are mutagenic in human cells; Voitkun V et al.; Carcinogenic Cr(VI) compounds were previously found to induce amino acid/glutathione-Cr(III)-DNA crosslinks with the site of adduction on the phosphate backbone . Utilizing the pSP189 shuttle vector plasmid we found that these ternary DNA adducts were mutagenic in human fibroblasts . The Cr(III)-glutathione adduct was the most potent in this assay, followed by Cr(III)-His and Cr(III)-Cys adducts . Binary Cr(III)-DNA complexes were only weakly mutagenic, inducing a significant response only at a 10 times higher number of adducts compared with Cr(III)-glutathione . Single base substitutions at the G:C base pairs were the predominant type of mutations for all Cr(III) adducts . Cr(III), Cr(III)-Cys and Cr(III)-His adducts induced G:C-->A:T transitions and G:C-->T:A transversions with almost equal frequency, whereas the Cr(III)-glutathione mutational spectrum was dominated by G:C-->T:A transversions . Adduct-induced mutations were targeted toward G:C base pairs with either A or G in the 3' position to the mutated G, while spontaneous mutations occurred mostly at G:C base pairs with a 3' A . No correlation was found between the sites of DNA adduction and positions of base substitution, as adducts were formed randomly on DNA with no base specificity . The observed mutagenicity of Cr(III)-induced phosphotriesters demonstrates the importance of a Cr(III)-dependent pathway in Cr(VI) carcinogenicity.

Nucleic Acids Res, 1998 Apr 15, 26(8), 2001 - 7
Up-regulation of base excision repair correlates with enhanced protection against a DNA damaging agent in mouse cell lines; Chen KH et al.; DNA polymerase beta is required in mammalian cells for the predominant pathway of base excision repair involving single nucleotide gap filling DNA synthesis . Here we examine the relationship between oxidative stress, cellular levels of DNA polymerase beta and base excision repair capacity in vitro , using mouse monocytes and either wild-type mouse fibroblasts or those deleted of the DNA polymerase beta gene . Treatment with an oxidative stress-inducing agent such as hydrogen peroxide, 3-morpholinosydnonimine, xanthine/xanthine oxidase or lipopolysaccharide was found to increase the level of DNA polymerase beta in both monocytes and fibroblasts . Base excision repair capacity in vitro , as measured in crude cell extracts, was also increased by lipopolysaccharide treatment in both cell types . In monocytes lipopolysaccharide-mediated up-regulation of the base excision repair system correlated with increased resistance to the monofunctional DNA alkylating agent methyl methanesulfonate . By making use of a quantitative PCR assay to detect lesions in genomic DNA we show that lipopolysaccharide treatment of fibroblast cells reduces the incidence of spontaneous DNA lesions . This effect may be due to the enhanced DNA polymerase beta-dependent base excision repair capacity of the cells, because a similar decrease in DNA lesions was not observed in cells deficient in base excision repair by virtue of DNA polymerase beta gene deletion . Similarly, fibroblasts treated with lipopolysaccharide were more resistant to methyl methanesulfonate than untreated cells . This effect was not observed in cells deleted of the DNA polymerase beta gene . These results suggest that the DNA polymerase beta-dependent base excision repair pathway can be up-regulated by oxidative stress-inducing agents in mouse cell lines.

Nucleic Acids Res, 1998 Apr 15, 26(8), 1996 - 2000
A high throughput method to investigate oligodeoxyribonucleotide hybridization kinetics and thermodynamics; Mazumder A et al.; We describe a high throughput microtiter-based assay to measure binding of oligodeoxyribonucleotides to nucleic acid targets . The assay utilizes oligodeoxyribonucleotide probes labeled with a highly chemiluminescent acridinium ester (AE) . Reaction of AE with sodium sulfite renders it non-chemiluminescent . When an AE-labeled probe hybridizes to a target nucleic acid AE is protected from reaction with sodium sulfite and thus remains chemiluminescent . In contrast, unhybridized probe readily reacts with sodium sulfite and is rendered non-chemiluminescent . Hybridization of an AE-labeled probe to a target nucleic acid can therefore be detected without physical separation of unhybridized probe by treatment of the hybridization reaction with sodium sulfite and measurement of the remaining chemiluminescence . Using this method we measured hybridization rate constants and thermodynamic affinities of oligodeoxyribonucleotide probes binding to simple synthetic targets as well as large complex biological targets . The kinetic and thermodynamic parameters were measured with a high degree of accuracy and were in excellent agreement with values measured by other established techniques.

Nucleic Acids Res, 1998 Apr 15, 26(8), 1991 - 5
NMR evidence for a base triple in the HIV-2 TAR C-G.C+ mutant-argininamide complex; Brodsky AS et al.; Formation of a specific complex between the HIV Tat protein and the small RNA element TAR is critical for activation of viral transcription . A model complex for this interaction composed of HIV-2 TAR and the amide derivative of arginine has been developed to study how Tat and TAR interact specifically . We have previously determined a high resolution NMR structure of the HIV-2 TAR-argininamide complex . The argininamide guanidium group hydrogen bonds to the major groove face of G26 and is stacked between U23 and A22, forming an arginine sandwich . This structure also provided evidence for formation of a U38-A27.U38 base triple, as U23 is positioned in the major groove within hydrogen bonding distance to A27 . However, the expected U23 imino proton was not observed, preventing unambiguous identification of the base triple . Previous work on an isomorphic C38-G27.C23+ base triple mutant of the three base bulge HIV-1 TAR-argininamide complex demonstrated that the base triple is required for specific argininamide binding . Here we investigate the same C38-G27.C23+ base triple mutant in the context of two base bulge HIV-2 TAR . The improved NMR spectral properties of HIV-2 TAR allowed observation of the C23 amino and imino protons for the first time, providing direct evidence that a hydrogen bonding interaction is occurring . The NOEs observed correspond to those observed in the high resolution structure of the HIV-2 TAR-argininamide complex, confirming that a base triple is an important feature of the TAR-argininamide interaction.

Nucleic Acids Res, 1998 Apr 15, 26(8), 1980 - 4
Mismatched nucleotides may facilitate expansion of trinucleotide repeats in genetic diseases; Nakayabu M et al.; We have studied the contribution of mismatch sequences to the trinucleotide repeat expansion that causes hereditary diseases . Using an oligonucleotide duplex, (CAG)5/(CTG)5, as a template-primer, DNA synthesis was carried out using either Escherichia coli DNA polymerase I (Klenow fragment) or human immunodeficiency virus type I reverse transcriptase (HIV-RT) . Both enzymes expanded the repeat sequence longer than 27 nucleotides (nt), beyond the maximum length expected from the template size . The expansion was observed under conditions in which extension occurs either in both strands or in one strand . In contrast, with another template-primer that contains a non-repetitive flanking sequence 5'-upstream of the repetitive sequence, the reaction products were not extended beyond the template size (45 nt) by these DNA polymerases . We then used mismatched template-primers, in which either 1, 2 or 6 non-complementary nucleotides were introduced to the repeat sequence that is flanked by a non-repetitive sequence . In this case, primers were efficiently expanded over the expected length of 45 nt, in a mismatch-dependent manner . One of the primers with six mismatches extended as long as 72 nt . These results imply that the misincorporation of non-complementary deoxyribonucleoside monophosphates (dNMPs) into the repeat sequence makes double-stranded DNA unstable and triggers the slippage and expansion of trinucleotide repeats by forming loops or hairpin structures during DNA synthesis.

Nucleic Acids Res, 1998 Apr 15, 26(8), 1974 - 9
The use of sequence comparison to detect 'identities' in tRNA genes; Sagara JI et al.; We have developed a computational method that detects 'identities' in tRNA genes by using principal component analysis to classify the sequences of bases in tRNA genes into groups of similar sequences and then comparing the distribution of sequences of bases, in order to extract characteristic bases that are conserved within a group but differ between groups . These classification and comparison procedures are applied recursively to classify the sequences into hierarchical groups, so that multiple levels of characteristic sites can be detected . By using this computational method, we were able to detect many characteristic sites in the T and D domains of tRNAs, as well as the characteristic sites that had already been detected experimentally . This suggests that bases not only in the contact regions but also in the elbow regions, which determine the structure and dynamics of the whole tRNA molecule, are important to the tRNA-aminoacyl tRNA synthetase recognition.

Nucleic Acids Res, 1998 Apr 15, 26(8), 1959 - 64
The -104G nucleotide of the human CYP21 gene is important for CYP21 transcription activity and protein interaction; Chin KK et al.; CYP21 gene encodes the steroid 21-hydroxylase (P450c21) that is involved in steroidogenesis in the adrenal cortex . Mutations occurring on CYP21 which convert it to the neighboring pseudogene, CYP21P, are found in patients with congenital adrenal hyperplasia (CAH), an autosomal recessive disease . We previously reported that the CYP21P pseudogene had lower transcription activity when compared with the active CYP21 gene . The sequences determining the basal transcription activity of the human CYP21 gene are located within the 166 bp region upstream from the transcriptional start site . Within this region, only 4 nucleotides are different between the active CYP21 and the CYP21P pseudogene; they are located at the -117, -104, -101 and -94 positions from the start site of the gene . Here, we report that the CYP21 gene-specific G nucleotide sequence at the -104 position is required for its basal transcription activity driven by the native TATA box of the human CYP21 gene . When this single nucleotide is changed to the CYP21P sequence, the basal transcription activity decreases by approximately 80% in transient transfection assay . In addition, our data from gel retardation assay show that this sequence is also critical for interaction with nuclear proteins from adrenal cells . These results therefore suggest that the single G sequence of the human CYP21 gene is crucial for the expression of its basal transcription activity, and this may be influenced by the interaction with specific nuclear proteins from the adrenal gland.

Nucleic Acids Res, 1998 Apr 15, 26(8), 1927 - 33
Simple and efficient generation in vitro of nested deletions and inversions: Tn5 intramolecular transposition; York D et al.; We have exploited the intramolecular transposition preference of the Tn 5 in vitro transposition system to test its effectiveness as a tool for generation of nested families of deletions and inversions . A synthetic transposon was constructed containing an ori, an ampicillin resistance (Ampr) gene, a multi-cloning site (MCS) and two hyperactive end sequences . The donor DNA that adjoins the transposon contains a kanamycin resistance (Kanr) gene . Any Amprreplicating plasmid that has undergone a transposition event (Kans) will be targeted primarily to any insert in the MCS . Two different size targets were tested in the in vitro system . Synthetic transposon plasmids containing either target were incubated in the presence of purified transposase (Tnp) protein and transformed . Transposition frequencies (Ampr/Kans) for both targets were found to be 30-50%, of which >95% occur within the target sequence, in an apparently random manner . By a conservative estimate 10(5) or more deletions/inversions within a given segment of DNA can be expected from a single one-step 20 microl transposition reaction . These nested deletions can be used for structure-function analysis of proteins and for sequence analysis . The inversions provide nested sequencing templates of the opposite strand from the deletions.

Nucleic Acids Res, 1998 Apr 15, 26(8), 1863 - 9
Inhibition of DNA polymerase reactions by pyrimidine nucleotide analogues lacking the 2-keto group; Guo MJ et al.; To investigate the influence of the pyrimidine 2-keto group on selection of nucleotides for incorporation into DNA by polymerases, we have prepared two C nucleoside triphosphates that are analogues of dCTP and dTTP, namely 2-amino-5-(2'-deoxy-beta-d-ribofuranosyl)pyridine-5'-triphosphate (d*CTP) and 5-(2'-deoxy- beta-d-ribofuranosyl)-3-methyl-2-pyridone-5'-triphosphate (d*TTP) respectively . Both proved strongly inhibitory to PCR catalysed by Taq polymerase; d*TTP rather more so than d*CTP . In primer extension experiments conducted with either Taq polymerase or the Klenow fragment of Escherichia coli DNA polymerase I, both nucleotides failed to substitute for their natural pyrimidine counterparts . Neither derivative was incorporated as a chain terminator . Their capacity to inhibit DNA polymerase activity may well result from incompatibility with the correctly folded form of the polymerase enzyme needed to stabilize the transition state and catalyse phosphodiester bond formation.

J Mol Biol, 1998 Mar 27, 277(2), 199 - 213
Missense mutations that inactivate Escherichia coli lac permease; Bailey J et al.; Although missense mutations that inactivate integral membrane proteins cause a variety of diseases, the mechanisms by which they act are poorly understood . To establish a model for investigating this issue, we identified 51 missense mutations arising in vivo that inactivate Escherichia coli lac permease, a well-characterized membrane transport protein . The mutants were isolated using a genetic screening procedure which eliminates mutations that block expression of the lac permease gene, such as nonsense and frameshift mutations . The majority of the 51 missense mutations caused highly non-conservative changes in membrane-spanning sequences, such as the introduction of charged residues . Nevertheless, the greatest clustering of substitutions occurred in the two regions of lac permease thought to be most important for transport function . The existence of this clustering indicates that even highly non-conservative substitutions may cause relatively localized structural defects . Conservative inactivating substitutions were scattered throughout lac permease and may affect residues that make contacts required for normal folding . Two unexpected phenotypes were observed in the collection of mutants: about 20% of the substitutions led to cold-sensitive lactose utilization, and one substitution made the mutant lac permease toxic to cells . This relatively unbiased collection of mutants should provide a resource for further studies of how missense mutations inactivate membrane proteins in vivo .

J Mol Biol, 1998 Mar 27, 277(2), 257 - 71
Site-directed mutations in motif VI of Escherichia coli DNA helicase II result in multiple biochemical defects: evidence for the involvement of motif VI in the coupling of ATPase and DNA binding activities via conformational changes; Hall MC et al.; Two site-directed mutants of Escherichia coli DNA helicase II (UvrD) were constructed to examine the functional significance of motif VI in a superfamily I helicase . Threonine 604 and arginine 605, representing two of the most highly conserved residues in motif VI, were replaced with alanine, generating the mutant alleles uvrD-T604A and uvrD-R605A . Genetic complementation studies indicated that UvrD-T604A, but not UvrD-R605A, functioned in methyl-directed mismatch repair and UvrABC-mediated nucleotide excision repair . Both mutant enzymes were purified and single-stranded DNA (ssDNA)-stimulated ATP hydrolysis, duplex DNA unwinding, and ssDNA binding were studied in the steady-state and compared to wild-type UvrD . UvrD-T604A exhibited a serious defect in ssDNA binding in the absence of nucleotide . However, in the presence of a non-hydrolyzable ATP analog, DNA binding was only slightly compromised . Limited proteolysis experiments suggested that UvrD-T604A had a "looser" conformation and could not undergo conformational changes normally associated with ATP binding/hydrolysis and DNA binding . UvrD-R605A, on the other hand, exhibited nearly normal DNA binding but had a severe defect in ATP hydrolysis (kcat=0.063 s-1 compared to 162 s-1 for UvrD) . UvrD-T604A exhibited a much less severe decrease in ATPase activity (kcat=8.8 s-1) . The Km for ATP for both mutants was not significantly changed . The results suggest that residues within motif VI of helicase II are essential for multiple biochemical properties associated with the enzyme and that motif VI is potentially involved in conformational changes related to the coupling of ATPase and DNA binding activities .

J Mol Biol, 1998 Mar 27, 277(2), 273 - 84
An endo-1,4-beta-xylanase-encoding gene from Agaricus bisporus is regulated by compost-specific factors; De Groot PW et al.; Compost is the preferred substrate for growth of the edible fungus Agaricus bisporus . Utilization of compost requires the production of enzymes involved in degradation of lignocellulolytic components . For molecular characterization of these processes we are isolating the encoding genes . By applying heterologous screening techniques, we have cloned such a gene, which is specifically induced on compost encoding an endo-1,4-beta-xylanase (xlnA) belonging to glycosyl hydrolase family 10 . The gene encodes a pre-protein of 333 amino acid residues with a predicted molecular mass of 34,946 for the mature protein . The open reading frame is interrupted by ten introns of which introns 5 and 6 are separated by an exon of only two base-pairs . High expression of the xlnA gene was observed in vegetative mycelium grown on sterilized compost while xlnA messengers were not detected in fruit bodies . Addition of glucose or xylose to compost repressed xlnA expression . When glucose-grown colonies were transferred to a medium containing cellulose, xylan or xylose as sole carbon source, the organism responded by expressing xlnA at a high level for a short period . Transfer from glucose to compost yielded a much stronger and constant xlnA induction . A similar pattern of expression was found for the cel3 gene encoding a cellulase, suggesting that these genes are induced by compost-specific factors rather than by the substrates they act upon . Antiserum raised against XLNA protein, which was heterologously expressed in Escherichia coli, detected, when the fungus was grown on compost, an extracellular protein of 33 kDa with endo-xylanase activity .

J Mol Biol, 1998 Mar 27, 277(2), 363 - 77
Structural principles for the inhibition of the 3'-5' exonuclease activity of Escherichia coli DNA polymerase I by phosphorothioates; Brautigam CA et al.; A two-metal-ion catalytic mechanism has previously been proposed for several phosphoryl-transfer enzymes . In order to extend the structural basis of this mechanism, crystal structures of three single-stranded DNA substrates bound to the 3'-5' exonucleolytic active site of the large fragment of DNA polymerase I from Escherichia coli have been elucidated . The first is a 2.1 A resolution structure of a Michaelis complex between the large fragment (or Klenow fragment, KF) and a single-stranded DNA substrate, stabilized by low pH and flash-freezing . The positions and identities of the catalytic metal ions, a Zn2+ at site A and a Mg2+ at site B, have been clearly established . The structural and kinetic consequences of sulfur substitutions in the scissile phosphate have been explored . A complex with the Rp isomer of phosphorothioate DNA, refined at 2.2 A resolution, shows Zn2+ bound to both metal sites and a mispositioning of the substrate and attacking nucleophile . The complex with the Sp phosphorothioate at 2 . 3 A resolution reveals that metal ions do not bind in the active site, having been displaced by a bulky sulfur atom . Steady-state kinetic experiments show that catalyzed hydrolysis of the Rp isomer was reduced only about 15-fold, while no enzyme activity could be detected with the Sp phosphorothioate, consistent with the structural observations . Furthermore, Mn2+ could not rescue the activity of the exonuclease on the Sp phosphorothioate . Taken together, these studies confirm and extend the proposed two-metal-ion exonuclease mechanism and provide a structural context to explain the effects of sulfur substitutions on this and other phosphoryl-transfer enzymes . These experiments also suggest that the possibility of metal-ion exclusion be taken into account when interpreting the results of Mn2+ rescue experiments .

J Chromatogr B Biomed Sci Appl, 1998 Apr 10, 707(1-2), 121 - 30
Endotoxin removal with poly(ethyleneimine)-immobilized adsorbers: Sepharose 4B versus flat sheet and hollow fibre membranes; Petsch D et al.; Poly(ethyleneimine) was immobilized on poly(vinyl alcohol)-coated nylon flat sheet membranes, poly(vinyl alcohol) and poly(ethylenevinyl alcohol) hollow fibre membranes as well as Sepharose 4B . The resulting poly(ethyleneimine)-immobilized adsorbers were used for removal of E . coli derived endotoxin from buffers and bovine serum albumin solutions . The efficiency of poly(ethyleneimine) proved to be constant over a wide pH range, including phosphate buffered saline . The performance depended upon the matrix type employed: endotoxin clearance factors varied from 100 to 120,000 in protein-free solutions and 40 to 33,000 in solutions of bovine serum albumin using 6000 EU/ml as feed concentration . The best adsorber was the flat sheet membrane-immobilized poly(ethyleneimine), followed by the hollow fibre-immobilized poly(ethyleneimine) and poly(ethyleneimine)-Sepharose . The factors influencing endotoxin clearance were the mass transport (convective systems were superior to the diffusive system), the chemical composition and the surface structure of the underlying matrix.

Protein Eng, 1998 Mar, 11(3), 233 - 41
Construction of mono- and bivalent human single-chain Fv fragments against the D antigen in the Rh blood group: multimerization effect on cell agglutination and application to blood typing; Furuta M et al.; An expression system for mono- and bivalent single-chain Fv fragments (scFv) of a human antibody against D antigen in the Rh blood group system was established in Escherichia coli . The cDNA encoding the Fv fragment of the anti-D monoclonal antibody D10 was cloned using the polymerase chain reaction and expressed in E.coli by fusing with a peptide tag link in the C-terminus of the light chain variable region . The scFv fragment expressed by the bacteria produced specific agglutination of human D positive red cells in the presence of an anti-peptide tag antibody . Flow cytometric analysis clearly indicated that the bacterially prepared scFv showed high specificity and affinity for D antigen, which was identical with that of the parental IgG . In order to construct bivalent D10 scFv for use in direct cell agglutination, the scFv was fused with a dimeric protein, bacterial alkaline phosphatase (BAP) . The fusion protein produced significant agglutination of human red blood cells with D antigen, confirming that the bacterially expressed fusion protein is a functional bivalent antibody fragment . Specific agglutination of D positive red cells by D10 scFv-BAP was enhanced in the presence of anti-BAP antibody, suggesting that further multimerization of scFv led to highly efficient cell agglutination . By grafting BAP enzymatic activity into the scFv fragment (enzyme-linked scFv), blood typing could conveniently be performed . These results indicate that bacterially expressed scFv and scFv-BAP would be of practical use in blood typing . The system reported here could also be applied to the examination of other cell surface antigens and cell agglutination.

Protein Eng, 1998 Mar, 11(3), 219 - 24
Chimeric small subunit inhibitors of mammalian ribonucleotide reductase: a dual function for the R2 C-terminus?
Hamann CS, Lentainge S, Li LS, Salem JS, Yang FD, Cooperman BS.
Here we report on the formation and activity of complexes between the large subunit (mR1) dimer of mouse ribonucleotide reductase (mRR) and small subunit chimeric dimers (cR2) derived from Escherichia coli and mouse small subunits . cR2 subunits were constructed by substituting mouse C-terminal gene sequences, coding for either 7 or 33 amino acid residues, for the corresponding E.coli R2 (eR2) sequences, with the remainder of the gene corresponding to eR2 . The purified cR2s contained the micro-oxo bridged diferric center and tyrosine radical necessary for reductase activity, although the radical signal was broadened compared with wild-type eR2 . Neither chimera formed an active complex with mR1, but each was a competitive inhibitor, with respect to mR2, of mRR activity . The inhibition constants for both chimeras were similar, and were sevenfold higher than the dissociation constant of mR2 dimer to mR1 dimer (0.24 +/- 0.02 microM) . Analysis of inhibition data showed that chimeric R2 subunits bind to mammalian R1 with a 1:1 (R1:R2) stoichiometry and permit the inference that both C-termini in a cR2 dimer bind to the two sites per mR1 dimer . The lack of enzymatic activity in the mR1-cR2 complex is attributed to perturbation or elimination of interactions linking the tyrosine radical/dinuclear iron center and the C-terminus within R2.

Protein Eng, 1998 Mar, 11(3), 205 - 12
Synthesis and characterization of a stable analog of the phosphorylated form of the chemotaxis protein CheY; Silversmith RE et al.; The bacterial chemotaxis protein CheY is activated in vivo by the covalent phosphorylation of a single aspartate residue at position 57 . However, this phosphate linkage is unstable (t1/2 approximately 20 s at room temperature), thereby precluding many biochemical analyses . Here we present a synthetic scheme to prepare an analog of CheY-phosphate (Che Y-P) with chemical stability of the phosphate linkage enhanced by several orders of magnitude relative to the native protein . Starting with CheY D57C, a site-specific mutant of CheY with a unique cysteine residue in place of the aspartate at position 57, two sequential disulfide exchange reactions were performed to form the final product 'CheY D57C-SPO3' with a thiophosphate moiety covalently bonded to the protein in a disulfide linkage . Mass spectral analysis showed that the desired analog was present at 70-80% of the total protein . The disulfide linkage had a t1/2 of 8 days at 4 degrees C . Biochemical characterization of CheY D57C-SPO3 included assessment of conformational properties using tryptophan fluorescence, evaluation of metal binding properties and measurement of binding interactions with the chemotaxis proteins CheZ and FliM . Despite possessing a phosphoryl group at a nearly identical location as native CheY-phosphate, the analog was unable to emulate CheY-phosphate function, thereby supporting the idea that there are very precise geometric requirements for successful CheY activation.

Arch Immunol Ther Exp (Warsz), 1998, 46(2), 105 - 11
Interferon and tumor necrosis factor production during endotoxemia in sheep; Bieniek K et al.; In humans endotoxemia has often been associated with the development of adult respiratory distress syndrome (ARDS) . Sheep have an abundant population of pulmonary intravascular macrophages, therefore they are a popular animal model for ARDS . In this study we characterized the temporal sequence and duration of the release of two cytokines: tumor necrosis factor (TNF) and interferon (IFN) and evaluated the effect of lipopolysaccharide (LPS) dose . Rectal temperature and white blood cell (WBC) count were also measured . Twenty four adult sheep were given E . coli endotoxin at a dose of 0 (saline solution) 0.05, 0.1 and 1.0 microg/kg of body weight by intravenous (i.v.) bolus . In all groups, TNF-alpha was produced earlier (3-4.5 h) after injection than IFN (4-5 h) . No correlation between increased rectal temperature, the magnitude of leukopenia and time course of both cytokines production was observed . No straight relationship between LPS dose and the titer of cytokines was seen, but lower doses of LPS-induced delayed cytokine response in comparison to the dose 1 microg/kg of LPS . As IFN, present in the circulation of sheep, was mainly alpha/beta type, the role of this class of IFN in endotoxemia is discussed.

FEBS Lett, 1998 May 1, 427(1), 64 - 8
Reconstitution of F1-ATPase activity from Escherichia coli subunits alpha, beta and subunit gamma tagged with six histidine residues at the C-terminus; Ekuni A et al.; An engineered gamma subunit of Escherichia coli F1-ATPase with extra 14 and 20 amino acid residues at the N- and C-termini (His-tag gamma), respectively, was overproduced in E . coli and purified . Six histidines are included in the C-terminal extension . The reconstituted F1 containing alpha, beta, and His-tagged gamma exhibited sixty percent of the wild-type ATPase activity . The reconstituted alphabeta His-tag gamma complex was subjected to affinity chromatography with nickel-nitrilotriacetic acid (Ni-NTA) agarose resin . ATPase activity was eluted specifically with imidazole . These results implied that the tag sequence protruded to the surface of the complex and did not seriously impair the activity . The reconstituted alphabeta His-tag gamma complex, even after its binding to the resin, exhibited ATPase activity suggesting that the gamma subunit, when fixed to a solid phase, may rotate the alphabeta complex . This system may provide a new approach for analysis of the rotation mechanisms in F1-ATPase.

FEBS Lett, 1998 May 1, 427(1), 51 - 4
Characterization of random-sequence proteins displayed on the surface of Escherichia coli RNase HI; Doi N et al.; In a previous study, random-sequence proteins of 120-130 amino acid residues were inserted into the surface loop region of the enzyme, Escherichia coli RNase HI {Doi et al . (1997) FEBS Lett . 402, 177-1801 . Here we established that the RNase H activity of the insertion mutants is correlated with their secondary structure contents evaluated by circular dichroism measurement at 222 nm . The random-sequence insert of a mutant enzyme possessing relatively high RNase H activity was detached from the RNase HI scaffold, and its characterization indicated that the random-sequence protein maintains its secondary structure after separation from the scaffold . Thus, the structural features of random-sequence proteins were suggested to be monitored by measuring the activity of the scaffold enzyme into which these proteins have been inserted.

Mol Gen Genet, 1998 Apr, 258(1-2), 123 - 32
Use of the ARG7 gene as an insertional mutagen to clone PHON24, a gene required for derepressible neutral phosphatase activity in Chlamydomonas reinhardtii; Adam M et al.; In Chlamydomonas reinhardtii, transforming DNA apparently integrates at random locations in the nuclear genome and generates a high number of mutants by gene inactivation . Twenty-four phoN mutants lacking the derepressible neutral (DN) phosphatase activity were isolated following transformation of the cw15arg7 strain with plasmid pASL harbouring the ARG7 gene encoding argininosuccinate lyase . In all mutants resulting from the transformation with linearised pASL, a functional ARG7 copy was found to be closely linked to a phoN mutation but additional ARG7 copies were present elsewhere in the genome . Of the 13 mutants submitted to allelism analysis, four were allelic or tightly linked to the previously isolated MNNG-induced phoN mutants (phoN2, phoN3, phoN24), the remaining mutants were distributed among seven additional loci . To learn more about the function of the genes involved in DN phosphatase production, we cloned PHON24 by plasmid rescue and screening of a wild-type genomic library . One clone complemented the phoN24 mutation in cotransformation experiments, as did several subcloned fragments . In all phoN24+ transformants, the DN phosphatase activity was 2-3 times lower than in the wild-type strain but about 10 times higher than in the untransformed control . In wild-type, PHON24 transcript accumulation was independent of inorganic phosphate deficiency . The transcripts were present in the MNNG-induced phoN24 mutant but were lacking in the two insertional phoN24 mutants . Insertional mutagenesis has thus permitted the isolation of novel mutants which were missing after induction with a chemical mutagen and the cloning of a gene which is probably involved in the regulation of the DN phosphatase.

Mol Gen Genet, 1998 Apr, 258(1-2), 69 - 77
The O6-methylguanine-DNA methyltransferase from the hyperthermophilic archaeon Pyrococcus sp . KOD1: a thermostable repair enzyme; Leclere MM et al.; The enzyme O6-methylguanine-DNA methyltransferase (MGMT) is the most common form of cellular defense against the biological effects of O6-methylguanine (O6-MeG) in DNA . Based on PCR amplification using primers derived from conserved amino acid sequences of MGMTs from 11 species, we isolated the DNA region coding for MGMT from the hyperthermophilic archaeon Pyrococcus sp . KOD1 . The MGMT gene from KOD1 (mgtk) comprises 522 nucleotides, encoding 174 amino acid residues; its product shows considerable similarity to the corresponding mammalian, yeast and bacterial enzymes, especially around putative methyl acceptor sites . Phylogenetic analysis of MGMTs showed that archaeal MGMTs were grouped with their bacterial counterparts . The location of the MGMT gene on the KOD1 chromosome was also determined . The cloned KOD1 MGMT gene was overexpressed using the T7 RNA polymerase expression system, and the recombinant protein was purified by ammonium sulfate fractionation, heat treatment, ion-exchange chromatography and gel filtration chromatography . The purified recombinant protein was assayed for its enzyme activity by monitoring transfer of {3H}methyl groups from the substrate DNA to the MGMT protein; the activity was found to be stable at 90 degrees C for at least 30 min . When the mgtk gene was placed under the control of the lac promoter and expressed in the methyltransferase-deficient Escherichia coli strain KT233 (delta ada, delta ogt) cells, a MGMT was produced . The enzyme was functional in vivo and complemented the mutant phenotype, making the cells resistant to the cytotoxic properties of the alkylating agent N-methyl-N'-nitro-N-nitrosoguanidine.

Vet Immunol Immunopathol, 1998 Feb 27, 61(2-4), 265 - 77
Quantitative detection of porcine interferon-gamma in response to mitogen, superantigen and recall viral antigen; Mateu de Antonio E et al.; Five monoclonal antibodies (mAbs) specific for porcine interferon-gamma (PoIFN-gamma) were isolated and utilized to develop a PoIFN-gamma sandwich ELISA . Specific reactivity of each mAb with E . coli derived recombinant PoIFN-gamma, but not with rPoIL-2 or rPolL-10, was confirmed in an indirect ELISA and in Western blots . Competitive ELISAs showed that mAbs P2A4 and P2C11 bound an epitope which was not recognized by mAbs P2G10, P1B7 or P2F6 . The latter three mAbs were able to neutralize the ability of natural and recombinant PoIFN-gamma to induce the de novo expression of class II MHC antigens on porcine endothelial cells . To simplify the detection of biologically active porcine IFN-gamma, a sandwich ELISA was developed using the mAb P2G10 as a capture antibody and mAb P2C11 as the detecting reagent . The sensitivity of the assay for PolFN-gamma ranged from 1 to 50 ng/ml . Peripheral blood mononuclear cells (PBMC) from all pigs tested produced IFN-gamma when stimulated with either mitogen (PHA) or superantigen (SEB) . In contrast, only PBMC obtained from pigs which had previously been vaccinated against PrV produced IFN-gamma in response to stimulation with this virus . Interestingly, cultures with the highest lymphoproliferative response did not necessarily have the highest level of IFN-gamma production.Furthermore, for recall viral antigen, the lymphoproliferative response decreased with time after immunization, whereas the IFN-gamma response increased . Thus, measurement of IFN-gamma production appears to be a good indicator of anti-viral immunological memory.

Nippon Koshu Eisei Zasshi, 1998 Feb, 45(2), 129 - 41
{Maximum likelihood estimation of date of infection in an outbreak of diarrhea due to contaminated foods assuming lognormal distribution for the incubation period}; Tango T; This paper proposes a new method for estimating the date of infection in an outbreak of diarrhea caused by eating foods contaminated by some sort of foodborne pathogens such as Escherichia coli O-157:H7 . Simple methods for calculating maximum likelihood estimator and confidence interval are proposed assuming (1) a common date of infection and (2) the incubation period has a log-normal distribution . Further, this paper shows that all three methods proposed so far for estimating the date of infection under the same assumptions are theoretically inappropriate or computationally imprecise . The proposed methods are illustrated with data from three outbreaks of Escherichia coli O-157:H7 and outbreak due to Escherichia coli O-118:H2 in Japan.

Microbiology, 1998 May, 144 ( Pt 5), 1417 - 22
Spacing and orientation requirements of GcvA-binding sites 3 and 2 and the Lrp-binding region for gcvT::lacZ expression in Escherichia coli; Stauffer LT et al.; Both GcvA and Lrp are required for normal regulation of the gcv operon . Moving the GcvA-binding sites 3 and 2 and the Lrp-binding region either closer to, or further away from, the gcv promoter by approximately one helical turn of DNA resulted in a less than twofold decrease in glycine-mediated activation or inosine-mediated repression of a gcvT::IacZ fusion . Moving these sites approximately two helical turns of DNA away from the gcv promoter resulted in a further loss of both activation and repression; moving these sites approximately three helical turns of DNA from the gcv promoter resulted in an essentially complete loss of both glycine-mediated activation and inosine-mediated repression . However, when these sites were moved by approximately 1.5 and 2.5 helical turns of DNA away from the gcv promoter, there was a complete loss of both glycine-mediated activation and inosine-mediated repression of the gcvT::IacZ fusion . The flexibility in the absolute distance of the GcvA- and Lrp-binding sites relative to the gcv promoter, but strict orientation dependence of these sites is consistent with a possible protein-protein interaction of either GcvA, Lrp, or both of these proteins with RNA polymerase . Because of the location of these target sites relative to the gcv promoter, it is also likely that DNA looping is required for this mechanism of regulation.

Microbiology, 1998 May, 144 ( Pt 5), 1309 - 17
Fused nucleoids resegregate faster than cell elongation in Escherichia coli pbpB(Ts) filaments after release from chloramphenicol inhibition; Van Helvoort JM et al.; The course of nucleoid movement during and upon release from protein synthesis inhibition by chloramphenicol in filaments of Escherichia coli pbpB(Ts) was analysed . Cells were grown at 42 degrees C in glucose minimal medium for two mass doublings and were treated with chloramphenicol to generate fusion (coalescence) of the nucleoids . Upon release from protein synthesis inhibition, the large distance between the border of the fused nucleoids and the cell poles immediately decreased, before full recovery of the rates of mass growth and length increase at 30 degrees C . This indicates that nucleoids can reoccupy the DNA-free cell ends independently of cell elongation . During filamentation at 42 degrees C, the pbpB cells established initial constrictions at midcell and at one-quarter and three-quarter positions . Nevertheless, divisions only started 75 min after chloramphenicol removal at 30 degrees C, when most nucleoids had moved back into the vacated cell ends . No 'guillotine-like' constrictions at the site of the nucleoids occurred . This suggests that segregating nucleoids postpone division recovery at previously established sites . The results are discussed in the light of a working model for transcription/translation-mediated chromosome segregation and nucleoid occlusion of cell division.

Microbiology, 1998 May, 144 ( Pt 5), 1205 - 11
Characterization of a haemolysin from Mycobacterium tuberculosis with homology to a virulence factor of Serpulina hyodysenteriae; Wren BW et al.; Scrutiny of sequence data from the Mycobacterium leprae genome sequencing project identified the presence of a gene encoding a 268-amino-acid polypeptide which is highly similar to a pore-forming haemolysin/cytotoxin virulence determinant, TlyA, from the swine pathogen Serpulina hyodysenteriae . Using degenerate oligonucleotide primers based on the TlyA sequences, the Mycobacterium tuberculosis homologue was amplified and this product was used to obtain the clone and sequence a 2.5 kb fragment containing the whole M . tuberculosis tlyA gene . tlyA encodes a 267-amino-acid protein with a predicted molecular mass of 28 kDa . TlyA homologues were identified by PCR in M . leprae, Mycobacterium avium and Mycobacterium bovis BCG, but appeared absent in Mycobacterium smegmatis, Mycobacterium vaccae, Mycobacterium kansasii, Mycobacterium chelonae and Mycobacterium phlei . The M . tuberculosis gene appeared to be the first gene in an operon containing at least two other genes . Introduction of the M . tuberculosis tlyA gene into M . smegmatis using a mycobacterial shuttle expression plasmid converted non-haemolytic cells into those exhibiting significant haemolytic activity . Similarly, inducible haemolytic activity was observed in sonicated bacteria when tlyA was expressed as a His6-tagged fusion protein in Escherichia coli . tlyA mRNA was detected in both M . tuberculosis and M . bovis BCG using RT-PCR, confirming that this gene is expressed in organisms cultured in vitro.

Int J Biochem Cell Biol, 1998 Mar, 30(3), 369 - 78
Heterologous expression of human transketolase; Schenk G et al.; Transketolase belongs to the family of thiamin diphosphate dependent enzymes . The aim of this study was to establish a bacterial expression system for human transketolase in order to investigate the functional characteristics of mammalian transketolases . The level of recombinant human enzyme expressed in Escherichia coli was modest . Purification of recombinant transketolase and separation from the E . coli enzyme has been greatly simplified by means of a non-cleavable hexa-histidine tag . The highest specific activity was 13.5 U/mg and the K(m) values were 0.27 +/- 0.02 and 0.51 +/- 0.05 mM for the substrates D-xylulose 5-phosphate and D-ribose 5-phosphate, respectively . Binding of cofactors to the apoenzyme showed the expected hysteresis . Without preincubation, the K(m) values for thiamin diphosphate and for Mg2+ were, respectively, 4.1 +/- 0.8 and 2.5 +/- 0.4 microM, but after 1 h of preincubation these values were 85 +/- 16 nM and 0.74 +/- 0.23 microM . The kinetic constants are similar to those of the native enzyme purified from human erythrocytes . Despite the modest expression level the reported system is well suited to a variety of functional studies.

Mol Gen Mikrobiol Virusol, 1998, (2), 29 - 32
{Isolation and cloning of gene for hepatitis delta antigen . The use of recombinant antigen for serodiagnosis of delta infection}; Ryzhova EV et al.; Hepatitis delta virus antigen was isolated from a patient by reverse transcription and polymerase chain reaction . Gel-purified cDNA was cloned in E . coli expression vectors . High expression of the recombinant HDAg in bacteria was observed . The minor and major forms of HDV antigens were simultaneously expressed in bacterial strains carrying SupE mutation . A laboratory method is developed for detecting anti-HD in patients' blood . It is based on the use of minor and major forms of recombinant HDV antigen immobilized on the same membrane filter . The method can be used for creating an original highly specific test system.

Gene, 1998 Jun 8, 212(2), 221 - 8
PCR isolation of catechol 2,3-dioxygenase gene fragments from environmental samples and their assembly into functional genes; Okuta A et al.; A method was developed to isolate central segments of catechol 2, 3-dioxygenase (C23O) genes from environmental samples and to insert these C23O gene segments into nahH (the structural gene for C23O encoded by catabolic plasmid NAH7) by replacing the corresponding nahH sequence with the isolated segments . To PCR-amplify the central C23O gene segments, a pair of degenerate primers was designed from amino acid sequences conserved among C23Os . Using these primers, central regions of the C23O genes were amplified from DNA isolated from a mixed culture of phenol-degrading or crude oil-degrading bacteria . Both the 5' and 3' regions of nahH were also PCR-amplified by using appropriate primers . These three PCR products, the 5'-nahH and 3'-nahH segments and the central C23O gene segments, were mixed and PCR-amplified again . Since the primers for the amplification of the central C23O gene segments were designed so that the 20 nucleotides at both ends of the segments are identical to the 3' end of the 5'-nahH segment and the 5' end of the 3'-nahH segment, respectively, the central C23O gene segments could anneal to both the 5'- and 3'-nahH segments . After the second PCR, hybrid C23O genes in the form of (5'-nahH segment-central C23O gene segment-3'-nahH segment) were amplified to full length . The resulting products were cloned into a vector and used to transform Escherichia coli . This method enabled divergent C23O sequences to be readily isolated, and more than 90% of the hybrid plasmids expressed C23O activity . Thus, the present method is useful to create, without isolating bacteria, a library of functional hybrid genes.

Nucleic Acids Res, 1998 Jun 15, 26(12), 2917 - 22
Mitochondrial targeting of human DNA glycosylases for repair of oxidative DNA damage; Takao M et al.; Oxidative damage to mitochondrial DNA has been implicated in human degenerative diseases and aging . Although removal of oxidative lesions from mitochondrial DNA occurs, the responsible DNA repair enzymes are poorly understood . By expressing the epitope-tagged proteins in COS-7 cells, we examined subcellular localizations of gene products of human DNA glycosylases: hOGG1, hMYH and hNTH1 . A gene encoding for hOGG1 which excises 7,8-dihydro-8-oxoguanine (8-oxoG) from DNA generates four isoforms by alternative splicing (types 1a, 1b, 1c and 2) . Three tagged isoforms (types 1b, 1c and 2) were localized in the mitochondria . Type 1a protein, which exclusively contains a putative nuclear localization signal, was sorted to the nucleus and lesser amount to the mitochondria . hMYH, a human homolog gene product of Escherichia coli mutY was mainly transported into the mitochondria . hNTH1 protein excising several pyrimidine lesions was transported into both the nucleus and mitochondria . In contrast to the three DNA glycosylases, translocation of the human major AP endonuclease (hAPE) into the mitochondria was hardly observed in COS-7 cells . These results suggest that the previously observed removal of oxidative base lesions in mitochondrial DNA is initiated by the above DNA glycosylases.

Am J Physiol, 1998 Jun, 274(6 Pt 1), E992 - 7
Endotoxin-induced alteration in the expression of leptin and beta3-adrenergic receptor in adipose tissue; Berkowitz DE et al.; Cytokines, such as tumor necrosis factor (TNF) and interleukin-6, may contribute to the anorexia and cachexia of infection, cancer, and AIDS . The present study tests the hypothesis that endotoxin alters the expression of two key fat cell proteins, leptin and beta3-adrenergic receptor (beta3-AR), through a mechanism involving TNF-alpha . Increasing doses of Escherichia coli endotoxin (lipopolysaccharide, LPS) resulted in dose-dependent elevations of plasma leptin (maximal response approximately 7-fold, half-maximal effective dose of approximately 16 microg/100 g body wt) and white fat leptin mRNA in C3/HeOUJ mice . LPS also produced a large decrease in adipose tissue beta3-AR mRNA and a parallel reduction in beta-agonist-induced activation of adenylyl cyclase . Changes in plasma leptin and beta3-AR mRNA were preceded by an approximately threefold increase in white fat TNF mRNA . TNF administration resulted in changes similar to those seen with LPS . We conclude that endotoxemia results in an induction of leptin mRNA and a decrease in beta3-AR mRNA in adipose tissue, an effect that may be mediated by alterations in TNF-alpha.

Am J Physiol, 1998 Jun, 274(6 Pt 1), C1686 - 98
Inflammatory cytokines (IL-1alpha, TNF-alpha) and LPS modulate the Ca2+ signaling pathway in osteoblasts; Tam VK et al.; Locally derived growth factors and cytokines in bone play a crucial role in the regulation of bone remodeling, i.e., bone formation and bone resorption processes . We studied the effect of interleukin (IL)-1alpha, tumor necrosis factor (TNF)-alpha, and Escherichia coli lipopolysaccharide (LPS) on the hormone-activated Ca2+ message system in the osteoblastic cell line UMR-106 and in osteoblastic cultures derived from neonatal rat calvariae . In both cell preparations, IL-1alpha, TNF-alpha, and LPS did not alter basal intracellular Ca2+ concentration ({Ca2+}i) but attenuated Ca2+ transients evoked by parathyroid hormone (PTH) and PGE2 in a dose (1-100 ng/ml)- and time (8-24 h)-dependent fashion . The cytokines modulated hormonally induced Ca2+ influx (estimated by using Mn2+ as a surrogate for Ca2+) as well as Ca2+ mobilization from intracellular stores . The latter was linked to suppressed production of hormonally induced inositol 1,4,5-trisphosphate . The effect of cytokines on {Ca2+}i was abolished by the tyrosine kinase inhibitor herbimycin A (50 ng/ml) . The cytokine's effect was, however, independent of nitric oxide (NO) production, since NO donors (sodium nitroprusside) as well as permeable cGMP analogs augment, rather than attenuate, hormonally induced Ca2+ transients in osteoblasts . Given the stimulatory role of cytokines on NO production in osteoblasts, the disparate effects of cytokines and NO on the Ca2+ signaling pathway may serve an autocrine/paracrine mechanism for modulating the effect of calciotropic hormones on bone metabolism.

J Biol Chem, 1998 May 29, 273(22), 13925 - 32
DNA topoisomerase I from Mycobacterium smegmatis . An enzyme with distinct features; Bhaduri T et al.; A type I topoisomerase has been purified to homogeneity from Mycobacterium smegmatis . It is the largest single subunit enzyme of this class having molecular mass of 110 kDa . The enzyme is Mg2+ dependent and can relax negatively supercoiled DNA, catenate, and knot single-stranded DNA, thus having typical properties of type I topoisomerases . Furthermore, the enzyme makes single-stranded nicks and the 5'-phosphoryl end of the nicked DNA gets covalently linked with a tyrosine residue of the enzyme . However, M . smegmatis enzyme shows some distinctive features from the prototype Escherichia coli topoisomerase I . The enzyme is relatively stable at higher temperatures and not inhibited by spermidine . It apparently does not contain any bound Zn2+ and on modification of cysteine residues retains the activity, suggesting the absence of the zinc-finger motif in DNA binding . Partially purified Mycobacterium tuberculosis topoisomerase I exhibits very similar properties with respect to size, stability, and reaction characteristics . Sequence comparison of topoisomerase I from E . coli and M . tuberculosis shows the absence of zinc-finger motifs in mycobacterial enzyme . Using a two-substrate assay system, we demonstrate that the enzyme acts processively at low ionic strength and switches over to distributive mode at high Mg2+ concentration . Significantly, the enzyme activity is stimulated by single strand DNA-binding protein . There is a potential to exploit the characteristics of the enzyme to develop it as a molecular target against mycobacterial infections.

J Biol Chem, 1998 May 29, 273(22), 13898 - 904
Requirements for the translocation of elongation-arrested, ribosome-associated OmpA across the plasma membrane of Escherichia coli; Behrmann M et al.; An oligodeoxynucleotide-dependent method to generate nascent polypeptide chains was adopted for use in a cell-free translation system prepared from Escherichia coli . In this way, NH2-terminal pOmpA fragments of distinct sizes were synthesized . Because most of these pOmpA fragments could be covalently linked to puromycin, precipitated with cetyltrimethylammonium bromide, and were enriched by sedimentation, they represent a population of elongation-arrested, ribosome-associated nascent chains . Translocation of these nascent pOmpA chains into inside-out membrane vesicles of E . coli required SecA and (depending on size) SecB . Whereas their translocation was strictly dependent on the H+-motive force of the vesicles, no indication for the involvement of the bacterial signal recognition particle was obtained . SecA and SecB, although required for translocation, did not mediate binding of the ribosome-associated pOmpA to membrane vesicles . However, SecA and SecB cotranslationally associated with nascent pOmpA, since they could be co-isolated with the ribosome-associated nascent chains and as such catalyzed translocation subsequent to the release of the ribosome . These results indicate that in E . coli, SecA also functionally interacts with preproteins before they are targeted to the translocase of the plasma membrane.

J Biol Chem, 1998 May 29, 273(22), 13669 - 74
The Rho-deamidating cytotoxic necrotizing factor 1 from Escherichia coli possesses transglutaminase activity . Cysteine 866 and histidine 881 are essential for enzyme activity; Schmidt G et al.; Recently, it has been reported that cytotoxic necrotizing factor 1 (CNF1) from Escherichia coli induces formation of stress fibers by deamidation of glutamine 63 of RhoA (Schmidt, G., Sehr, P., Wilm, M., Selzer, J., Mann, M., and Aktories, K . (1997) Nature 387, 725-729); Flatau, G., Lemichez, E., Gauthier, M., Chardin, P., Paris, S., Fiorentini, C., and Boquet, P . (1997) Nature 387, 729-733) . By using mass spectrometric analysis, we show now that the toxin transfers ethylenediamine, putrescine, and dansylcadaverine specifically onto glutamine 63 of RhoA . RhoA was also a substrate for guinea pig liver transglutaminase, which modified not only glutamine 63, but also glutamine residues at positions 52 and 136 . Treatment of the fully active N-terminal fragment of CNF1 (amino acid residues 709-1014) with iodoacetamide inhibited both deamidation and transglutamination activities . Moreover, exchange of cysteine 866 with serine blocked the enzyme activity of the N-terminal CNF1 fragment . In addition, we identified histidine 881 to be essential for the enzyme activity of CNF1 . The data indicate that CNF1 shares a catalytic dyad of cysteine and histidine residues with eukaryotic transglutaminases and cysteine proteases.

Biochem Biophys Res Commun, 1998 May 19, 246(2), 453 - 6
Effect of gabexate mesylate (FOY), a drug for serine proteinase-mediated diseases, on the nitric oxide pathway; Colasanti M et al.; Considering the structural similarity between gabexate mesylate (FOY), a drug for serine proteinase-mediated diseases, and L-arginine, the effect of gabexate mesylate on the nitric oxide (NO) pathway has been investigated . Gabexate mesylate inhibits competitively constitutive and inducible NO synthase (cNOS and iNOS, respectively), with Ki values of 1.0 x 10(-4) M and 5.0 x 10(-3) M, respectively, at pH 7.4 and 37.0 degrees C . However, gabexate mesylate is not an NO precursor . Moreover, like other NOS inhibitors, gabexate mesylate increases iNOS mRNA expression in rat C6 glioma cells, as induced by E . coli lipopolysaccharide plus interferon-gamma . Finally, gabexate mesylate inhibits dose-dependently nitrite production (i.e . NO release) in rat C6 glioma cells, as induced by E . coli lipopolysaccharide plus interferon-gamma . Thus, this drug should be administered under careful control, since enzyme inhibition may occur also in vivo.

Biochem Biophys Res Commun, 1998 May 19, 246(2), 342 - 6
Isolation and crystallization of functionally competent Escherichia coli peptide deformylase forms containing either iron or nickel in the active site; Groche D et al.; Three metallo forms of peptide deformylase (PDF, EC 3.5.1.31) of Escherichia coli were prepared and crystallized (space group C2, diffraction limit 1.9 A) for initiating the X-ray structure determination of the metal center in correlation with the catalytic functionality of this enzyme . The native Fe2+ containing enzyme species was directly isolated from overproducing bacteria by using catalase as a buffer additive, which stabilizes the catalytic activity against oxidative destruction . The Ni2+ containing form, which is oxygen-insensitive, was obtained by metal exchange with free Ni2+ and found to be catalytically equally effective (kcat/KM = 10(5) M-1 s-1 for N-formyl-Met-Ala) . The Zn2+ form, prepared from the apoenzyme or by displacement of bound Ni2+ by free Zn2+, proved virtually inactive.

J Radiat Res (Tokyo), 1998 Mar, 39(1), 21 - 33
3-Amino-1,4-dimethyl-5H-pyrido{4,3-b}indole (Trp-P-1) sensitizes mammalian cells to UV radiation by causing the S-phase arrest, not by inhibiting the repair of DNA damage as observed in Escherichia coli; Mori T et al.; 3-Amino-1,4-dimethyl-5H-pyrido{4,3-b}indole (Trp-P-1) is known to be a mutagen and carcinogen isolated from the charred parts of cooked foods . We found previously that Trp-P-1 enhanced UV-induced lethality and mutation frequency in Escherichia coli by inhibiting the repair of UV-induced DNA damage . In the present study, we investigated whether Trp-P-1 also potentiated UV-induced lethality by inhibiting the repair of UV-induced DNA damage in cultured mammalian cells . As a result, Trp-P-1 enhanced UV-induced lethality in a concentration-dependent manner in human and Chinese hamster cells . However, Trp-P-1 was unable to inhibit the repair of the two major photolesions (cyclobutane pyrimidine dimers and (6-4)photoproducts) from the genomic DNA, as determined using monoclonal antibodies specific for each type of lesion . On the other hand, Trp-P-1, with or without UV irradiation, efficiently suppressed DNA synthesis and arrested cells in S phase in concentration- and time-dependent manners, as measured by pulse-labelling with 3H-thymidine and flow cytometry . Thus, the present results suggest that Trp-P-1 potentiates UV-induced lethality in cultured mammalian cells by causing the S-phase arrest, not by inhibiting the repair of UV-induced DNA damage as observed in Escherichia coli.

Biochemistry, 1998 May 19, 37(20), 7478 - 86
Physicochemical and biochemical properties of 2',5'-linked RNA and 2',5'-RNA:3',5'-RNA "hybrid" duplexes; Wasner M et al.; In recent publications, oligonucleotides joined by 2',5'-linkages were found to bind to complementary single-stranded RNA but to bind weakly, or not at all, to single-stranded DNA {e.g., P . A . Giannaris and M . J . Damha (1993) Nucleic Acids Res . 21, 4742-4749} . In this work, the biochemical and physicochemical properties of 2',5'-linked oligoribonucleotides containing mixed sequences of the four nucleobases (A, G, C, and U) were evaluated . CD spectra of RNA:2', 5'-RNA duplexes were compared with the spectra of DNA:DNA, RNA:RNA, and DNA:RNA duplexes of the same base sequence . The CD results indicated that the RNA:2',5'-RNA duplex structure more closely resembles the structure of the RNA:DNA hybrid, being more A-form than B-form in character . The melting temperature (Tm) values of the backbone-modified duplexes were compared with the Tm values of the unmodified duplexes . The order of thermal stability was RNA:RNA > DNA:DNA approximately RNA:DNA approximately DNA:RNA > RNA:2',5'-RNA > 2',5'-RNA:2',5'-RNA >> DNA:2',5'-RNA (undetected) . RNA:2',5'-RNA duplexes are not substrates of the enzyme RNase H (Escherichia coli, or HIV-1 reverse transcriptase), but they can inhibit the RNase H-mediated cleavage of a natural DNA:RNA substrate . Structural models that are consistent with the selective association properties of 2',5'-linked oligonucleotides are discussed.

Biochemistry, 1998 May 19, 37(20), 7431 - 43
Thermodynamics of the interaction of the Escherichia coli regulatory protein TyrR with DNA studied by fluorescence spectroscopy; Bailey MF et al.; Fluorescence quenching was used to study the site-specific binding of the Escherichia coli regulatory protein TyrR to a fluoresceinated oligonucleotide (9F30A/30B) containing a TyrR binding site . The equilibrium constant for the interaction (KL) was measured as a function of temperature and salt concentration in the presence and absence of ATPgammaS, a specific ligand for TyrR . Fluorescence titrations yielded a KL value of 1.20 x 10(7) M-1 at 20 degrees C, which was independent of the acceptor (9F30A/30B) concentration in the range 5-500 nM, indicating that the system exhibits true equilibrium binding . Clarke and Glew analysis of the temperature dependence of binding revealed a linear dependence of R ln KL on temperature in the absence of ATPgammaS . The thermodynamic parameters obtained at 20 degrees C (theta) were = -35.73 kJ mol-1, = 57.41 kJ mol-1, and = 93.14 kJ mol-1 . Saturating levels of ATPgammaS (200 microM) strengthened binding at all temperatures and resulted in a nonlinear dependence of Rln KL on temperature . The thermodynamic parameters characterizing binding under these conditions were = -39.32 kJ mol-1, = 37.16 kJ mol-1, = 76.40 kJ mol-1, and = -1.03 kJ mol-1 K-1 . Several conclusions were drawn from these data . First, binding is entropically driven at 20 degrees C in both the presence and absence of ATPgammaS . This can partly be accounted for by counterions released from the DNA upon TyrR binding; in the absence of ATPgammaS and divalent cations, the TyrR-9F30A/30B interaction results in the release of two to three potassium ions . Second, the more favorable value, and hence tighter binding observed in the presence of ATPgammaS, is primarily due to a decrease in (-20.3 kJ mol-1), which overcomes an unfavorable decrease in (-16.7 kJ mol-1) . Third, the negative value obtained in the presence of ATPgammaS indicates that the binding of ATPgammaS favors a conformational change in TyrR upon binding to 9F30A/30B, yielding a more stable complex.

Biochemistry, 1998 May 19, 37(20), 7363 - 70
Molybdenum cofactor properties and {Fe-S} cluster coordination in Escherichia coli nitrate reductase A: investigation by site-directed mutagenesis of the conserved his-50 residue in the NarG subunit; Magalon A et al.; Most of the molybdoenzymes contain, in the amino-terminal region of their catalytic subunits, a conserved Cys group that in some cases binds an {Fe-S} cluster . In dissimilatory nitrate reductases, the first Cys residue of this motif is replaced by a conserved His residue . Site-directed mutagenesis of this residue (His-50) was performed on the NarG subunit from Escherichia coli nitrate reductase A . The results obtained by EPR spectroscopy enable us to exclude the implication of this residue in {Fe-S} binding . Additionally, we showed that the His-50 residue does not coordinate the molybdenum atom, but its substitution by Cys or Ser introduces a perturbation of the hydrogen bonding network around the molybdenum cofactor . From potentiometric studies, it is proposed that the high-pH and the low-pH forms of the Mo(V) are both involved during the redox turnover of the enzyme . Perturbation of the Mo(V) pKV value might be responsible for the low activity reported in the His-50-Cys mutant enzyme . A catalytic model is proposed in which the protonation/deprotonation of the Mo(V) species is an essential step . Thus, one of the two protons involved in the catalytic cycle could be the one coupled to the molybdenum atom in the dissimilatory nitrate reductase of E . coli.

Biochemistry, 1998 May 19, 37(20), 7351 - 62
Effect of iron-sulfur cluster environment in modulating the thermodynamic properties and biological function of ferredoxin from Pyrococcus furiosus; Brereton PS et al.; The ferredoxin (7.5 kDa) of the hyperthermophilic archaeon, Pyrococcus furiosus, contains a single {4Fe-4S}1+,2+ cluster that is coordinated by three Cys and one Asp residue rather than the expected four Cys . The role of this Asp residue was investigated using a series of mutants, D14X, where X = C, S, H, N, V, and Y, prepared by heterologous gene expression in Escherichia coli . While the recombinant form of the wild-type and the D14S and D14C mutants contained a {4Fe-4S}1+,2+ cluster, the D14V, D14H, D14Y, and D14N proteins contained a {3Fe-4S}0,+ center, as determined by visible spectroscopy and electrochemistry . The redox potentials (at pH 7.0, 23 degrees C) of the D14C and D14S mutants were decreased by 58 and 133 mV, respectively, compared to those of the wild-type 4Fe-ferredoxin (Em -368 mV), while those of the 3Fe-protein mutants (including the 3Fe-form of the D14S, generated by chemical oxidation) were between 15 and 118 mV more positive than that of wild-type 3Fe-form (obtained by chemical oxidation, Em -203 mV) . The reduction potentials of all of the 3Fe-forms, except the D14S mutant, showed a pH response over the range 3.0-10.0 with a pK of 3.3-4.7, and this was assigned to cluster protonation . The D14H mutant and the wild-type 3Fe-proteins showed an additional pK (both at 5.9) assumed to arise from protonation of the amino acid side chain . With the 4Fe-proteins, there was no dramatic change in the potentials of the wild-type or D14C form, while the pH response of the D14S mutant (pK 4.75) was ascribed to protonation of the serinate . While the ferredoxin variants exhibited a range of thermal stabilities (measured at 80 degrees C, pH 2.5), none of them showed any temperature-dependent transitions (0-80 degrees C) in their reduction potentials, and there was no correlation between the calculated DeltaS degrees' values and the absorbance maximum, reduction potential, or hydrophobicity of residue 14 . In contrast, there was a linear correlation between the DeltaH degrees' value and reduction potential . Kinetic analyses were carried out at 80 degrees C using the ferredoxin as either an electron acceptor to pyruvate oxidoreductase (POR) or as an electron donor to ferredoxin:NADP oxidoreductase (FNOR, both from P . furiosus) . The data showed that the reduction potential of the ferredoxin, rather than cluster type or the nature of the residue at position 14, appears to be the predominant factor in determining efficiency of electron transfer in both systems . However, compared to all the variants, the reduction potential of WT Fd makes it the most appropriate protein to both accept electrons from POR and donate them to FNOR.

Biochemistry, 1998 May 19, 37(20), 7328 - 39
Chimeric beta-EF3-alpha hemoglobin (Psi): energetics of subunit interaction and ligand binding; Kiger L et al.; Among the numerous strategies to design an oxygen carrier, we outline in this work the engineering of a stable homotetrameric hemoglobin, expressed in Escherichia coli . The chimeric globin (Psi) consists of the first 79 residues of human beta globin (corresponding to positions NA1 --> EF3) followed by the final 67 residues of human alpha globin (corresponding to positions EF3 --> HC3) . The molecular mass for beta-EF3-alpha (Psi) globin was measured using mass spectrometry to be equal to its theoretical value: 15782 Da . Correct protein folding was assessed by UV/visible and fluorescence spectra . The subunit interaction free energies were estimated by HPLC gel filtration . In the cyanometHb species, the formation of the dimer-tetramer interface is 2 kcal/mol less favorable (Delta G = -7 kcal/mol) than that of Hb A (Delta G = -9 kcal/mol), whereas the dimer-monomer interface is tightly assembled (< -10 kcal/mol) as for the Hb A alpha 1 beta 1 interface . In contrast to Hb A, oxygen binding to Psi Hb is not cooperative . The free energy for binding four oxygen molecules to a Psi homotetramer is slightly increased compared to a Hb A heterotetramer (-28 and -27.5 kcal/4 mol of O2, respectively) . The intrinsic O2 affinity of a Psi homodimer is 6-fold higher than that of a homotetramer . The linkage scheme between dimer-tetramer subunit assembly and the noncooperative oxygenation of Psi Hb predicts a stabilization of the tetramer after ligand release . This protein mechanism resembles that of Hb A for which the dimers exhibit a 100-fold higher O2 affinity relative to deoxy tetramers (which are 10(5) times more stable than oxy tetramers) . A potent allosteric effector of Hb A, RSR4, binds to Psi Hb tetramers, inducing a decrease of the overall O2 affinity . Since RSR4 interacts specifically with two binding sites of deoxy Hb A, we propose that the chimeric tetramer folding is close to this native structure.

Biochemistry, 1998 May 19, 37(20), 7313 - 20
UTP is a cofactor for the DNA strand exchange reaction performed by the RecA protein of Escherichia coli; Bianco PR et al.; The RecA protein of Escherichia coli is required for homologous genetic recombination and induction of the SOS regulon . In order for RecA protein to function in these two roles, a nucleoside triphosphate cofactor, usually ATP or dATP, is required . We have examined the ability of UTP to substitute for (r,d)ATP as nucleoside triphosphate cofactor . We have found that although UTP is hydrolyzed by RecA protein in the presence of long DNA molecules, it is not hydrolyzed in reactions in which the cofactors are oligodeoxyribonucleotides less than approximately 50 nt in length . We show that UTP can efficiently substitute for ATP as nucleoside triphosphate cofactor for the DNA strand exchange reaction in vitro . The RecA1332 protein (Cys129 --> Met), which was originally shown to be defective for homologous recombination in vivo, is able to perform DNA strand exchange in vitro with ATP, but is unable to do so with UTP . These results suggest that UTP may be a cofactor for DNA strand exchange in vivo . The inability of RecA protein to hydrolyze UTP with oligodeoxyribonucleotides as cofactor and the ability of RecA to utilize UTP as cofactor in DNA strand exchange suggest a separation of the functions of RecA protein into those that require exclusively ATP and those which can utilize additional nucleoside triphosphate cofactors.

Biochemistry, 1998 May 19, 37(20), 7089 - 95
A structural role for glutamine 214 in human thymidylate synthase; Steadman DJ et al.; Studies of the crystal structures of thymidylate synthase (TS) have revealed that a kink is present in beta-sheets that form the core of the enzyme . The beta-kink is proposed to serve as a "hinge" during conformational changes that occur in the enzyme after ligand binding at the active site . A residue in one of the beta-bulges that form the kink, glutamine at position 214 of human TS, is highly conserved in all TSs and is postulated to interact with nucleotide ligands that bind at the active site . To examine the role of this residue, glutamine at position 214 was replaced by residues that differ in volume, hydrophobicity, electrostatic charge, and hydrogen bonding potential . Genetic complementation studies utilizing a TS-deficient bacterial strain revealed that residues with large side chain volumes or that are prohibited in beta-bulges created loss of function proteins . Kinetic studies indicated that residue hydrophobicity is not correlated with catalytic activity . Residues that are predicted to alter the charge at position 214 created enzymes with kcat/Km values at least 10(3) lower than those of the wild type . Kinetic and ligand binding studies indicated that residue 214 is involved in nucleotide binding; however, hydrogen bonding potential does not contribute significantly to nucleotide binding energy . The data are consistent with the hypothesis that residue 214 is involved in maintaining the enzyme in a conformation that facilitates nucleotide binding and catalysis.

Biochemistry (Mosc), 1998 Mar, 63(3), 253 - 8
The role of active site flexibility in enzyme catalysis; Tsou CL; It has been shown in this and other laboratories that during the unfolding of a number of enzymes inactivation generally precedes global unfolding of the enzyme molecule, leading to the suggestion that enzyme active sites are usually more "fragile" and more easily "perturbed" than the molecule as a whole and are therefore conformationally more flexible than the rest of the molecule . However, the role of active site flexibility in enzyme catalysis still remains to be explored . In the induced fit hypothesis originally proposed by Koshland, the presence of the substrate induces a conformational change at the active site so as to fit with the structure of the substrate . By X-ray crystallographic structural analysis of E . coli dihydrofolate reductase liganded with cofactors and substrates, Sawaya and Kraut showed the enzyme in different conformational states indeed while complexed with different ligands, suggesting that the enzyme molecule passes through different conformational states through the whole process of catalysis . Muscle lactate dehydrogenase can be stabilized either in concentrated ammonium sulfate or by cross-linking with glutaraldehyde together with a decrease in enzyme activity which can be restored to the original level in dilute guanidine hydrochloride possibly by increased flexibility at the active site . It is known that a number of enzymes can be activated by chaotropic agents such as urea or guanidine hydrochloride . The activation of dihydrofolate reductase by either urea or guanidine hydrochloride is accompanied by an increase in susceptibility to proteolysis . Isolation of the tryptic peptides of the activated enzyme and sequence analysis allowed identification of the sites of proteolysis to be at or near the active site of the enzyme, indicating an opening up of the active site conformation in the activated state . All the above indicate that active site flexibility plays an important role in enzyme catalysis . It is possible that during the catalytic cycle, the enzyme molecule passes through different stages and each stage requires the molecule to be in a different conformation, especially at the active site . Rapid transition between the different conformational states, and hence the flexibility of the active site, is therefore mandatory for the maximal expression of enzyme activity.

Anal Chem, 1998 May 15, 70(10), 2019 - 24
Detection of conformational changes in an immobilized protein using surface plasmon resonance; Sota H et al.; Utilizing surface plasmon resonance (SPR), we have developed novel methodology for the detection of conformational change(s) in immobilized proteins . A genetically altered E . coli dihydrofolate reductase (DHFR-ASC) was attached to a carboxymethyldextran matrix layer covering the sensor surface of an SPR biosensor through a disulfide linkage at the engineered protein's C-terminus . The DHFR-ASC-immobilized surface exhibited a larger response to acid treatment than reference surfaces lacking immobilized proteins . The SPR signal of the tethered protein and the molar ellipticity of DHFR-ASC in solution responded similarly to pH changes, consistent with the interpretation that changes in the SPR signal reflect conformational changes occurring during acid denaturation . A pH shift observed between the SPR signal and ellipticity changes may reflect a difference between surface and bulk pH . The tethered protein sensor surface was stable to repeated acid treatment using solutions in the pH range of 0.12-7.80 and yielded reproducible measurements . This is the first demonstration of detection of conformational changes in an immobilized protein using an SPR biosensor . This technique has potential for developing novel sensors and/or switching devices in response to protein conformational changes.

J Biotechnol, 1998 Feb 26, 60(3), 183 - 93
Recombinant flounder growth hormone from Escherichia coli: overexpression, efficient recovery, and growth-promoting effect on juvenile flounder by oral administration; Jeh HS et al.; An efficient production method for recombinant flounder growth hormone (r-fGH) from Escherichia coli was developed and the biological activity of purified r-fGH was examined using juvenile flounder . The use of bicistronic construction in the expression plasmid resulted in the production of over 40% of the E . coli cellular protein as r-fGH . The r-fGH was recovered from cell lysates following inclusion body washing, solubilization and refolding in sodium dodecylsulfate (SDS) solution, and removal of contaminated proteins with secondary butanol treatment . The SDS content in purified r-fGH solution was adjusted to appropriate levels by diafiltration . More than 47% of the r-fGH was recovered from the E . coli cell lysates and the purity of recovered r-fGH was 98% . The oral administration of purified r-fGH to juvenile flounder, once a week for 4 weeks at a dosage of 40 micrograms r-fGH g-1 fish body weight, resulted in significant increases both in weight and length . These results of overexpression, simple purification with high recovery yield and purity, and good growth-promoting activity of the r-fGH suggest that the production scheme described in this study is useful for the potential application of r-fGH in fish farming.

Mol Genet Metab, 1998 Mar, 63(3), 168 - 75
Cloning and characterization of the mouse and rat type II arginase genes; Iyer RK et al.; Two forms of arginase, both catalyzing the hydrolysis of arginine to ornithine and urea, are found in animals ranging from amphibians to mammals . In humans, inherited deficiency of hepatic or type I arginase results in hyperargininemia, a syndrome characterized by periodic episodes of hyperammonemia, spasticity, and neurological deterioration . In these patients, a second extrahepatic or type II arginase activity is significantly increased, an induction that may partially compensate for the lack of AI activity and apparently mitigates some of the clinical effects of the condition . Cloning and characterization of the human AII cDNA was recently accomplished . The cloning, sequencing, and partial characterization of the mouse and rat AII cDNAs are reported herein . The DNA sequences predicted polypeptides of 354 amino acids, including a N-terminal mitochondrial import signal . Sequence homology to the human type II arginase, arginase activity data, and immunoprecipitation with an anti-AII antibody confirm the identity of these cloned genes as rodent extrahepatic type II arginases.

Nature, 1998 May 21, 393(6682), 280 - 4
Protein-primed RNA synthesis by purified poliovirus RNA polymerase; Paul AV et al.; A small protein, VPg, is covalently linked to the 5' end of the plus-stranded poliovirus genomic RNA . Poliovirus messenger RNA, identical in nucleotide sequence to genomic RNA, is not capped at its 5' end by the methylated structure that is common to most eukaryotic mRNAs . These discoveries presented two problems . First, as cap structures are usually required for translation of mRNA into protein, how does this uncapped viral RNA act as a template for translation? Second, what is the function of VPg? The identification of the internal ribosomal-entry site, which allows the entry of ribosomes into viral mRNA independently of the 5' mRNA end, has solved the first conundrum . Here we describe the resolution of the second problem . VPg is linked to the genomic RNA through the 5'-terminal uridylic acid of the RNA . We show that VPg can be uridylylated by the poliovirus RNA polymerase 3Dpol . Uridylylated VPg can then prime the transcription of polyadenylate RNA by 3Dpol to produce VPg-linked poly(U) . Initiation of transcription of the poliovirus genome from the polyadenylated 3' end therefore depends on VPg.

J Pediatr Surg, 1998 May, 33(5), 778 - 80
Retroperitoneal necrotizing fasciitis in a 4-year-old girl; Paya K et al.; Necrotizing fasciitis is a rare but serious condition with a poor prognosis both in adults and in children . Retroperitoneal localization is mostly associated with fatal outcome . Early diagnosis, extensive and repeated surgical debridement, and use of antibiotics are necessary . Herein the authors report on a 4-year-old girl in whom retroperitoneal necrotizing fasciitis developed after she suffered from pyelonephritis . In this case, the outcome was favorable because of early surgical intervention, confirming the diagnosis.

FEBS Lett, 1998 May 8, 427(2), 203 - 8
Features of replicative senescence induced by direct addition of antennapedia-p16INK4A fusion protein to human diploid fibroblasts; Kato D et al.; The p16INK4A cyclin-dependent kinase (Cdk) inhibitor is now recognized as a major tumor suppressor that is inactivated by a variety of mechanisms in a wide range of human cancers . It is also implicated in the mechanisms underlying replicative senescence since p16INK4A RNA and protein accumulate as cells approach their proscribed limit of population doublings in tissue culture . To obtain further evidence of its role in senescence, we have sought ways of overexpressing p16INK4A in primary human diploid fibroblasts (HDF) . To circumvent the low transfection efficiency of primary cells we have exploited a recombinant form of the full-length p16INK4A protein fused to a 16 amino acid peptide from the Drosophila antennapedia protein . This peptide has the capacity to cross both cytoplasmic and nuclear membranes allowing the direct introduction of the active protein to primary cells . Here, we show that antennapedia-tagged wild-type p16INK4A protein, but not a functionally compromised tumor-specific variant, causes G1 arrest in early passage HDFs by inhibiting the phosphorylation of the retinoblastoma protein . Significantly, the arrested cells display several phenotypic features that are considered characteristic of senescent cells . These data support a role for p16INK4A in replicative senescence and raise the possibility of using the antennapedia-tagged protein therapeutically.

Vaccine, 1998 Jan-Feb, 16(2-3), 255 - 60
Safety and immunogenicity of an oral inactivated enterotoxigenic Escherichia coli vaccine; Jertborn M et al.; The safety and immunogenicity of two different lots, 001 and 003, of an oral inactivated enterotoxigenic Escherichia coli (ETEC) vaccine consisting of a mixture of formalin-killed whole bacteria expressing the most prevalent colonisation factor antigens, i.e . CFA/I, CFA/II and CFA/IV and recombinantly produced cholera B subunit (rCTB) have been evaluated in Swedish volunteers . Neither of the two vaccine preparations, containing different CFA/II-expressing strains but otherwise identical, gave rise to any significant side-effects . Mucosal immune responses, as reflected in antibody-secreting cell (ASC) responses in peripheral blood, were studied after two doses of vaccine and did not differ significantly for the two vaccine lots . Vaccination induced high levels of CTB-specific IgA ASCs in 100% of the volunteers, and significant IgA ASC responses (9- to 36-fold) were noted in 84% of them against CFA/I, in 87% against CFA/II subcomponents CS1-CS3 and in 91% against CFA/IV subfactors CS4 and/or CS5 . The frequencies and magnitudes of CFA IgA ASC responses were similar when giving the vaccine with a 1 or 2 week interval . Results from serological analyses showed that the local IgA responses against CFAs are only infrequently associated with serum antibody titre rises.

Vaccine, 1998 Jan-Feb, 16(2-3), 248 - 54
Mutants of Escherichia coli heat-labile enterotoxin as an adjuvant for nasal influenza vaccine; Komase K et al.; The effectiveness and safety of known mutants of Escherichia coli heat-labile enterotoxin (LT) as an adjuvant for nasal influenza vaccine were examined . Six mutants, called LT7K (Arg to Lys), LT61F (Ser to Phe), LT112K (Glu to Lys), LT118E (Gly to Glu), LT146E (Arg to Glu) and LT192G (Arg to Gly) were constructed by the replacement of one amino acid at one position of the A1 subunit to another using site-directed mutagenesis . All mutants were confirmed to be less toxic than wild-type LT when analyzed using Y-1 adrenal cells in vitro . When influenza vaccine was administered intranasally with LT7K and LT192G, BALB/c mice developed high levels of serum and local antibodies to the HA molecules . The adjuvant activity of these mutant LTs corresponded to that of wild-type LT when 1 microgram of these mutant LTs (or wild-type LT) was coadministered with the vaccine . From the point of view of safety, LT7K was considered to be the most potent mucosal adjuvant and was examined in more detail . The adjuvant activity of the mutant was lowered more rapidly with a decrease in dose than was that of wild-type LT . The low level of adjuvant of a relatively small amount of LT7K was heightened by adding LTB to the mutant LT . These results suggest that LT7K supplemented with LTB can be used as a less toxic, effective adjuvant for nasal influenza vaccine.

Vaccine, 1998 Jan-Feb, 16(2-3), 240 - 7
Immunisation with recombinant AMA-1 protects mice against infection with Plasmodium chabaudi; Anders RF et al.; The Plasmodium merozoite surface antigen apical membrane antigen-1 (AMA-1) has previously been shown to provide partial protection to Saimiri and rhesus monkeys immunised with recombinant Plasmodium fragile or parasite-derived Plasmodium knowlesi AMA-1, respectively . In the study reported here we have used the Plasmodium chabaudi/mouse model system to extend our pre-clinical assessment of an AMA-1 vaccine . We describe here the expression of the full-length Plasmodium chabaudi adami AMA-1 and the P . chabaudi adami AMA-1 ectodomain using both baculovirus and Escherichia coli . The ectodomain expressed in E . coli, which contained an N-terminal hexa-his tag, was purified by Ni-chelate chromatography and refolded in vitro in the presence of oxidised and reduced glutathione to generate intramolecular disulphide bonds . In a series of vaccine trials, in both inbred and outbred mice, highly significant protection was obtained by immunising with the refolded AMA-1 ectodomain . Protection was shown to correlate with antibody response and was dependent on intact disulphide bonds . Passive transfer of antibodies raised in rabbits against the refolded AMA-1 ectodomain was also protective . In view of this demonstration that E . coli expression of a soluble P . chabaudi AMA-1 domain can generate a vaccine that is effective in mice, we are pursuing a similar approach to generating a vaccine against P . falciparum for testing in human volunteers.

Vaccine, 1998 Jan, 16(1), 9 - 15
Epitope specificity of antibodies raised against enterotoxigenic Escherichia coli CFA/I fimbriae in mice immunized with naked DNA; Alves AM et al.; The cfaB gene, coding for the CFA/I fimbrial adhesin of enterotoxigenic Escherichia coli (ETEC), was cloned and expressed as a fusion peptide with the glycoprotein D (gD) from herpes simplex virus type 1 (HSV) in the pRE4 eukaryotic expression vector, resulting in the recombinant plasmid pRECFA . All BALB/c mice injected intramuscularly (i.m.) with a single dose (100 micrograms) of the purified plasmid developed antibodies against epitopes found on dissociated CFA/I subunits as well as other homologous ETEC fimbriae . Surface-exposed epitopes found on intact CFA/I fimbriae were also recognized by antibodies derived from DNA immunization, but they did not overlap with those generated with purified CFA/I fimbriae . None of the sera raised in mice immunizated with pRECFA were able to agglutinate bacterial cells or inhibit haemagglutination promoted by CFA/I bearing ETEC cells . These results show that pRECFA can elicit CFA/I-specific antibodies, which may have different epitope specificities and functional properties compared with those generated with purified bacterial protein.

Arch Biochem Biophys, 1998 May 15, 353(2), 337 - 48
Cloning, expression, and biochemical characterization of a functionally novel alpha class glutathione S-transferase with exceptional activity in the glutathione conjugation of (+)-anti-7,8-dihydroxy-9,10-oxy-7,8,9,10-tetrahydrobenzo(a)pyrene; Xia H et al.; The present study describes cDNA cloning, expression, and kinetic characterization of the two subunits of a murine alpha-class glutathione (GSH) S-transferase (GST) isoenzyme (previously designated as GST 9.5), which, unlike other alpha-class mammalian GSTs, is exceptionally efficient in the GSH conjugation of (+)-anti-7,8-dihydroxy-9,10-oxy-7,8,9,10-tetrahydrobenzo(a)pyrene {(+)-anti-BPDE} {X . Hu, S . K . Srivastava, H . Xia, Y . C . Awasthi, and S . V . Singh (1996) J . Biol . Chem . 271, 32684-32688} . The cDNAs for both subunits of GST 9.5 (GST 9.5-1 and GST 9.5-2) were cloned by RT-PCR . The deduced amino acid sequences of GST 9.5-1 and GST 9.5-2 clones were identical to those of mGSTA1 and mGSTA2, respectively . Both these subunits were expressed in Escherichia coli to determine the relationships between recombinant mGSTA1-1 and mGSTA2-2 and corresponding subunits of tissue-isolated GST 9.5 . The pI values of recombinant mGSTA1-1 and mGSTA2-2 (9.49 and 9.45, respectively) were similar to that of the tissue-isolated isoenzyme (pI 9.5) . The reverse-phase HPLC elution profiles and immunological cross-reactivities of recombinant mGSTA1-1 and mGSTA2-2 were also similar to those of the corresponding subunits of tissue-isolated GST 9.5 . The catalytic efficiency of recombinant mGSTA1-1 toward (+)-anti-BPDE, 131 mM-1.s-1, was approximately 9.5-to 655-fold higher compared with tissue-isolated mGSTP1-1, mGSTA3-3, mGSTM1-1, and mGSTA4-4 . Moreover, the catalytic efficiency of mGSTA1-1 toward (+)-anti-BPDE was about 3.3-fold higher compared with recombinant mGSTA2-2 . The mGSTA1 and/or mGSTA2 subunits were expressed to varying degrees in female A/J mouse tissues . For example, mGSTA1, but not mGSTA2, subunit expression was observed in the skin, which is a target organ for benzo(a)pyrene (BP)-induced cancer in mice . On the other hand, the expression of either mGSTA1 or mGSTA2 subunit could not be detected in the lung, which is another target organ for BP-induced cancer in mice . Interestingly, relatively large amounts of both mGSTA1 and mGSTA2 subunits were detected in the kidney . In conclusion, the results of the present study clearly indicate that the A1-type subunit of GST 9.5 is responsible for its exceptional catalytic efficiency in the GSH conjugation of (+)-anti-BPDE, which is the ultimate carcinogen of widespread environmental pollutant BP.

Arch Biochem Biophys, 1998 May 15, 353(2), 285 - 96
Cytochrome P450cam substrate specificity: relationship between structure and catalytic oxidation of alkylbenzenes; Sibbesen O et al.; The oxidation by cytochrome P450cam (CYP101) of ethylbenzene and a series of substrates derived from it by addition of one, two, three, or four carbon atoms has been examined . For each of the 18 substrates, the shift in spin state due to substrate binding, the extent of coupled turnover to give organic products and uncoupled turnover to give H2O2 and H2O, and the identities of the organic products have been determined . The same studies have been carried out with the T185L and T185F mutants of P450cam in which the active site volume is decreased . The results show that no detectable correlation exists between the observed spin state change and any other parameter studied . For substrates of equal size, coupled and uncoupled turnover vary widely but both are maximized when the phenyl ring bears one large alkyl substituent or a methyl ortho to the largest alkyl substituent . The presence of substituents in addition to these decreases activity . In the absence of other changes, coupled turnover is correlated with the size of the largest substituent, but no such correlation exists for uncoupled turnover . Decreasing the size of the active site cavity by a T185L mutation generally increases coupled turnover without altering the dependence on the alkyl group size . A T185F mutation causes too great an active site perturbation for structure-activity studies . Substrate oxidation occurs preferentially at 2 degrees or 3 degrees C-H bonds of the largest substituent or on the benzylic methyl ortho to it . Aromatic hydroxylation only competes with the oxidation of nonbenzylic 1 degree C-H bonds . The extent of coupled turnover is a function of substrate shape, substrate size, and cavity size, but still elusive parameters control the extent of uncoupled turnover.

Arch Microbiol, 1998 May, 169(5), 434 - 44
Molecular genetic evidence for extracytoplasmic localization of sulfur globules in Chromatium vinosum; Pattaragulwanit K et al.; Purple sulfur bacteria store sulfur as intracellular globules enclosed by a protein envelope . We cloned the genes sgpA, sgpB, and sgpC, which encode the three different proteins that constitute the sulfur globule envelope of Chromatium vinosum D (DSMZ 180(T)) . Southern hybridization analyses and nucleotide sequencing showed that these three genes are not clustered in the same operon . All three genes are preceded by sequences resembling sigma70-dependent promoters, and hairpin structures typical for rho-independent terminators are found immediately downstream of the translational stop codons of sgpA, sgpB, and sgpC . Insertional inactivation of sgpA in Chr . vinosum showed that the presence of only one of the homologous proteins SgpA and SgpB suffices for formation of intact sulfur globules . All three sgp genes encode translation products which - when compared to the isolated proteins - carry amino-terminal extensions . These extensions meet all requirements for typical signal peptides indicating an extracytoplasmic localization of the sulfur globule proteins . A fusion of the phoA gene to the sequence encoding the proposed signal peptide of sgpA led to high specific alkaline phosphatase activities in Escherichia coli, further supporting the envisaged targeting process . Together with electron microscopic evidence these results provide strong indication for an extracytoplasmic localization of the sulfur globules in Chr . vinosum and probably in other Chromatiaceae . Extracytoplasmic formation of stored sulfur could contribute to the transmembranous Deltap that drives ATP synthesis and reverse electron flow in Chr . vinosum.

Arch Microbiol, 1998 May, 169(5), 417 - 23
Characterization of HetR protein turnover in Anabaena sp . PCC 7120; Zhou R et al.; The hetR gene plays an important role in heterocyst development and pattern formation in heterocystous cyanobacteria . The hetR gene from Anabaena sp . PCC 7120 was overexpressed in Escherichia coli . Antibodies raised against the recombinant HetR protein (rHetR) were used to characterize metabolism of the HetR of Anabaena sp . PCC 7120 in vivo . HetR was present at a low level when Anabaena sp . PCC 7120 was grown in the presence of combined nitrogen . Shifting from nitrogen repletion conditions to nitrogen depletion conditions led to a two fold increase of HetR in total cell extracts, and most of HetR was located in heterocysts . The amount of HetR in total cellular extracts increased rapidly after shifting to nitrogen depletion conditions and reached a maximum level 3 h after the shift . Isoelectrofocusing electrophoresis revealed that the native HetR had a more acidic isoelectric point than did rHetR . After combined nitrogen was added to the nitrogen-depleted cultures, the degradation of HetR depended on culture conditions: before heterocysts were fully developed, HetR was rapidly degraded; after heterocysts were fully developed, HetR was degraded much more slowly . The distribution of HetR in other species of cyanobacteria was also studied.

Arch Microbiol, 1998 May, 169(5), 393 - 6
The Helicobacter felis ftsH gene encoding an ATP-dependent metalloprotease can replace the Escherichia coli homologue for growth and phage lambda lysogenization; Melchers K et al.; Cloning and sequencing of an approximately 6.0-kb chromosomal DNA fragment from Helicobacter felis revealed five complete open reading frames . The deduced amino acid sequence of one ORF exhibited sequence similarity to the FtsH protein, an ATP-dependent metalloprotease, from various bacterial species . The encoded protein consists of 638 amino acid residues with a molecular mass of 70.2 kDa . The hydropathy profile of the FtsH protein predicted two N-terminal transmembrane regions that were confirmed experimentally . Insertion of ftsH into a new versatile expression vector resulted in overexpression of FtsH protein in Escherichia coli . In addition, the E . coli ftsH gene could be replaced by the H . felis homologue to allow reduced growth and tenfold increased lysogenization by temperate phage lambda.

Biochemistry (Mosc), 1998 Feb, 63(2), 224 - 34
Comparative structural and immunochemical characterization of recombinant and natural cytochrome p450scc (CYPXIAI); Lepesheva GI et al.; Optimization of the conditions for heterologous expression of recombinant cytochrome P450scc in E . coli provided an expression level of about 420 nmoles of cytochrome P450scc per liter of bacterial culture . A new procedure for purification of recombinant protein in substrate-bound high-spin and substrate-free low-spin form is described . Highly purified electrophoretically homogeneous recombinant cytochrome P450scc contains 12.3 and 16.7 nmoles heme per mg protein for substrate-free and substrate-bound forms, respectively . The recombinant and natural cytochrome P450scc from bovine adrenocortical mitochondria were compared functionally and immunochemically . The dissociation constants for the complexes of cytochrome P450scc with cholesterol and adrenodoxin, the efficiency of enzymatic reduction in the reconstituted system (NADPH--adrenodoxin reductase--adrenodoxin), and cholesterol side-chain cleavage activity were determined . It was found that limited proteolysis of the recombinant cytochrome P450scc with trypsin forms two main fragments which are electrophoretically and immunochemically identical with the fragments F1 (29.8 kD) and F2 (26.6 kD) formed during proteolysis of bovine adrenocortical cytochrome P450scc . The quantitative values of the studied parameters are practically identical in natural and substrate-bound recombinant cytochrome P450scc, while there were great differences between substrate-bound and substrate-free forms of recombinant cytochrome P450scc both of functional (decrease of cholesterol side-chain cleavage activity, efficiency of enzymatic reduction in the reconstituted system, and affinity to adrenodoxin for substrate-free cytochrome P450scc) as well as structural (increase in accessibility to exogenous and endogenous proteolysis) character . The identity of the folding process for recombinant and natural proteins as well as the nature of a stabilizing and activating effect of cholesterol on cytochrome P450scc is discussed.

Cell Motil Cytoskeleton, 1998, 40(1), 71 - 86
FtsZ from Escherichia coli, Azotobacter vinelandii, and Thermotoga maritima--quantitation, GTP hydrolysis, and assembly; Lu C et al.; We have cloned the ftsZ genes from Thermotoga maritima and Azotobacter vinelandii and expressed the proteins (TmFtsZ and AzFtsZ) in Escherichia coli . We compared these proteins to E . coli FtsZ (EcFtsZ), and found that several remarkable features of their GTPase activities were similar for all three species, implying that these characteristics may be universal among FtsZs . Using a calibrated protein assay, we found that all three FtsZs bound 1 mole guanine nucleotide per mole FtsZ and hydrolyzed GTP at high rates (> 2 GTP per FtsZ per min) . All three required magnesium and a monovalent cation for GTP hydrolysis . Previous reports showed that EcFtsZ (and some other species) required potassium . We confirmed this specificity for EcFtsZ but found that potassium and sodium both worked for Az- and TmFtsZ . Specific GTPase activity had a striking dependence on FtsZ concentration: activity (per FtsZ molecule) was absent or low below 50 microg/ml, rose steeply from 50 to 300 microg/ml and plateaued at a constant high value above 300 microg/ml . This finding suggests that the active state requires a polymer that is assembled cooperatively at 50-300 microg/ml . A good candidate for the active polymer was visualized by negative stain electron microscopy--straight protofilaments and protofilament pairs were seen under all conditions with active GTPase . We suggest that the GTP hydrolysis of FtsZ may be coupled to assembly, as it is for tubulin, with hydrolysis occurring shortly after an FtsZ monomer associates onto a protofilament end . As a part of this study, we determined the concentration of EcFtsZ and TmFtsZ by quantitative amino acid analysis and used this to standardize the bicinchonic acid colorimetric assay . This is the first accurate determination of FtsZ concentration . Using this standard and quantitative Western blotting, we determined that the average E . coli cell has 15,000 molecules of FtsZ, at a concentration of 400 microg/ml . This is just above the plateau for full GTPase activity in vitro.

Anal Biochem, 1998 May 15, 259(1), 68 - 73
Strep-tag II affinity purification: an approach to study intermediates of metalloenzyme biosynthesis; Maier T et al.; Complex metalloenzymes (e.g., nitrogenase, hydrogenase, urease) are synthesized starting from the apoprotein via several intermediates by the action of accessory proteins . The isolation and biochemical characterization of such intermediates is hampered by their low abundance and their lability . Here we describe a technique for efficient single-step purification of a hydrogenase precursor under mild conditions using a N-terminal Strep-tag II affinity peptide and a novel StrepTactin Sepharose matrix . The tag was fused to the large subunit of {NiFe} hydrogenase 3 (HycE) of Escherichia coli . No significant influence of the affinity peptide on maturation or activity of the protein was observed when the modified gene was integrated into the chromosome by homologous recombination . A tagged nickel-free precursor form of HycE bound quantitatively to a recombinant StrepTactin Sepharose column . More than 90% pure subunit could be obtained after elution with desthiobiotin . The procedure was shown to be more efficient than purification by immobilized metal affinity chromatography using a N-terminal His-tag . General advantages of the novel Strep-tag II affinity purification especially for applications with metalloenzymes are discussed.

J Med Primatol, 1998 Feb, 27(1), 38 - 43
Secnidazole vs . paromomycin: comparative antiprotozoan treatment in captive primates; Gracenea M et al.; The antiprotozoan activity of secnidazole was studied in Cercocebus t . torquatus, Cercopithecus campbelli, Erythrocebus patas (Cercopithecidae), and Gorilla gorilla (Pongidae) compared with that of paromomycin in Cercocebus t . lunulatus (Cercopithecidae), E . patas, and G . gorilla (Pongidae) by coprological analysis . The antiprotozoan activity of both drugs depended on the parasite species and the host species . The drugs acted in a similar way on Entamoeba coli parasitising C . t . torquatus, and E . patas . This activity was different from that observed on I . buestchlii from the same host species . Nevertheless, E . coli parasitising cercopithecids and pongids responded to drugs differently.

Naunyn Schmiedebergs Arch Pharmacol, 1998 Apr, 357(4), 371 - 7
Effects of phosducin on the GTPase cycle of Go; Bauer PH et al.; The cytosolic phosphoprotein phosducin is an inhibitor of G-protein GTPase activity and G-protein-mediated signalling . Here we investigate the effects of phosducin on individual steps of the GTPase cycle of Go, and the role of the G-protein betagamma subunits in mediating these effects . Phosducin was expressed in E . coli and purified to apparent homogeneity . Phosducin inhibited the MAS-7-stimulated as well as basal steady-state GTPase activity of Go, but did not affect the GTP-hydrolytic step . It slowed the release of GDP from Go in the presence of high Mg2+ concentrations (25 mM), and enhanced GDP release at low Mg2+ concentrations (100 microM) . Likewise, phosducin inhibited basal GTPase activity at 25 mM Mg2+ and stimulated at 100 microM Mg2+ . All of these effects were lost following phosphorylation of phosducin by protein kinase A (PKA) . These observations are compatible with the hypothesis that phosducin antagonizes the influence of betagamma subunits on alpha(o) . Titration of the effects of phosducin on the GDP release and GTPase activity of Go and on the betagamma subunit-dependent ADP-ribosylation of alpha(o) by pertussis toxin indicated an apparent affinity of approximately 20 nM . We conclude that via high-affinity interactions with G-protein betagamma subunits phosducin decreases the proportion of active GTP-bound G-proteins by slowing GDP-release without affecting GTP-hydrolysis, and that thereby it inhibits G-protein-mediated signalling.

Acta Anat (Basel), 1997, 159(4), 218 - 21
Localization of beta-D-galactosidase activity in semithin epon sections of embryonic tissues using differential interference contrast optics; Poggi P et al.; In the present study we describe a method for the histochemical demonstration of beta-D-galactosidase activity on tissue sections processed for light microscopy at high resolution . 5-Bromo-indolyl-beta-D-galactopyranoside (Bluo-Gal) was utilized as an indigogenic method for the demonstration of Escherichia coli beta-D-galactosidase reporter gene activity whose expression was studied in a transgenic line where the enzyme, with a nuclear localization signal (nlacZ), is under the transcriptional control of a striated muscle-specific promoter . At the light-microscopic level, by using Differential Interference Contrast (DIC) optics, the reaction product was detected as precipitates in the form of fine birefringent crystals . These were located around and inside the nuclei of beta-gal-expressing cells . This simple method allows an easy and rapid identification of few or even one labeled cell(s) within large microscopic fields (whole embryos) and the labeled cell(s) can be evaluated both morphologically and quantitatively.

Protein Eng, 1998 Feb, 11(2), 143 - 52
Engineering the steroid-specificity of an anti-17beta-estradiol Fab by random mutagenesis and competitive phage panning; Saviranta P et al.; We have employed random mutagenesis and phage display to improve the steroid-specificity of an anti-17beta-estradiol Fab fragment . The VH domain was mutated using error-prone PCR; the mutation rate was controlled by adjusting the number of effective duplications . A phage library of 2 x 10(6) independent mutants was generated, each mutant containing on average 24 amino acid changes . We selected for decreased testosterone (TES) cross-reactivity by adding a large excess TES as a competitor to the panning reactions . After four panning rounds, the cross-reactivities of the individual mutant clones ranged from 19 to 4%, showing up to 20-fold improvement over the original value (78%) . Estradiol affinities were mainly unchanged . Sequencing of the VH regions revealed two hot spots, one located around Ser32 in CDR1 and the other around Thr52A in CDR2, while no mutations were found in CDR3 . Although most clones had multiple mutations, it was possible to deduce the residues relevant to the improved specificity by comparing the sequences and binding data of the mutants . We demonstrated that controlled error-prone PCR mutagenesis is a rapid method to identify such key residues, lending itself to the scanning of 'lead' positions for further mutagenesis by other methods.

Protein Eng, 1998 Feb, 11(2), 101 - 8
The role of Glu259 in Escherichia coli elongation factor Tu in ternary complex formation; Pedersen GN et al.; Determination of the crystal structure of the ternary complex formed between elongation factor Tu:GTP and aminoacylated tRNA revealed three regions of interaction between elongation factor Tu and tRNA . The structure indicates that the conserved glutamic acid at position 271 in Thermus aquaticus EF-Tu could be involved in the binding of the 3' CCA-Phe end of the aminoacylated tRNA . Therefore, the corresponding residue, Glu259, of Escherichia coli EF-Tu was mutated into alanine, aspartic acid, glutamine and tyrosine, in order to substantiate the crystallographic structural evidence and to obtain further knowledge of the importance of this residue . All of the mutated proteins showed nucleotide binding properties similar to the wild type . In addition the GTPase activities were similar to the wild type . The mutation of Glu259 to either alanine or aspartic acid resulted in a reduced strength of interaction with tRNA, while mutation to tyrosine abolished completely the interaction with tRNA . Finally, mutation to glutamine resulted in an elongation factor Tu variant behaving like the wild type . In conclusion, the environment around the site binding the CCA-Phe end of the tRNA is very restricted spatially and chemically so that only a residue with almost the same size and chemical properties as glutamic acid fulfils the requirements with regard to size, salt bridge-formation potential and maintenance of the backbone conformation at the 259 position.

Genes Cells, 1998 Feb, 3(2), 79 - 97
Characterization of RecA1332 in vivo and in vitro . A role for alpha-helix E as a liaison between the subunit-subunit interface and the DNA and ATP binding domains of RecA protein; Bianco PR et al.; BACKGROUND: The RecA protein of Escherichia coli is essential for homologous recombination and induction of the SOS response . RecA has three cysteines located at positions 90, 116 and 129 . Chemical modification of these residues abolishes ATP hydrolysis and repressor cleavage, and causes a reduction in the DNA strand exchange and DNA strand annealing activities . Several mutants at each of these positions were isolated and partially characterized . One of these, recA1332, replaces cysteine 129 with methionine . Although this is a relatively conservative mutation based on hydrophobicity, recA1332 was completely defective for DNA repair but the purified protein was active for ATPase in vitro . RESULTS: In vivo, strains containing this mutant allele were shown to be defective when assayed for all RecA-dependent activities . In vitro, RecA1332 protein possessed DNA-dependent ATP hydrolysis activity that showed an increased sensitivity to inhibition by monovalent cations, and whose k(cat) was reduced 3- to 12-fold . In addition, RecA1332 was unable to use oligodeoxyribonulceotides as ssDNA cofactors in the ATPase reaction . RecA1332 showed altered binding to single- and double-stranded DNA and, although it was able to perform DNA strand exchange, it was slowed in its ability to both form joint molecule intermediates and to convert these species to product . CONCLUSIONS: Our results are consistent with a defect in intermolecular interactions between RecA monomers . We propose that alpha-helix E (which includes C129M) is a liaison that connects the subunit-subunit interactions to DNA and ATP binding, thereby creating filament stability and cooperativity.

Acta Anaesthesiol Scand, 1998 May, 42(5), 558 - 64
The inflammatory cytokine response after autotransfusion of shed mediastinal blood; Schmidt H et al.; BACKGROUND: The inflammatory response in patients undergoing cardiac surgery with cardiopulmonary bypass is well known and increased levels of inflammatory cytokines have been shown . High levels of cytokines have been reported in blood drained from the surgical field . The present study aimed to elucidate whether autotransfusion of shed mediastinal blood in itself causes increased cytokine levels in coronary artery bypass graft (CABG) patients . METHODS: A prospective, randomized controlled study was performed in 23 patients having elective uncomplicated CABG . Autotransfusion of shed mediastinal blood was done every hour for 18 h in group I . In group II, the shed mediastinal blood was accumulated for 4 h in the cardiotomy reservoir and then autotransfused every hour for the next 14 h . Plasma levels of tumour necrosis factor-alpha (TNFalpha) and interleukin (IL)-1alpha, IL-1beta, IL-6 were measured . In vitro study of cytokine production was performed with or without stimulation (phytohaemagglutinin (PHA) and Escherichia coli (E . coli) lipopolysaccharide (LPS)) . RESULTS: We found high levels of IL-6 in the shed mediastinal blood . However, autotransfusion of shed mediastinal blood did not lead to increased level of cytokines (TNFalpha, IL-1alpha, IL-1beta and IL-6) in plasma in group I nor in group II . In vitro study showed activation of the leucocytes in the shed mediastinal blood with a significantly increased production of TNFalpha and IL-6 both in the stimulated and non-stimulated samples . CONCLUSION: Shed mediastinal blood contains high levels of IL-6 . However, autotransfusion of shed mediastinal does not cause measurable elevations in plasma levels of IL-6 . In vitro study shows that autotransfusion activates leucocytes, which may enhance production of inflammatory cytokines.

Acta Anaesthesiol Scand, 1998 May, 42(5), 536 - 44
The effects of nitric oxide inhalation on respiratory mechanics and gas exchange during endotoxaemia in the pig; Dahm PL et al.; BACKGROUND: In the adult respiratory distress syndrome, nitric oxide (NO) inhalation improves oxygenation through reducing ventilation-perfusion mismatching, but detailed information on the pulmonary effects of NO inhalation in septic shock is scarce . The present study investigated the effects of inhaled NO on alveolar dead space (Vdalv) and venous admixture as well as on respiratory system compliance (Crs) and respiratory system resistance (Rrs) in a porcine model of septic shock . Protective effects of NO are discussed . METHODS: Thirteen anaesthetised and ventilated pigs were given an infusion of endotoxin for an observation time of 220 min to induce acute lung injury (ALI) . In the NO-early group (n=6), an inhalation of 60 ppm NO was started simultaneously with the endotoxin infusion and continued for 190 min . In 7 control/NO-late animals, 60 ppm NO was administered for 30 min following 190 min of endotoxin infusion . Haemodynamics, single-breath CO2-, pressure-, and flow signals were recorded . RESULTS: Endotoxin induced haemoconcentration, pulmonary vasoconstriction, and a decrease in Crs, while venous admixture, Vdalv, and Rrs increased . In the NO-early group, the pulmonary vasoconstriction was attenuated, no increase in pulmonary venous admixture or in Vdalv was seen before cessation of NO, and the improvements in oxygenation outlasted the NO inhalation . In the control/NO-late group, the NO inhalation reversed the changes in dead space and venous admixture . NO had no effect on the changes in respiratory mechanics . CONCLUSION: In porcine ALI, 60 ppm NO diminishes pulmonary vasoconstriction and improves gas exchange by reducing pulmonary venous admixture and alveolar dead space, but does not prevent a fall in Crs . NO inhalation may help prevent long-lasting pulmonary failure.

Protein Sci, 1998 May, 7(5), 1245 - 9
Assembly and crystallization of the complex between the human T cell coreceptor CD8alpha homodimer and HLA-A2; Gao GF et al.; A strategy for overexpression in Escherichia coli of the extracellular immunoglobulin domain of human CD8alpha was devised using codon usage alterations in the 5' region of the gene, designed so as to prevent the formation of secondary structures in the mRNA . A fragment of CD8alpha, comprising residues 1-120 of the mature protein, excluding the signal peptide and the membrane-proximal stalk region, was recovered from bacterial inclusion bodies and refolded to produce a single species of homodimeric, soluble receptor . HLA-A2 heavy chain, beta2-microglobulin and a synthetic peptide antigen corresponding to the pol epitope from HIV-1 were also expressed in E . coli, refolded and purified . CD8alpha/HLA-A2 complexes were formed in solution and by co-crystallization with a stoichiometry of one CD8alpha alpha dimer to one HLA-A2-peptide unit.

Protein Sci, 1998 May, 7(5), 1233 - 44
Characterization of Escherichia coli thioredoxin variants mimicking the active-sites of other thiol/disulfide oxidoreductases; Mossner E et al.; Thiol/disulfide oxidoreductases like thioredoxin, glutaredoxin, DsbA, or protein disulfide isomerase (PDI) share the thioredoxin fold and a catalytic disulfide bond with the sequence Cys-Xaa-Xaa-Cys (Xaa corresponds to any amino acid) . Despite their structural similarities, the enzymes have very different redox properties, which is reflected by a 100,000-fold difference in the equilibrium constant (K(eq)) with glutathione between the most oxidizing member, DsbA, and the most reducing member, thioredoxin . Here we present a systematic study on a series of variants of thioredoxin from Escherichia coli, in which the Xaa-Xaa dipeptide was exchanged by that of glutaredoxin, PDI, and DsbA . Like the corresponding natural enzymes, all thioredoxin variants proved to be stronger oxidants than the wild-type, with the order wild-type < PDI-type < DsbA-type < glutaredoxin-type . The most oxidizing, glutaredoxin-like variant has a 420-fold decreased value of K(eq), corresponding to an increase in redox potential by 75 mV . While oxidized wild-type thioredoxin is more stable than the reduced form (delta deltaG(ox/red) = 16.9 kJ/mol), both redox forms have almost the same stability in the variants . The pH-dependence of the reactivity with the alkylating agent iodoacetamide proved to be the best method to determine the pKa value of thioredoxin's nucleophilic active-site thiol (Cys32) . A pKa of 7.1 was measured for Cys32 in the reduced wild-type . All variants showed a lowered pKa of Cys32, with the lowest value of 5.9 for the glutaredoxin-like variant . A correlation of redox potential and the Cys32 pKa value could be established on a quantitative level . However, the predicted correlation between the measured delta deltaG(ox/red) values and Cys32 pKa values was only qualitative.

Protein Sci, 1998 May, 7(5), 1221 - 32
Mutational analysis of the active site of indoleglycerol phosphate synthase from Escherichia coli; Darimont B et al.; Indoleglycerol phosphate synthase catalyzes the ring closure of 1-(2-carboxyphenylamino)-1-deoxyribulose 5'-phosphate to indoleglycerol phosphate, the fifth step in the pathway of tryptophan biosynthesis from chorismate . Because chemical synthesis of indole derivatives from arylamino ketones requires drastic solvent conditions, it is interesting by what mechanism the enzyme catalyzes the same condensation reaction . Seven invariant polar residues in the active site of the enzyme from Escherichia coli have been mutated directly or randomly, to identify the catalytically essential ones . A strain of E . coli suitable for selecting and classifying active mutants by functional complementation was constructed by precise deletion of the trpC gene from the genome . Judged by growth rates of transformants on selective media, mutants with either S58 or S60 replaced by alanine were indistinguishable from the wild-type, but R186 replaced by alanine was still partially active . Saturation random mutagenesis of individual codons showed that E53 was partially replaceable by aspartate and cysteine, whereas K114, E163, and N184 could not be replaced by any other residue . Partially active mutant proteins were purified and their steady-state kinetic and inhibitor binding constants determined . Their relative catalytic efficiencies paralleled their relative complementation efficiencies . These results are compatible with the location of the essential residues in the active site of the enzyme and support a chemically plausible catalytic mechanism . It involves two enzyme-bound intermediates and general acid-base catalysis by K114 and E163 with the support of E53 and N184.

Protein Sci, 1998 May, 7(5), 1195 - 200
Calorimetric analyses of the interaction between SecB and its ligands; Randall LL et al.; SecB is a chaperone in Escherichia coli dedicated to export of proteins from the cytoplasm to the periplasm and outer membrane . It functions to bind and deliver precursors of exported proteins to the translocation apparatus before they fold into their native structures, thus maintaining them in a competent state for translocation across the membrane . The natural ligands of SecB are precursor proteins containing leader sequences . There are numerous reports in the literature indicating that SecB does not specifically recognize the leader peptides . However, two published investigations have concluded that the leader peptide is the recognition element (Watanabe M, Blobel G . 1989 . Cell 58:685-705; Watanabe M, Blobel G . 1995 . Proc Natl Acad Sci USA 92:10133-10136) . In this work we use titration calorimetry to show that SecB binds two physiological ligands, which contain leader sequences, with no higher affinity than the same molecules lacking their leader sequences . Indeed, for one ligand the presence of the leader sequence reduces the affinity . Therefore, it can be concluded that the leader sequence provides no positive contribution to the binding energy.

Protein Sci, 1998 May, 7(5), 1186 - 94
Isolation and characterization of a DnaJ-like protein in rats: the C-terminal 10-kDa domain of hsc70 is not essential for stimulating the ATP-hydrolytic activity of hsc70 by a DnaJ-like protein; Leng CH et al.; A DnaJ-like protein, RDJ1, was isolated from a rat brain cDNA library . The protein is predicted to have 397 amino acid residues and shares 99% identity to that of HDJ2, a human DnaJ-like protein . RDJ1 was also shown to rescue the temperature-sensitive lethality of a strain containing a mutated cytosolic DnaJ in yeast, ydj1-151 . Fragments containing the J-domain of RDJ1 either with or without the G/F motif were expressed in Escherichia coli . The purified proteins stimulated the ATPase activity of hsc70 and of the 60-kDa N-terminal fragment of hsc70 . These results imply that RDJ1 can interact with the N-terminal 60-kDa fragment of hsc70 to activate ATP hydrolysis by hsc70.

J Biochem (Tokyo), 1998 Jun, 123(6), 1088 - 96
Identification of genes affecting lycopene formation in Escherichia coli transformed with carotenoid biosynthetic genes: candidates for early genes in isoprenoid biosynthesis; Hemmi H et al.; Although isopentenyl diphosphate is a precursor of isoprenoids in Escherichia coli, the genes and enzymes involved in its biosynthesis have not been identified . Thus, we tried to isolate E . coli mutants deficient in the biosynthesis and their complementary genes by use of an artificial phenotypic screening system employing three carotenoid biosynthetic genes, crtE, crtB, and crtI . Cells were mutagenized with ethylmethanesulfonate, then transformed with a plasmid for expression of the carotenogenic genes . Mutants deficient in biosynthesis of isopentenyl diphosphate were expected to form white colonies, because they are unable to produce enough lycopene, whereas wild-type cells form red colonies . Among large numbers of red colonies, we identified 117 white colonies . Next, we transformed each mutant with an E . coli genomic library . Twenty-nine complementary genes that restore red color of host colonies were isolated . A homology search and further complementation study using subcloned genes revealed that the true complementary genes encode isopentenyl diphosphate isomerase, subunits of ATP synthase, enzymes of the Krebs cycle, some aldehyde dehydrogenases, phosphate acetyltransferase, and enzymes which relate to the biosynthesis of ubiquinones and menaquinones . Two unknown genes were also found, designated elb1 and 2, which may be involved in the early steps of isoprenoid biosynthesis.

Proc Natl Acad Sci U S A, 1998 May 26, 95(11), 6442 - 7
Restriction-modification gene complexes as selfish gene entities: roles of a regulatory system in their establishment, maintenance, and apoptotic mutual exclusion; Nakayama Y et al.; We have reported some type II restriction-modification (RM) gene complexes on plasmids resist displacement by an incompatible plasmid through postsegregational host killing . Such selfish behavior may have contributed to the spread and maintenance of RM systems . Here we analyze the role of regulatory genes (C), often found linked to RM gene complexes, in their interaction with the host and the other RM gene complexes . We identified the C gene of EcoRV as a positive regulator of restriction . A C mutation eliminated postsegregational killing by EcoRV . The C system has been proposed to allow establishment of RM systems in new hosts by delaying the appearance of restriction activity . Consistent with this proposal, bacteria preexpressing ecoRVC were transformed at a reduced efficiency by plasmids carrying the EcoRV RM gene complex . Cells carrying the BamHI RM gene complex were transformed at a reduced efficiency by a plasmid carrying a PvuII RM gene complex, which shares the same C specificity . The reduction most likely was caused by chromosome cleavage at unmodified PvuII sites by prematurely expressed PvuII restriction enzyme . Therefore, association of the C genes of the same specificity with RM gene complexes of different sequence specificities can confer on a resident RM gene complex the capacity to abort establishment of a second, incoming RM gene complex . This phenomenon, termed "apoptotic mutual exclusion," is reminiscent of suicidal defense against virus infection programmed by other selfish elements . pvuIIC and bamHIC genes define one incompatibility group of exclusion whereas ecoRVC gene defines another.

Proc Natl Acad Sci U S A, 1998 May 26, 95(11), 6394 - 8
Human sulfite oxidase R160Q: identification of the mutation in a sulfite oxidase-deficient patient and expression and characterization of the mutant enzyme; Garrett RM et al.; Sulfite oxidase catalyzes the terminal reaction in the degradation of sulfur amino acids . Genetic deficiency of sulfite oxidase results in neurological abnormalities and often leads to death at an early age . The mutation in the sulfite oxidase gene responsible for sulfite oxidase deficiency in a 5-year-old girl was identified by sequence analysis of cDNA obtained from fibroblast mRNA to be a guanine to adenine transition at nucleotide 479 resulting in the amino acid substitution of Arg-160 to Gln . Recombinant protein containing the R160Q mutation was expressed in Escherichia coli, purified, and characterized . The mutant protein contained its full complement of molybdenum and heme, but exhibited 2% of native activity under standard assay conditions . Absorption spectroscopy of the isolated molybdenum domains of native sulfite oxidase and of the R160Q mutant showed significant differences in the 480- and 350-nm absorption bands, suggestive of altered geometry at the molybdenum center . Kinetic analysis of the R160Q protein showed an increase in Km for sulfite combined with a decrease in kcat resulting in a decrease of nearly 1,000-fold in the apparent second-order rate constant kcat/Km . Kinetic parameters for the in vitro generated R160K mutant were found to be intermediate in value between those of the native protein and the R160Q mutant . Native sulfite oxidase was rapidly inactivated by phenylglyoxal, yielding a modified protein with kinetic parameters mimicking those of the R160Q mutant . It is proposed that Arg-160 attracts the anionic substrate sulfite to the binding site near the molybdenum.

Proc Natl Acad Sci U S A, 1998 May 26, 95(11), 6134 - 8
Visualization of elongation factor G on the Escherichia coli 70S ribosome: the mechanism of translocation; Agrawal RK et al.; During protein synthesis, elongation factor G (EF-G) binds to the ribosome and promotes the step of translocation, a process in which tRNA moves from the A to the P site of the ribosome and the mRNA is advanced by one codon . By using three-dimensional cryo-electron microscopy, we have visualized EF-G in a ribosome-EF-G-GDP-fusidic acid complex . Fitting the crystal structure of EF-G-GDP into the cryo density map reveals a large conformational change mainly associated with domain IV, the domain that mimics the shape of the anticodon arm of the tRNA in the structurally homologous ternary complex of Phe-tRNAPhe, EF-Tu, and a GTP analog . The tip portion of this domain is found in a position that overlaps the anticodon arm of the A-site tRNA, whose position in the ribosome is known from a study of the pretranslocational complex, implying that EF-G displaces the A-site tRNA to the P site by physical interaction with the anticodon arm.

Proc Natl Acad Sci U S A, 1998 May 26, 95(11), 6114 - 7
Fourier transform infrared spectroscopy reveals a rigid alpha-helical assembly for the tetrameric Streptomyces lividans K+ channel; le Coutre J et al.; The structure of the tetrameric K+ channel from Streptomyces lividans in a lipid bilayer environment was studied by polarized attenuated total reflection Fourier transform infrared spectroscopy . The channel displays approximately 43% alpha-helical and 25% beta-sheet content . In addition, H/D exchange experiments show that only 43% of the backbone amide protons are exchangeable with solvent . On average, the alpha-helices are tilted 33 degrees normal to the membrane surface . The results are discussed in relationship to the lactose permease of Escherichia coli, a membrane transport protein.

Proc Natl Acad Sci U S A, 1998 May 26, 95(11), 6108 - 13
Role of the J-domain in the cooperation of Hsp40 with Hsp70; Greene MK et al.; The Escherichia coli Hsp40 DnaJ and Hsp70 DnaK cooperate in the binding of proteins at intermediate stages of folding, assembly, and translocation across membranes . Binding of protein substrates to the DnaK C-terminal domain is controlled by ATP binding and hydrolysis in the N-terminal ATPase domain . The interaction of DnaJ with DnaK is mediated at least in part by the highly conserved N-terminal J-domain of DnaJ that includes residues 2-75 . Heteronuclear NMR experiments with uniformly 15N-enriched DnaJ2-75 indicate that the chemical environment of residues located in helix II and the flanking loops is perturbed on interaction with DnaK or a truncated DnaK molecule, DnaK2-388 . NMR signals corresponding to these residues broaden and exhibit changes in chemical shifts in the presence of DnaK(MgADP) . Addition of MgATP largely reversed the broadening, indicating that NMR signals of DnaJ2-75 respond to ATP-dependent changes in DnaK . The J-domain interaction is localized to the ATPase domain of DnaK and is likely to be dominated by electrostatic interactions . The results suggest that the J-domain tethers DnaK to DnaJ-bound substrates, which DnaK then binds with its C-terminal peptide-binding domain.

Proc Natl Acad Sci U S A, 1998 May 26, 95(11), 6079 - 84
Identification of an additional negative regulatory region for p53 sequence-specific DNA binding; Muller-Tiemann BF et al.; The DNA binding activity of p53 is crucial for its tumor suppressor function and is subject to tight regulation . Previous studies revealed that the inhibitory function of the p53 C terminus is implicated in the latent, low affinity sequence-specific DNA binding activity of p53 in the uninduced state . Sequence-specific DNA binding of p53 has been shown to be activated by several posttranslational modifications and interacting proteins that target predominantly the C terminus . Moreover, several authors have shown that synthetic peptides corresponding to p53 C-terminal sequences activate p53 sequence-specific DNA binding . In an effort to identify the interaction site of p53 with these activating peptides we assessed complex formation between p53 deletion constructs and C-terminal activating peptides by peptide affinity precipitation . This study revealed that two distal regions of the p53 molecule contribute synergistically to the interaction with activating C-terminal peptides: amino acids 80-93 and 364-393 . The C-terminal residues 364-393 are already well characterized as having negative regulatory function . DNA binding analyses with these deletion constructs reveal a comparable negative regulatory activity for residues 80-93, defining this region as a previously unidentified negative regulatory domain of p53 . Furthermore, synthetic peptides spanning this newly identified proline-rich negative regulatory region (residues 80-93) are able to activate p53 sequence-specific DNA binding in vitro . We suggest that both negative regulatory regions, residues 80-93 and 364-393, contribute cooperatively to the maintenance of the latent, low-affinity DNA binding conformation of p53.

Proc Natl Acad Sci U S A, 1998 May 26, 95(11), 6049 - 54
DNA annealing by RAD52 protein is stimulated by specific interaction with the complex of replication protein A and single-stranded DNA; Sugiyama T et al.; Homologous recombination in Saccharomyces cerevisiae depends critically on RAD52 function . In vitro, Rad52 protein preferentially binds single-stranded DNA (ssDNA), mediates annealing of complementary ssDNA, and stimulates Rad51 protein-mediated DNA strand exchange . Replication protein A (RPA) is a ssDNA-binding protein that is also crucial to the recombination process . Herein we report that Rad52 protein effects the annealing of RPA-ssDNA complexes, complexes that are otherwise unable to anneal . The ability of Rad52 protein to promote annealing depends on both the type of ssDNA substrate and ssDNA binding protein . RPA allows, but slows, Rad52 protein-mediated annealing of oligonucleotides . In contrast, RPA is almost essential for annealing of longer plasmid-sized DNA but has little effect on the annealing of poly(dT) and poly(dA), which are relatively long DNA molecules free of secondary structure . These results suggest that one role of RPA in Rad52 protein-mediated annealing is the elimination of DNA secondary structure . However, neither Escherichia coli ssDNA binding protein nor human RPA can substitute in this reaction, indicating that RPA has a second role in this process, a role that requires specific RPA-Rad52 protein interactions . This idea is confirmed by the finding that RPA, which is complexed with nonhomologous ssDNA, inhibits annealing but the human RPA-ssDNA complex does not . Finally, we present a model for the early steps of the repair of double-strand DNA breaks in yeast.

Proc Natl Acad Sci U S A, 1998 May 26, 95(11), 6021 - 6
Mapping the sigma70 subunit contact sites on Escherichia coli RNA polymerase with a sigma70-conjugated chemical protease; Owens JT et al.; The core enzyme of Escherichia coli RNA polymerase acquires essential promoter recognition and transcription initiation activities by binding one of several sigma subunits . To characterize the proximity between sigma70, the major sigma for transcription of the growth-related genes, and the core enzyme subunits (alpha2 beta beta'), we analyzed the protein-cutting patterns produced by a set of covalently tethered FeEDTA probes {FeBABE: Fe (S)-1-(p-bromoacetamidobenzyl)EDTA} . The probes were positioned in or near conserved regions of sigma70 by using seven mutants, each carrying a single cysteine residue at position 132, 376, 396, 422, 496, 517, or 581 . Each FeBABE-conjugated sigma70 was bound to the core enzyme, which led to cleavage of nearby sites on the beta and beta' subunits (but not alpha) . Unlike the results of random cleavage {Greiner, D . P., Hughes, K . A., Gunasekera, A . H . & Meares, C . F . (1996) Proc . Natl . Acad . Sci . USA 93, 71-75}, the cut sites from different probe-modified sigma70 proteins are clustered in distinct regions of the subunits . On the beta subunit, cleavage is observed in two regions, one between residues 383 and 554, including the conserved C and Rif regions; and the other between 854 and 1022, including conserved region G, regions of ppGpp sensitivity, and one of the segments forming the catalytic center of RNA polymerase . On the beta' subunit, the cleavage was identified within the sequence 228-461, including beta' conserved regions C and D (which comprise part of the catalytic center).

Proc Natl Acad Sci U S A, 1998 May 26, 95(11), 6010 - 4
A scrapie-like unfolding intermediate of the prion protein domain PrP(121-231) induced by acidic pH; Hornemann S et al.; The infectious agent of transmissible spongiform encephalopathies is believed to consist of an oligomeric isoform, PrPSc, of the monomeric cellular prion protein, PrPC . The conversion of PrPC to PrPSc is characterized by a decrease in alpha-helical structure, an increase in beta-sheet content, and the formation of PrPSc amyloid . Whereas the N-terminal part of PrPC comprising residues 23-120 is flexibly disordered, its C-terminal part, PrP(121-231), forms a globular domain with three alpha-helices and a small beta-sheet . Because the segment of residues 90-231 is protease-resistant in PrPSc, it is most likely structured in the PrPSc form . The conformational change of the segment containing residues 90-120 thus constitutes the minimal structural difference between PrPC and a PrPSc monomer . To test whether PrP(121-231) is also capable to undergo conformational transitions, we analyzed its urea-dependent unfolding transitions at neutral and acidic pH . We identified an equilibrium unfolding intermediate of PrP(121-231) that is exclusively populated at acidic pH and shows spectral characteristics of a beta-sheet protein . The intermediate is in rapid equilibrium with native PrP(121-231), significantly populated in the absence of urea at pH 4.0, and may have important implications for the presumed formation of PrPSc during endocytosis.

Blood, 1998 Jun 1, 91(11), 4127 - 35
Growth inhibition of granulocyte-macrophage colony-forming cells by human cytidine deaminase requires the catalytic function of the protein; Gran C et al.; Previous studies have indicated that cytidine deaminase (CDD) is a potent growth inhibitor of granulocyte-macrophage colony-forming cells (GM-CFC) . In this study, we have undertaken molecular cloning and purification of recombinant human CDD to elucidate the growth regulatory potential and mechanism behind the growth suppressive effect . The purified protein had a specific activity of 1.35 x 10(5) U/mg and a Km value of 30 micromol/L . In the GM-CFC assay, the recombinant protein was shown to reduce colony formation to 50% at 16 pmol/L concentration . Similarly, as was observed with CDD derived from granulocyte extract, the effect depended on the presence of thymidine (>/= 4 x 10(-5) mol/L) . These results imply that CDD is an extremely potent inhibitor of GM-CFC and that no additional factor from the granulocyte extract is required for the growth inhibitory effect . Modification of CDD by truncation from the C-terminal end, or by amino acid substitution of an active site glutamate residue, eliminated both the enzyme activity and the growth regulatory potential of CDD . Furthermore, CDD from Escherichia coli was found to be even more effective than human CDD in growth suppression of GM-CFC, with 10-fold higher inhibitory activity corresponding to a 10-fold higher enzymatic activity . Taken together, these results show that the catalytic nucleoside deaminating function of the protein is essential for the growth suppressive effect of CDD . Most probably, CDD exerts growth inhibition by depleting the cytidine and deoxycytidine pool required for DNA synthesis, as addition of deoxycytidine monophosphate, which is not a substrate for CDD, neutralizes the inhibiting effect.

J Virol, 1998 Jun, 72(6), 4911 - 7
Transcription factor YY1 represses cell-free replication from human papillomavirus origins; Lee KY et al.; We have established cell-free replication for the human papillomavirus type 18 (HPV-18) origin of replication (ori)-containing DNA by using purified HPV-18 E1 and E2 gene products expressed as fusion proteins in Escherichia coli . The transcription factor YY1 has been shown to regulate RNA transcription by binding to a sequence overlapping the putative E1 protein binding site in the HPV-18 ori . We show that exogenously added YY1 fusion protein inhibited HPV-18 ori replication . Cotransfection of YY1 expression vectors also inhibited transient replication in 293 cells . However, inhibition did not appear to be mediated by binding to its cognate site in the ori as YY1 also inhibited the replication of the HPV-11 ori, which does not have a known or suspected YY1 binding site . Moreover, inhibition was not alleviated by the inclusion of YY1 binding oligonucleotides in the replication reaction mixtures . Rather, we demonstrated a direct interaction between purified fusion E2 protein and fusion YY1 protein by the pull-down assay and a partial restoration of replication activity by an elevated E2 protein concentration . These results suggest that YY1 can inhibit HPV ori replication by interfering with E2 protein functions.

J Virol, 1998 Jun, 72(6), 4798 - 810
N-Terminal extension of human immunodeficiency virus capsid protein converts the in vitro assembly phenotype from tubular to spherical particles; Gross I et al.; Expression of retroviral Gag polyproteins is sufficient for morphogenesis of virus-like particles with a spherical immature protein shell . Proteolytic cleavage of Gag into the matrix (MA), capsid (CA), nucleocapsid (NC), and p6 domains (in the case of human immunodeficiency virus {HIV}) leads to condensation to the mature cone-shaped core . We have analyzed the formation of spherical or cylindrical particles on in vitro assembly of purified HIV proteins or inside Escherichia coli cells . CA protein alone yielded cylindrical particles, while all N-terminal extensions of CA abolished cylinder formation . Spherical particles with heterogeneous diameters or amorphous protein aggregates were observed instead . Extending CA by 5 amino acids was sufficient to convert the assembly phenotype to spherical particles . Sequences C-terminal of CA were not required for sphere formation . Proteolytic cleavage of N-terminally extended CA proteins prior to in vitro assembly led to the formation of cylindrical particles, while proteolysis of in vitro assembly products caused disruption of spheres but not formation of cylinders . In vitro assembly of CA and extended CA proteins in the presence of cyclophilin A (CypA) at a CA-to-CypA molar ratio of 10:1 yielded significantly longer cylinders and heterogeneous spheres, while higher concentrations of CypA completely disrupted particle formation . We conclude that the spherical shape of immature HIV particles is determined by the presence of an N-terminal extension on the CA domain and that core condensation during virion maturation requires the liberation of the N terminus of CA.

Biochim Biophys Acta, 1998 Apr 22, 1371(1), 24 - 30
Sequence and phylogenetic analysis of the Borrelia burgdorferi secA gene; Guina T et al.; A Borrelia burgdorferi secA homologue was cloned and the complete DNA sequence was determined . The deduced protein sequence consists of 899 amino acids and shows a high degree of homology to SecA homologues from other Bacteria and photosynthetic plastids . The presence of the secA gene in Spirochetes suggests that this gene is present in most if not all major lineages within Bacteria . The ease of isolation of secA by conservation of its ATP-binding motifs combined with its extreme conservation in protein secretion pathways and the presence of a phylogenetic sequence marker in one of its ATP-binding domains makes this gene useful for phylogenetic analysis of Bacteria and photosynthetic plastids .

Biochim Biophys Acta, 1998 Apr 14, 1364(1), 73 - 83
Isolation of mutants of the Arabidopsis thaliana alternative oxidase (ubiquinol:oxygen oxidoreductase) resistant to salicylhydroxamic acid; Berthold DA; The plant-type ubiquinol:oxygen oxidoreductase, commonly called the alternative oxidase, is a respiratory enzyme thought to contain non-heme iron at its active site . To explore the structure of the enzyme by identifying amino acids involved in inhibitor-binding, a library of random mutants of the Arabidopsis thaliana alternative oxidase was constructed using error-prone polymerase chain reaction and expressed in the heme-deficient Escherichia coli SASX41B . Selection for resistance to salicylhydroxamic acid (SHAM) resulted in the recovery of four mutations . Three of these, F215L, M219I, and M219V, confer a small, but measurable resistance to SHAM of between 1.4- and 1.7-fold relative to the wild type alternative oxidase . These changes are located in a putative amphipathic helix following the second transmembrane helix . The fourth mutation, G303E, is found three residues from the C-terminus of the protein, and results in 4 . 6-fold resistance to SHAM . None of the mutations have any effect on the sensitivity of the alternative oxidase to propyl gallate . The identification of distant residues involved in SHAM resistance suggests that the poorly conserved C-terminal region is in spatial proximity to the amphipathic helix, and thus located in the vicinity of the iron-binding motif .

Eur J Nucl Med, 1998 Apr, 25(4), 347 - 52
Imaging of infection in rabbits with radioiodinated interleukin-1 (alpha and beta), its receptor antagonist and a chemotactic peptide: a comparative study; van der Laken CJ et al.; Previous studies have reported the favourable characteristics of chemotactic peptides and interleukins for imaging of infection and inflammation . In the present study, the potential of two species of interleukin 1 (IL-1), IL-1alpha and IL-1beta, the IL-1 receptor antagonist (IL-1ra) and the synthetic chemotactic peptide N-formyl-methionyl-leucyl-phenylalanyl-lysine (fMLFK) were directly compared in a rabbit model of infection . IL-1alpha, IL-1beta, IL-1ra and fMLFK were labelled with iodine-123 according to the Bolton-Hunter method . Twenty-four hours after induction of Escherichia coli abscesses in the left thigh muscle, rabbits were injected intravenously with 0.5 mCi of 123I-labelled agent . Gamma camera images were obtained at 5 min and 1, 4, 8 and 20 h p.i . Biodistribution was determined at 20 h p.i . Although all agents rapidly cleared from the blood, at 20 h p.i . blood levels and the levels in most organs of 123I-fMLFK were significantly lower than those of the other three agents (P<0.05) . The abscesses were clearly visualized with all agents from 4 h p.i . onwards . After 1 h p.i., the abscess uptake of 123I-IL-1beta was significantly higher than that of the other agents (P<0.05), with the highest uptake observed at 8 h p.i . (1.3%+/-0.3%) . After 20 h p.i., the highest abscess-to-contralateral muscle ratios were obtained with 123I-IL-1beta, i.e . 39.0+/-11.5 vs 18.7+/-5.4, 18.1+/-2.3 and 29 . 9+/-7.0 for 123I-IL-1alpha, 123I-IL-1ra and 123I-fMLFK, respectively . In conclusion, all agents localized in the infectious focus . The potential of radiolabelled IL-1beta for imaging of infection was better than that of the other agents: higher absolute uptake in the infection and higher abscess-to-contralateral muscle ratios were obtained . The observation of localization of radiolabelled IL-1ra in infection was important since this protein can be administered to humans without any side-effects.

J Biol Chem, 1998 Apr 24, 273(17), 10602 - 8
Tenascin-C hexabrachion assembly is a sequential two-step process initiated by coiled-coil alpha-helices; Kammerer RA et al.; We have investigated the oligomerization process of tenascin-C using a variety of recombinant wild-type and mutant polypeptide chain fragments produced by heterologous gene expression in Escherichia coli . Biochemical and biophysical analyses of the structures and assemblies of these fragments indicated a sequential two-step oligomerization mechanism of tenascin-C involving the concerted interaction of two distinct domains and cysteines 64, 111, and 113 . First, the sequence between alanine 114 and glutamine 139 initiates hexabrachion formation via a parallel three-stranded coiled coil . Subsequently, the tenascin assembly domain, which is unique to the tenascins, is responsible for the connection of two triplets to a hexamer . The oligomerization of the tenascin assembly domains by the three-stranded coiled coil increases their homophilic binding affinity and is an important prerequisite for tenascin-C hexamerization . Although formation of the characteristic hexabrachion structure involves the covalent linkage of the six subunits by cysteine residues, mutational analysis indicates that hexamer formation is not dependent on intermolecular disulfide bonds . Most interestingly, substitution of glutamate 130 within the coiled-coil domain by leucine or alanine resulted in the formation of parallel four-stranded helix structures, which further associated to dodecamers . Aside from supporting a sequential process of tenascin-C assembly, this finding provides experimental evidence that non-core residues can have profound effects on the oligomerization states of coiled coils.

J Biol Chem, 1998 Apr 24, 273(17), 10578 - 85
The M4M5 cytoplasmic loop of the Na,K-ATPase, overexpressed in Escherichia coli, binds nucleoside triphosphates with the same selectivity as the intact native protein; Gatto C et al.; Escherichia coli was used to overexpress the large cytoplasmic loop of the rat Na,K-ATPase . A 1260-base DNA segment encoding Lys354-Lys774 of the rat alpha1-subunit was constructed via polymerase chain reaction . The polymerase chain reaction product was successfully subcloned into the expression vector pET-28 (Novagen), which produces an N-terminal 6-histidine-tagged fusion protein . The pET-28 vector containing rat alpha-loop, i.e . pAN, was used to transform calcium-competent E . coli BL21(DE3) cells, and positive clones were selected by kanamycin resistance . Bacterial cultures were grown, and protein synthesis was induced with isopropyl beta-D-thiogalactoside . Cells were harvested and lysed, revealing production of the His-tagged fusion protein ( approximately 46 kDa) . The fusion protein was affinity-purified from other soluble cellular proteins via a Ni-NTA column, which routinely yielded approximately 20 mg of soluble His6-alpha-loop/L cell culture . The His6-alpha-loop retained significant native structure, as evidenced by the ability of ATP and ADP (but not AMP, CTP, GTP, or UTP) to protect against chemical modification by either fluorescein isothiocyanate or maleimidylanilinonapthalene sulfonic acid . More specifically, circular dichroism spectroscopy was used to estimate the secondary structure of the His6 loop, revealing an ordered folding composed of 23% alpha-helix, 23% antiparallel beta-sheet, 4% parallel beta-sheet, 19% beta-turn, and 32% random coil . The 6-histidine loop bound the fluorescent ATP analog trinitrophenyl-ATP with high affinity, as determined by measuring the fluorescence changes associated with binding . Affinities for ATP ( approximately 350 microM) and ADP ( approximately 550 microM) were determined by their ability to compete with and displace 2',3'-O-{2,4,6,-trinitrophenyl}-ATP . These nucleotide affinities are similar to those observed for the E2 conformation of the intact Na,K-ATPase.

J Biol Chem, 1998 Apr 24, 273(17), 10515 - 29
Does single-stranded DNA pass through the inner channel of the protein hexamer in the complex with the Escherichia coli DnaB Helicase? Fluorescence energy transfer studies; Jezewska MJ et al.; The structure of the complex of the Escherichia coli primary replicative helicase DnaB protein with single-stranded (ss) DNA and replication fork substrates has been examined using the fluorescence energy transfer method . In these experiments, we used the DnaB protein variant, R14C, which has arginine 14 replaced by cysteine in the small 12-kDa domain of the protein using site-directed mutagenesis . The cysteine residues have been modified with a fluorescent marker which serves as a donor or an acceptor to another fluorescence label placed in different locations on the DNA substrates . Using the multiple fluorescence donor-acceptor approach, we provide evidence that, in the complex with the enzyme, ssDNA passes through the inner channel of the DnaB hexamer . This is the first evidence of the existence of such a structure of a hexameric helicase-ssDNA complex in solution . In the stationary complex with the 5' arm of the replication fork, without ATP hydrolysis, the distance between the 5' end of the arm and the 12-kDa domains of the hexamer (R = 47 A) is the same as in the complex with the isolated ssDNA oligomer (R = 47 A) having the same length as the arm of the fork . These data indicate that both ssDNA and the 5' arm of the fork bind in the same manner to the DNA binding site . Moreover, in the complex with the helicase, the length of the ssDNA is similar to the length of the ssDNA strand in the double-stranded DNA conformation . In the stationary complex, the helicase does not invade the duplex part of the fork beyond the first 2-3 base pairs . This result corroborates the quantitative thermodynamic data which showed that the duplex part of the fork does not contribute to the free energy of binding of the enzyme to the fork . Implications of these results for the mechanism of a hexameric helicase binding to DNA are discussed.

J Biol Chem, 1998 Apr 24, 273(17), 10325 - 30
Identification and characterization of a novel cap-binding protein from Arabidopsis thaliana; Ruud KA et al.; Cap-binding proteins specifically bind to the 7-methyl guanosine (m7G) functional group at the 5' end of eukaryotic mRNAs . A novel Arabidopsis thaliana protein has been identified that has sequence similarity to cap-binding proteins but is clearly a different form of the protein . The most obvious primary sequence difference is the substitution of two of the eight conserved tryptophan residues with other aromatic amino acids in the novel protein . Analogous forms of this novel protein appear to be present in other higher eukaryotes but not in yeast . Analysis of the native and recombinant forms of the novel protein by retention on m7GTP-Sepharose indicate that it is a functional cap-binding protein . Measurements of the dissociation constant for this protein indicate that it binds m7GTP 5-20-fold tighter than eukaryotic initiation factor (eIF)(iso)4E . The novel protein also supports the initiation of translation of capped mRNA in vitro . Biochemical analysis and yeast two-hybrid data indicate that it interacts with eIF(iso)4G to form a complex . Based on these observations, this protein appears to be able to function as a cap-binding protein and is given the designation of novel cap-binding protein (nCBP).

J Biol Chem, 1998 Apr 24, 273(17), 10296 - 301
Alkyl-dihydroxyacetonephosphate synthase . Fate in peroxisome biogenesis disorders and identification of the point mutation underlying a single enzyme deficiency; de Vet EC et al.; Peroxisomes play an indispensible role in ether lipid biosynthesis as evidenced by the deficiency of ether phospholipids in fibroblasts and tissues from patients suffering from a number of peroxisomal disorders . Alkyl-dihydroxyacetonephosphate synthase, a peroxisomal enzyme playing a key role in the biosynthesis of ether phospholipids, contains the peroxisomal targeting signal type 2 in a N-terminal cleavable presequence . Using a polyclonal antiserum raised against alkyl-dihydroxyacetonephosphate synthase, levels of this enzyme were examined in fibroblast cell lines from patients affected by peroxisomal disorders . Strongly reduced levels were found in fibroblasts of Zellweger syndrome and rhizomelic chondrodysplasia punctata patients, indicating that the enzyme is not stable in the cytoplasm as a result of defective import into peroxisomes . In a neonatal adrenoleukodystrophy patient with an isolated import deficiency of proteins carrying the peroxisomal targeting signal type 1, the precursor form of alkyl-dihydroxyacetonephosphate synthase was detected at a level comparable to that of the mature form in control fibroblasts, in line with an intraperoxisomal localization . A patient with an isolated deficiency in alkyl-dihydroxyacetonephosphate (DHAP) synthase activity had normal levels of this protein . Analysis at the cDNA level revealed a missense mutation leading to a R419H substitution in the enzyme of this patient . Expression of a recombinant protein carrying this mutation in Escherichia coli yielded an inactive enzyme, whereas a comparable control recombinant enzyme was active, providing further proof that this substitution is responsible for the inactivity of the enzyme and the phenotype . In line with this result is the observation that wild-type alkyl-DHAP synthase activity can be inactivated by the arginine-modifying agent phenylglyoxal . The enzyme is efficiently protected against this inactivation when the substrate palmitoyl-DHAP is present at a saturating concentration . The gene encoding human alkyl-dihydroxyacetonephosphate synthase was mapped on chromosome 2q31.

J Biol Chem, 1998 Apr 24, 273(17), 10249 - 52
The N terminus of eukaryotic translation elongation factor 3 interacts with 18 S rRNA and 80 S ribosomes; Gontarek RR et al.; Elongation factor-3 (EF-3) is an essential fungal-specific translation factor which exhibits a strong ribosome-dependent ATPase activity and has sequence homologies that may predict domains critical for its role in protein synthesis, including a domain at the N terminus, which exhibits sequence homology with Escherichia coli ribosomal protein S5 . A portion of the N terminus of Saccharomyces cerevisiae EF-3 (spanning the S5 homology region) has been cloned, expressed, and purified from E . coli . UV cross-linking experiments revealed that the N-terminal EF-3 protein (N-term EF-3) can be specifically cross-linked to 18 S rRNA . Filter-binding assays confirmed these data, and also established that the interaction has a Kd approximately 238 nM . Additional evidence shows that N-term EF-3 is able to associate with yeast ribosomes and inhibit the ribosome-dependent ATPase activity of native EF-3 . These data taken together suggest that at least one of the ribosome-binding sites of EF-3 is located at the N terminus.

J Biol Chem, 1998 Apr 24, 273(17), 10196 - 201
Human tyrosine hydroxylase isoforms . Inhibition by excess tetrahydropterin and unusual behavior of isoform 3 after camp-dependent protein kinase phosphorylation; Alterio J et al.; Human tyrosine hydroxylase exists as four isoforms (hTH1-4), generated by alternative splicing of pre-mRNA, with tissue-specific distribution . Unphosphorylated hTH3 and hTH1 were produced in large amounts in Escherichia coli and purified to homogeneity . The phosphorylation sites were determined after labeling with {32P}phosphate in the presence of cAMP-dependent protein kinase (PKA) and calmodulin-dependent protein kinase II (CaM-PKII) . Ser40 was phosphorylated by PKA, and both Ser19 and Ser40 were phosphorylated by CaM-PKII . The enzyme kinetics of hTH3 were determined in the presence of various concentrations of the natural co-substrate (6R)-tetrahydrobiopterin and compared with those of recombinant hTH1 (similar to rat TH) . We show that, under initial velocity conditions, excess (6R)-tetrahydrobiopterin inhibits hTH3 and hTH1 . The TH catalytic constants (kcat) were determined for each of the two isoenzymes: hTH3 is about five times more active than hTH1 . Phosphorylation by CaM-PKII did not affect the kinetic parameters of hTH3 . The classical activation of TH by PKA phosphorylation, demonstrated for hTH1, was not observed with hTH3 . Furthermore, hTH3 escapes activity regulation by phosphorylation and is always more active than phosphorylated hTH1 . The properties of the hTH3 enzyme may be relevant to diseases affecting dopaminergic cells.

Genes Dev, 1998 Apr 15, 12(8), 1134 - 44
RecQ helicase, in concert with RecA and SSB proteins, initiates and disrupts DNA recombination; Harmon FG et al.; RecQ helicase is important to homologous recombination and DNA repair in Escherichia coli . We demonstrate that RecQ helicase, in conjunction with RecA and SSB proteins, can initiate recombination events in vitro . In addition, RecQ protein is capable of unwinding a wide variety of DNA substrates, including joint molecules formed by RecA protein . These data are consistent with RecQ helicase assuming two roles in the cell; it can be (1) an initiator of homologous recombination, or (2) a disrupter of joint molecules formed by aberrant recombination . These findings also shed light on the function of the eukaryotic homologs of RecQ helicase, the Sgs1, Blm, and Wrn helicases.

Curr Biol, 1998 Apr 9, 8(8), 452 - 8
Evidence for two modes of cooperative DNA binding in vivo that do not involve direct protein-protein interactions; Vashee S et al.; BACKGROUND: The promoter regions of most eukaryotic genes contain binding sites for more than one transcriptional activator and these activators often bind cooperatively to promoters . The most common type of cooperativity is supported by direct protein-protein interactions . Recent studies have shown that proteins that do not specifically interact with one another can bind cooperatively to chromatin in vitro . probably by the localized destabilization of nucleosome structure by one factor, facilitating binding of another to a nearby site . This mechanism does not require that the transcription factors have activation domains . We have examined whether this phenomenon occurs in vivo . RESULTS: Unrelated non-interacting proteins can bind DNA cooperatively in yeast cells; this cooperative binding can contribute significantly to transcriptional activation, does not require that both factors have activation domains and is only operative over relatively short distances . In addition to this 'short-range' mechanism, unrelated non-interacting proteins can bind cooperatively to sites separated by hundreds of base pairs, so long as both have potent activation domains . CONCLUSION: Cooperative binding of transcription factors in vivo can occur by several mechanisms, some of which do not require direct protein-protein interactions and which cannot be detected in vitro using naked DNA templates . These findings must be taken into account when evaluating mechanisms for synergistic transcriptional activation.

J Mol Biol, 1998 Apr 10, 277(4), 805 - 24
The arginine repressor of Escherichia coli K-12 makes direct contacts to minor and major groove determinants of the operators; Wang H et al.; In order to gain further insight into the molecular mechanism of arginine-dependent operator recognition by the hexameric Escherichia coli arginine repressor we have probed protein-DNA interactions in vitro and in vivo . We have extensively applied the chemical modification-protection and premodification-interference approach to two operators, the natural operator overlapping the P2 promoter of the carAB operon and a fully symmetrical consensus sequence . Backbone contacts were revealed by hydroxyl radical footprinting and phosphate ethylation interference . Base-specific contacts to purines and pyrimidines were revealed by methylation protection and premodification interference, KMnO4 and NH2OH.HCl-specific modification of thymine and cytosine residues, base-removal (depurination and depyrimidation), and base substitution (uracil and inosine) . Additional information on the groove specificity of repressor binding was obtained by small ligand binding interference (distamycin and methyl green) . In vivo, we measured the effects on the repressibility of 24 single base-pair substitutions obtained by saturation mutagenesis of half an Arg box in the carAB operator . The results of these experiments point to the conclusion that a hexameric arginine repressor molecule covers four turns of the helix, makes base-specific contacts to at least one guanine (G4 or G4') and two thymine (T3, T13', or T3', T13) residues in each one of four consecutive major grooves on one face of the helix and with four A-T/T-A base-pairs, comprising the adenine residues A9, 9', 12, 12' and the thymine residues T10, 10', 11, 11', in the two outermost minor grooves of the operator, on the very same face of the DNA molecule . The hydrophobic 5-methyl groups of four thymine residues (T3, 3', 13, 13') in each Arg box contribute to major groove-specific recognition via hydrophobic and/or van der Waals interactions . The importance of minor groove contacts was further supported by the drastic effect of distamycin binding interference . In vivo, the most pronounced drops in repressibility were occasioned by mutations at positions 10 (A-->G or C), 11 (T-->A or G) and 12 (A-->G, T or C) .

J Mol Biol, 1998 Apr 10, 277(4), 789 - 804
Transcription activation at promoters carrying tandem DNA sites for the Escherichia coli cyclic AMP receptor protein: organisation of the RNA polymerase alpha subunits; Belyaeva TA et al.; We have constructed a family of promoters carrying tandem DNA sites for the Escherichia coli cyclic AMP receptor protein (CRP), with one of the sites centred between base-pairs 41 and 42 upstream from the transcription start site, and the second site located further upstream . In vivo activity measurements show that the activity of these promoters is completely dependent on CRP and that, depending on the precise location, CRP bound at the upstream site increases transcription activation . Hydroxyl radical footprinting was exploited to investigate the binding of CRP and RNA polymerase holoenzyme (RNAP) to these promoters . The study shows that the C-terminal domains of the RNAP alpha subunits bind adjacent to the upstream CRP and that their precise positioning depends on the location of upstream-bound CRP . The C-terminal domains of the RNAP alpha subunits interact with both the upstream and downstream-bound CRP via activating region 1 of CRP .

J Mol Biol, 1998 Apr 10, 277(4), 771 - 7
Plugging interactions of HAP2 pentamer into the distal end of flagellar filament revealed by electron microscopy; Maki S et al.; Bacterial flagellum has a cap structure tightly attached to its distal end . The cap is an oligomeric assembly of HAP2 protein (also called FliD) and plays an essential role in the filament growth in vivo by preventing flagellin monomers from leaking out without polymerization . Electron micrographs of the HAP2 complex formed in solution showed exclusively a pentagonal shape, called "star-cap", which was thought to be the end-on view of the cap . The molecular mass roughly corresponded to a dodecamer of HAP2, and therefore a double-layered star-cap was modeled to be the cap . Here, we have observed the side view of the complex in electron micrographs . The images clearly show a rectangular shape, about 80 A wide and 180 A long, with a bipolar feature in its long axis, indicating that the complex is a bipolar pair of pentamers . A thin plate feature is identified at each end of the particle, which looks exactly like the one observed as the structure of the native filament cap . Together with the structure of the filament previously analyzed by electron cryomicroscopy, the results suggest that the cap is a pentamer with its thin plate exposed to the solvent and the other half plugged into the hole at the distal end of the filament, which is almost twice wider than its central channel . This also allows us to model the axial domain arrangement of flagellin subunit in the filament .

Foot Ankle Int, 1998 Mar, 19(3), 173 - 6
Risk of contamination of the wound in a hydrotherapeutic tank; Stanwood W et al.; Over a 4-week period, samples for culture were taken from active hydrotherapeutic tanks (whirlpools) from two institutions in a university medical center . Samples were obtained in the morning before treatments began, and in the evening after, the final patient had been treated . Specific attention was directed toward recovery of S . aureus, P . aeruginosa, and E . coli, organisms felt to be especially dangerous for the diabetic dysvascular patients utilizing the hydrotherapeutic tanks involved in this study . Only eleven of 96 cultures (11.5%) were positive for these prospective pathogens . Of the positive cultures, nine (9.4%) were taken from near the agitator-jet, and only two (2.1%) from the floor of the hydrotherapeutic tanks, where the extremity is likely to be placed . Our results reveal that hydrotherapeutic immersion is not likely to expose patients with open wounds to potential iatrogenic contamination of the wound.

J Biochem (Tokyo), 1998 Mar, 123(3), 499 - 507
Cloning and characterization of a cDNA encoding a novel heterogeneous nuclear ribonucleoprotein-like protein and its expression in myeloid leukemia cells; Tsuchiya N et al.; We isolated a cDNA encoding a novel heterogeneous nuclear ribonucleoprotein (hnRNP)-like protein on DNA affinity screening of a K562 cDNA expression library with an oligodeoxynucleotide (JKT41) derived from intron 9 of the human myeloperoxidase gene . The cDNA has a 1,305 bp sequence that encodes a polypeptide of 301 amino acid residues . The protein, named JKTBP, contains two repeats of a putative RNA binding domain (RBD), each composed of canonical RNP-2 and RNP-1 motifs, and a glycine- and tyrosine-rich carboxyl terminus . The sequences of these two repeats are highly homologous with those of the 2 x RBD-Gly rich group of hnRNPs . Northern blotting showed that two mRNAs of approximately 1.4 and 2.8 kb were present in most cultured cells examined . The recombinant protein expressed in Escherichia coli interacted with the double-stranded form of JKT41 as well as with its single-stranded form . This interaction was competitively inhibited by the same unlabeled JKT41 and to nearly the same extent by unrelated oligonucleotides . Moreover, the recombinant protein interacted with poly(G) and poly(A), but not with poly(U) or poly(C) . Transient expression of the protein in SKM-1 cells repressed the expression of chloramphenicol acetyltransferase reporter genes located downstream of the intron 9 element of JKT41 or intron 7 element of FERE27 . The implications of the protein in the biogenesis of mRNA are discussed.

J Biochem (Tokyo), 1998 Mar, 123(3), 479 - 86
Molecular cloning and expression of an amine sulfotransferase cDNA: a new gene family of cytosolic sulfotransferases in mammals; Yoshinari K et al.; A cDNA of amine sulfotransferase-RB1 (AST-RB1), which efficiently catalyzes 4-phenyl-1,2,3,6-tetrahydropyridine (PTHP) sulfation, has been isolated by immunoscreening of a rabbit liver cDNA library . The cDNA consisted of 1,117 base pairs and encoded a protein of 301 amino acids with a molecular weight of 35,876 . The deduced amino acid sequence matched at six positions those of peptide fragments obtained from purified AST-RB1 protein . The sequence had less than 38% identity at the amino acid level with cytosolic sulfotransferases in mammals, although high degrees of similarity were observed with regions conserved throughout mammalian sulfotransferases . These results indicate that AST-RB1, arbitrarily named sulfotransferase 3A1 (ST3A1), constitutes a new and third gene family of cytosolic sulfotransferases in mammals . ST3A1 expressed in Escherichia coli as a fused protein catalyzed sulfation of amines such as PTHP, aniline, 4-chloroaniline, 2-naphthylamine, and desipramine, but barely O-sulfation of typical aryl and hydroxysteroid sulfotransferase substrates . These data unequivocally demonstrate the existence of a cytosolic sulfotransferase showing a high selectivity for amine substrates, and indicate that multiple forms of sulfotransferase mediate sulfation of xenobiotics in mammalian livers.

J Biochem (Tokyo), 1998 Mar, 123(3), 450 - 7
RecA protein has extremely high cooperativity for substrate in its ATPase activity; Mikawa T et al.; The single-stranded DNA-dependent ATPase activity of Escherichia coli RecA protein, especially its cooperativity for ATP, was investigated . To measure the ATPase activity in detail, the methods and reaction conditions for the ATPase assay were reexamined . Under conditions where RecA protein always showed a maximal rate of ATP hydrolysis, its poly(dT)-dependent ATPase activity was measured . At 25 degrees C, increasing the concentration of RecA protein from 0.3 to 1.0 microM increased the turnover number (kcat) from 0.16 to 0.19 s-1 and the Hill coefficient (nH) for ATP from 9.3 to 11.6 . At 0.5 microM RecA protein, increasing the temperature from 25 to 37 degrees C increased kcat from 0.18 to 0.35 s-1 but decreased nH from 9.8 to 6.6 . Interestingly, the ATPase activity of RecA protein measured in this study showed much higher cooperativity for ATP than those reported to date . Furthermore, the nH value of 11.6 for ATP obtained here was the highest of any ATPase reported so far . These results suggest that the binding of an ATP molecule to a RecA molecule within a nucleoprotein helical filament causes structural change of many other neighboring RecA molecules . This implies that ATP binding induces structural change of the whole nucleoprotein helical filament . Finally, we demonstrated that analysis of cooperativity is useful for revealing how a protein composed of many subunits functions as a whole.

Microb Pathog, 1998 Mar, 24(3), 155 - 66
Genetic and immunological analyses of Vls (VMP-like sequences) of Borrelia burgdorferi; Kawabata H et al.; DNA fragments containing the VMP-like sequence (Vls) were cloned from Borrelia burgdorferi strain 297 . Analyses by PCR, PFGE, and Southern hybridization revealed that the Vls sequences existed in multi-copies on the 20-kb borrelial plasmid, but not on chromosomes or other plasmids . One Vls unit of the strain 297 was about 669 bases, and predicted peptides length was 223 amino acids . Homologues of the Vls fragment were detected in three B . burgdorferi strains, a B . garinii strain 20047, and a B . afzelii strain P/Gau . A recombinant VlsII protein prepared in Escherichia coli strain JM109 reacted with antibodies that existed in three of five patients, by immunoblotting . These results suggested that the Vls of B . burgdorferi is expressed in Lyme disease patients .

Microb Pathog, 1998 Mar, 24(3), 145 - 54
Involvement of glutamic acid residue at position 7 in the formation of the intramolecular disulfide bond of Escherichia coli heat-stable enterotoxin Ip in vivo; Yamanaka H et al.; Escherichia coli heat-stable enterotoxin Ip (STIp) is a small peptide toxin composed of 18 amino acid residues containing three intramolecular disulfide bonds . We found previously that the bonds are formed by the catalysis of DsbA (a oxidoreductase) in periplasm {1} . To interact with DsbA, the STIp in periplasm must have a structure suitable as substrate . However, the amino acid residues contributing to the construction of this structure have not been elucidated . We mutated the codon for the glutamic acid at position 7 of STIp by oligonucleotide site-specific mutagenesis in vivo and analysed the STIp produced from the mutant gene . The intramolecular disulfide bonds were not formed in mutant STIp (Glu-7-->Ala), but were formed in mutant STIp (Glu-7-->Asp) . Furthermore, we found that replacing the asparagine residue at position 11 and the proline residue at position 12 did not affect the disulfide bond formation of STIp . The results indicate that a negative charge at position 7 in the sequence of STIp is necessary for STIp to interact with DsbA in periplasm .

Nucleic Acids Res, 1998 Apr 1, 26(7), 1713 - 7
The processivity of DNA synthesis exhibited by drug-resistant variants of human immunodeficiency virus type-1 reverse transcriptase; Avidan O et al.; The reverse transcriptase (RT) of human immunodeficiency virus (HIV) undergoes rapid mutagenesis due to selective pressure by RT inhibitors which renders the mutated RT variants resistant to these inhibitors . Resistance to nucleoside analogs during drug therapy results from point mutations that lead to specific variations in the RT sequences . It was recently shown that several well-defined drug-resistant variants of HIV-1 RT (i.e . Leu74Val, Glu89Gly, Tyr183Phe, Met184Lue, Met184Val and Met184Ile) show enhanced accuracy of DNA synthesis relative to wild-type HIV-1 RT (as evident from a reduction in the capacity to introduce mispairs and to elongate them) . Since the last two Met184 variants were shown also to possess decreased processivity of DNA synthesis, it was recently suggested that there might be an inverse correlation between the apparent in vitro fidelity and processivity of DNA synthesis in drug-resistant HIV-1 RT mutants . In the present study we have conducted a comparative analysis of the processivity of DNA synthesis on both DNA and RNA templates of the Leu74Val, Glu89Gly, Tyr183Phe and Met184Leu drug-resistant mutants of HIV-1 RT in comparison with wild-type RT . Apart from the Met184 mutant, which shows reduced relative processivity (similar to the other mutants of residue 184 already studied), the other three variants have relative processivity at least as high as that of wild-type RT . This suggests that the inverse correlation between reduced processivity and increased fidelity is restricted only to mutants with modifications of Met184 . The results presented may bear on potential mechanistic and structural differences in the involvement of the various mutated residues studied in processivity, fidelity and sensitivity to nucleoside analogs.

Nucleic Acids Res, 1998 Apr 1, 26(7), 1707 - 12
Escherichia coli RNA and DNA polymerase bypass of dihydrouracil: mutagenic potential via transcription and replication; Liu J et al.; Dihydrouracil (DHU) is a DNA base damage product produced in significant amounts by ionizing radiation damage to cytosine under anoxic conditions . DHU represents a model for pyrimidine base damage (ring saturation products) of the type recognized and repaired by Escherichia coli endonuclease III and its homologs in other species . We have built this lesion into synthetic oligonucleotides, with DHU placed at a single location downstream from an E.coli RNA polymerase promoter . This construct was used to determine the effect of DHU when encountered on a DNA template strand by either E.coli RNA or DNA polymerase (Klenow fragment) . Single round transcription experiments or primer extension-type replication experiments were conducted in order to make a direct comparison between RNA and DNA polymerases with DHU placed within the same sequence context . Both DNA and RNA polymerase efficiently bypass DHU and insert adenine opposite this lesion . These results suggest that DHU is mutagenic with respect to both replication and transcription and have implications for DNA repair as well the routes leading to generation of mutant proteins in dividing and non-dividing cells.

Nucleic Acids Res, 1998 Apr 1, 26(7), 1700 - 6
Isolation and characterization of a human cDNA for mRNA 5'-capping enzyme; Yamada-Okabe T et al.; The human mRNA 5'-capping enzyme cDNA was identified . Three highly related cDNAs, HCE1 (human mRNAcappingenzyme1), HCE1A and HCE1B , were isolated from a HeLa cDNA library . The HCE1 cDNA has the longest ORF, which can encode a 69 kDa protein . A short region of 69 bp in the 3'-half of the HCE1 ORF was missing in HCE1A and HCE1B , and, additionally, HCE1B has an early translation termination signal, which suggests that the latter two cDNAs represent alternatively spliced product . When expressed in Escherichia coli as a fusion protein with glutathione S -transferase, Hce1p displayed both mRNA 5'-triphosphatase (TPase) and mRNA 5'-guanylyltransferase (GTase) activities, and it formed a cap structure at the 5'-triphosphate end of RNA, demonstrating that it indeed specifies an active mRNA 5'-capping enzyme . The recombinant proteins derived from HCE1A and HCE1B possessed only TPase activity . When expressed from ADH1 promoter, HCE1 but not HCE1A and HCE1B complemented Saccharomyces cerevisiae CEG1 and CET1 , the genes for GTase and TPase, respectively . These results demonstrate that the N-terminal part of Hce1p is responsible for TPase activity and the C-terminal part is essential for GTase activity . In addition, the human TPase domain cannot functionally substitute for the yeast enzyme in vivo.

Nucleic Acids Res, 1998 Apr 1, 26(7), 1588 - 96
Mechanisms of DNA damage by chromium(V) carcinogens; Bose RN et al.; Reactions of bis(2-ethyl-2-hydroxy-butanato)oxochromate(V) with pUC19 DNA, single-stranded calf thymus DNA (ss-ctDNA), a synthetic oligonucleotide, 5'-GATCTATGGACTTACTTCAAGGCCGGGTAATGCTA-3' (35mer), deoxyguanosine and guanine were carried out in Bis-Tris buffer at pH 7.0 . The plasmid DNA was only nicked, whereas the single-stranded DNA suffered extensive damage due to oxidation of the ribose moiety . The primary oxidation product was characterized as 5-methylene-2-furanone . Although all four bases (A, C, G and T) were released during the oxidation process, the concentration of guanine exceeds the other three . Orthophosphate and 3'-phosphates were also detected in this reaction . Likewise, the synthetic oliogomer exhibits cleavage at all bases with a higher frequecncy at G sites . This increased cleavage at G sites was more apparent after treating the primary oxidation products with piperidine, which may indicate base oxidation as well . DNA oxidation is shown to proceed through a Cr(V)-DNA intermediate in which chromium(V) is coordinated through the phosphodiester moiety . Two alternative mechanisms for DNA oxidation by oxochromate(V) are proposed to account for formation of 5-methylene-2-furanone, based on hydrogen abstraction or hydride transfer from the C1' site of the ribose followed by hydration and two successive beta-eliminations . It appears that phosphate coordination is a prerequisite for DNA oxidation, since no reactions between chromium(V) and deoxyguanosine or guanine were observed . Two other additional pathways, hydrogen abstraction from C4' and guanine base oxidation, are also discussed.

Nucleic Acids Res, 1998 Apr 1, 26(7), 1560 - 6
Recognition and manipulation of branched DNA by the RusA Holliday junction resolvase of Escherichia coli; Chan SN et al.; Homologous recombination is a fundamental cellular process that shapes and reshapes the genomes of all organisms and promotes repair of damaged DNA . A key step in this process is the resolution of Holliday junctions formed by homologous DNA pairing and strand exchange . In Escherichia coli , a Holliday junction is processed into recombinant products by the concerted activities of the RuvA and RuvB proteins, which together drive branch migration, and RuvC endonuclease, which resolves the structure . In the absence of RuvABC, recombination can be promoted by increasing the expression of the RusA endonuclease, a Holliday junction resolvase encoded by a cryptic prophage gene . Here, we describe the DNA binding properties of RusA . We found that RusA was highly selective for branched molecules and formed complexes with these structures even in the presence of a large excess of linear duplex DNA . However, it does bind weakly to linear duplex DNA . Under conditions where there was no detectable binding to duplex DNA, RusA formed a highly structured complex with a synthetic Holliday junction that was remarkably stable and insensitive to divalent metal ions . The duplex arms were found to adopt a specific alignment within this complex that approximated to a tetrahedral conformation of the junction.

Nature, 1998 May 14, 393(6681), 185 - 7
Regulation of adenovirus alternative RNA splicing by dephosphorylation of SR proteins; Kanopka A et al.; SR proteins are a family of essential splicing factors required for early recognition of splice sites during spliceosome assembly . They also function as alternative RNA splicing factors when overexpressed in vivo or added in excess to extracts in vitro . SR proteins are highly phosphorylated in vivo, a modification that is required for their function in spliceosome assembly and splicing catalysis . Here we show that SR proteins purified from late adenovirus-infected cells are inactivated as splicing enhancer or splicing repressor proteins by virus-induced dephosphorylation . We further show that the virus-encoded protein E4-ORF4 activates dephosphorylation by protein phosphatase 2A of HeLa SR proteins and converts their splicing properties into that of SR proteins purified from late adenovirus-infected cells . Taken together, our results suggest that E4-ORF4 is an important factor controlling the temporal shift in adenovirus alternative RNA splicing . We conclude that alternative pre-mRNA splicing, like many other biological processes, is regulated by reversible protein phosphorylation.

J Gen Virol, 1998 May, 79 ( Pt 5), 1289 - 98
Nucleotide sequence and genome organization of grapevine leafroll-associated virus-2 are similar to beet yellows virus, the closterovirus type member; Zhu HY et al.; The entire genome of grapevine leafroll-associated closterovirus-2 (GLRaV-2), except the exact 5' terminus, was cloned and sequenced . The sequence encompasses nine open reading frames (ORFs) which include, in the 5' to 3' direction, an incomplete ORF1a encoding a putative viral polyprotein and eight ORFs that encode proteins of 52 kDa (ORF1b), 6 kDa (ORF2), 65 kDa (ORF3), 63 kDa (ORF4), 25 kDa (ORF5), 22 kDa (ORF6), 19 kDa (ORF7) and 24 kDa (ORF8) respectively, and 216 nucleotides of the 3' untranslated region . An incomplete ORF1a potentially encoded a large polyprotein containing the conserved domains characteristic of a papain-like protease, methyltransferase and helicase . ORF1b potentially encoded a putative RNA-dependent RNA polymerase . The expression of ORF1b may be via a +1 ribosomal frameshift mechanism, similar to other closteroviruses . A unique gene array, which is conserved in other closteroviruses, was also identified in GLRaV-2; it includes genes encoding a 6 kDa small hydrophobic protein, 65 kDa heat shock protein 70, 63 kDa protein of function unknown, 25 kDa coat protein duplicate and 22 kDa coat protein . Identification of ORF6 (22 kDa) as the coat protein gene was further confirmed by in vivo expression in E . coli and immunoblotting . Phylogenetic analysis comparing different genes of GLRaV-2 with those of other closteroviruses demonstrated a close relationship with beet yellows virus (BYV), beet yellow stunt virus and citrus tristeza virus . GLRaV-2 is the only closterovirus, so far, that matches the genome organization of the type member of the group, BYV, and thus can be unambiguously classified as a definitive member of the genus Closterovirus.

J Gen Virol, 1998 May, 79 ( Pt 5), 1273 - 80
Nucleic acid-binding properties and subcellular localization of the 3a protein of brome mosaic bromovirus; Fujita M et al.; Brome mosaic bromovirus (BMV) 3a protein is required for cell-to-cell movement of the virus in host plants . The BMV 3a protein (B3a) was produced in Escherichia coli using an expression vector . Gel retardation analysis and UV cross-linking experiments demonstrated that B3a bound single-stranded RNA cooperatively without sequence specificity . Binding competition analysis showed that B3a bound to single-stranded nucleic acids more strongly than to double-stranded nucleic acids . Deletion mutagenesis located a nucleic acid-binding domain to amino acids 189-242 . Western blot analysis of fractionated proteins of BMV-infected barley using monoclonal antibodies against B3a indicated that B3a may interact with membrane materials and form complexes in the cytoplasm . Immunogold labelling of thin sections of infected barley tissues revealed that B3a was associated with plasmodesmata and cytoplasmic inclusions.

J Gen Virol, 1998 May, 79 ( Pt 5), 989 - 99
Immune response in pigs vaccinated with plasmid DNA encoding ORF5 of porcine reproductive and respiratory syndrome virus; Pirzadeh B et al.; The ORF5-encoded major envelope glycoprotein (GP5) of porcine reproductive and respiratory syndrome virus (PRRSV) is one of the three major structural proteins of this virus . While some porcine convalescent sera and monoclonal antibodies directed against GP4 and GP5 have the capacity to neutralize the virus in vitro, the protein specificity of porcine neutralizing sera has not yet been established . DNA immunization with a plasmid encoding GP5 of PRRSV, under the control of a human cytomegalovirus promoter, induced anti-GP5-specific neutralizing antibodies in pigs and BALB/c mice . The GP5 protein specificity of neutralizing sera was confirmed by immunoblotting and ELISA . Peripheral blood mononuclear cells obtained from DNA-vaccinated pigs underwent blastogenic transformation in the presence of E . coli-expressed recombinant ORF5-encoded protein, indicating the specificity of the cellular immune response to GP5 . Following a massive intratracheal challenge with the virulent IAF-Klop strain of PRRSV, DNA-vaccinated pigs were protected from generalized viraemia and the development of typical macroscopic lung lesions that were observed in unvaccinated, virus-challenged controls, as well as in pigs that were immunized with E . coli-expressed GST-ORF5 recombinant fusion protein . Interstitial pneumonitis and broncho-alveolitis were remarkably milder in DNA-vaccinated animals . These results suggest that the GP5 of PRRSV is a good candidate for a subunit recombinant-type vaccine.

Am J Respir Crit Care Med, 1998 May, 157(5 Pt 1), 1645 - 51
Contribution of macrophages to pulmonary nitric oxide production in septic shock; Fujii Y et al.; Bacterial lipopolysaccharide (LPS) is known to induce the expression of inducible nitric oxide synthase (iNOS) in the lung and to lead to increased pulmonary nitric oxide (NO) production . The contribution of various pulmonary cells to this phenomenon remains unclear . In this study, we used gadolinium chloride, a blocker of macrophage activation, to assess the role of macrophages in LPS-induced pulmonary NO production . Anesthetized, mechanically ventilated rats were injected with either saline or LPS (Escherichia coli endotoxin) and studied for 5 h . Two other groups of rats were pretreated 24 h earlier with gadolinium chloride . Unlike control rats, rats injected with LPS showed a progressive decline in arterial pressure and a several-fold rise in lung iNOS activity and exhaled NO concentration . Large numbers of alveolar macrophages also expressed iNOS after LPS injection . Gadolinium chloride pretreatment eliminated the rise in lung iNOS activity and protein expression and significantly attenuated the increase in pulmonary exhaled NO product, but it had no effect on arterial pressure . Fewer numbers of alveolar macrophages expressed iNOS protein after gadolinium pretreatment . We conclude that macrophage activation plays a critical role in enhancing NO production in the respiratory system, but it is of less importance in mediating hemodynamic alterations of acute endotoxemia.

Am J Respir Crit Care Med, 1998 May, 157(5 Pt 1), 1542 - 9
Endogenous nitric oxide and the pulmonary microvasculature in healthy sheep and during systemic inflammation; Hinder F et al.; Nitric oxide (NO) influences microvascular integrity . NO synthase inhibitors are regarded as therapeutic options, but their impact on the pulmonary microvasculature is not well defined . We studied the microvascular effects of the nonselective NO synthase inhibitor N(omega)-nitro L-arginine methylester (L-NAME) in healthy sheep and during systemic inflammation . Permeability analysis was performed in 30 adult ewes with chronic lung lymph fistulas and pulmonary venous occluders . Experiment 1: 20 sheep received Escherichia coli endotoxin (lipopolysaccharide, 10 ng/kg/min) for 32 h . After 24 h of endotoxemia, 10 sheep were given L-NAME (25 mg/kg), and 10 sheep received NaCl 0.9% . Experiment 2: six sheep were treated with L-NAME (25 mg/kg), and four animals received NaCl 0.9% . Endotoxin induced a phasic pulmonary microvascular response with early transiently increased endothelial permeability at 4 h and late normalization of microvascular integrity to large molecules after 24 h . At that time systemic vasodilation had occurred . L-NAME raised pulmonary artery pressure and pulmonary vascular resistance index without signs of increased permeability in either experiment . NO is involved in vascular tone in healthy sheep and during systemic inflammation, but it does not seem to play a role in the integrity of the pulmonary microvascular barrier function to large molecules.

Biomed Sci Instrum, 1997, 34, 157 - 62
Analysis of biological particles using dielectrophoresis and impedance measurement; Milner KR et al.; A novel electrical technique for detecting the collection of particles by dielectrophoresis (DEP) is described . The method is based on the impedance changes resulting from this collection in a microfabricated, integrated dual-channel electrode structure . The results show good agreement with measurements of DEP collection by optical method for suspensions of bacterial species B . subtilis, E . coli and abiotic latex beads, but with substantially reduced experimental uncertainties . The technique overcomes the restriction on particle size of optical techniques and can potentially be used to investigate highly sub-micron sized particles like viruses and DNA fragments . The dual-channel electrode cells can be integrated with other structures for analysing sub-micron scale particles, for example chip-based capillary electrophoresis.

Biochim Biophys Acta, 1998 Apr 2, 1383(2), 292 - 300
Analysis of the secondary structure of the catalytic domain of mouse Ras exchange factor CDC25Mm; Coccetti P et al.; The minimal active domain (GEF domain) of the mouse Ras exchange factor CDC25Mm was purified to homogeneity from recombinant Escherichia coli culture . The 256 amino acids polypeptide shows high activity in vitro and forms a stable complex with H-ras p21 in absence of guanine nucleotides . Circular dichroism (CD) spectra in the far UV region indicate that this domain is highly structured with a high content of alpha-helix (42%) . Near UV CD spectra evidenced good signal due to phenylalanine and tyrosine while a poor contribution was elicited by the three tryptophan residues contained in this domain . The tryptophan fluorescence signal was scarcely affected by denaturation of the protein or by formation of the binary complex with H-ras p21, suggesting that the Trp residues, which are well conserved in the GEF domain of several Ras-exchange factors, were exposed to the surface of the protein and they are not most probably directly involved in the interaction with Ras proteins.

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