Escherichia coli

J Bacteriol, 1997 Feb, 179(4), 987 - 97
Cloning and characterization of the Helicobacter pylori flbA gene, which codes for a membrane protein involved in coordinated expression of flagellar genes; Schmitz A et al.; Flagellar motility has been shown to be an essential requirement for the ability of Helicobacter pylori to colonize the gastric mucosa . While some flagellar structural components have been studied in molecular detail, nothing was known about factors that play a role in the regulation of flagellar biogenesis . We have cloned and characterized an H . pylori homolog (named flbA) of the lcrD/flbF family of genes . Many proteins encoded by these genes are known to be involved in flagellar biogenesis or secretion of virulence-associated proteins via type III secretion systems . The H . pylori flbA gene (2,196 bp) is capable of coding for a predicted 732-amino-acid, 80.9-kDa protein that has marked sequence similarity with other known members of the LcrD/FlbF protein family . An isogenic strain with a mutation in the flbA gene was constructed by disruption of the gene with a kanamycin resistance cassette and electroporation-mediated allelic exchange mutagenesis . The mutant strain expressed neither the FlaA nor the FlaB flagellin protein . The expression of the FlgE hook protein was reduced in comparison with the wild-type strain, and the extent of this reduction was growth phase dependent . The flbA gene disruption was shown to downregulate the expression of these flagellar genes on the transcriptional level . The flbA mutants were aflagellate and completely nonmotile . Occasionally, assembled hook structures could be observed, indicating that export of axial flagellar filament components was still possible in the absence of the flbA gene product . The hydrophilic part of the FlbA protein was expressed in Escherichia coli, purified, and used to raise a polyclonal rabbit antiserum against the FlbA protein . Western blot experiments with this antiserum indicated that the FlbA protein is predominantly associated with the cytoplasmic membrane in H . pylori . The antiserum cross-reacted with two other proteins (97 and 43 kDa) whose expression was not affected by the flbA gene disruption and which might represent further H . pylori homologs of the LcrD/FlbF protein family.

Curr Genet, 1997 Feb, 31(2), 133 - 8
Isolation and characterisation of a gene encoding protein disulphide isomerase, pdiA, from Aspergillus niger; Ngiam C et al.; Current strategies to improve the secretion of heterologous proteins from Aspergillus niger include the manipulation of chaperones and foldases specific to the endoplasmic reticulum (ER) . Here we report the isolation of a gene, pdiA, encoding a putative protein disulphide isomerase (PDI) from A . niger using the Saccharomyces cerevisiae PDI gene as a probe . Sequencing of a genomic clone and RT-PCR products predict a 515-aa protein comprising a 20-aa ER-translocation signal sequence and a 495-aa mature protein (Mr = 54.3 kDa) . The predicted protein also contains two thiol oxidoreductase active sites with a -CGHC- motif and a carboxy terminal -HDEL ER-retention signal . Three introns were identified within the pdiA gene and Southern- and dot-blot analysis indicates that the gene is present in a single copy . Northern-blot analysis shows a transcript of the predicted size . Sequence homology to a motif associated with protein trafficking and the induction of chaperones has been identified in the pdiA promoter . Transcription of pdiA is induced 3-4-fold after treatment with tunicamycin, an inhibitor of N-linked glycosylation . The kinetics of induction suggest that pdiA expression is not part of the primary stress response.

J Gen Virol, 1997 Feb, 78 ( Pt 2), 343 - 51
Monoclonal antibodies specific for Semliki Forest virus replicase protein nsP2; Kujala P et al.; A panel of monoclonal antibodies (MAbs) was raised against Semliki Forest virus (SFV) nonstructural protein nsP2, which is a protease, an NTPase, a putative RNA helicase, and a regulator of the synthesis of the subgenomic 26S mRNA encoding the structural proteins . nsP2, used for immunization, was expressed as a histidine fusion protein in Escherichia coli and purified by metal affinity chromatography . Dot-blot assay, using a membrane fraction from SFV-infected cells as antigen, gave 33 positive clones . Of these, 30 MAbs recognized nsP2 in Western immunoblotting, and 25 showed positive indirect immunofluorescence (IFAT) in SFV-infected cells; 15 MAbs stained the cytoplasmic vacuoles (CPVI), which are the sites of viral RNA synthesis in alphavirus-infected cells . MAb 3B5 recognized only CPVIs, as shown by double immunofluorescence staining with polyclonal anti-nsP3 antiserum . Most of the MAbs (20/33) recognized the nuclear form of nsP2, which may be associated with SFV neurovirulence . Immunoprecipitation with MAbs revealed that the SFV nonstructural proteins are associated with each other . None of the MAbs recognized Sindbis virus nsP2 in immunoblotting, indicating that they were directed to non-conserved sequences specific for SFV . Interestingly, these epitopes were located mostly within the N-terminal half of nsP2 . Unexpectedly, the anti-nsP2 MAb 1E9 cross-reacted strongly with a host protein of 78 kDa from uninfected human, murine, avian and insect cells . This protein was identified as the immunoglobulin binding protein, BiP, by 2-D gel mapping and reaction with anti-BiP antiserum.

J Gen Virol, 1997 Feb, 78 ( Pt 2), 337 - 41
Internal proteolysis of the NS3 protein specified by dengue virus 2; Teo KF et al.; The NS3 protein of flaviviruses is a multifunctional polypeptide required for virus replication . Enzymic activities that have been demonstrated or predicted from the presence of sequence motifs include protease, NTPase, helicase and RNA triphosphatase . Both full-length and truncated forms of NS3 have been identified in infected cells . To examine internal cleavage of the NS3 protein of dengue virus 2 (DEN-2), infected cells or COS cells transfected with cDNA encoding NS2B/3 were radiolabelled and immunoprecipitated with antiserum against NS3 or hyperimmune mouse ascitic fluid . The polypeptides detected were NS2B/3 (Mr 83000), NS3 (Mr 69000), NS3' (Mr 50000) and NS3" (Mr 19000) . The latter polypeptide has not been previously identified . For DEN-2, it has been proposed that NS3' results from cleavage at the site ...R457R / GR460.. . within an RNA helicase sequence motif of NS3 . Our results demonstrated that cleavage occurred at this site, and that prior cleavage between NS2B/NS3 was not necessary.

Biophys J, 1997 Feb, 72(2 Pt 1), 936 - 41
pH-dependent conformational changes in Escherichia coli dihydrofolate reductase revealed by Raman difference spectroscopy; Chen YQ et al.; The catalytic site of all dihydrofolate reductases contains an invariant carboxylic acid, equivalent to Asp-27 in Escherichia coli dihydrofolate reductase (ecDHFR) . It has been found that various kinetic and ligand binding properties of ecDHFR show a pH profile with a pKa of about 6.5 . The group responsible for this pKa is often assumed to be carboxyl group of Asp-27 . To determine the ionization state of this carboxyl and its pKa, we have employed a novel method, based on Raman difference spectroscopy, to obtain its vibrational spectrum in situ . The method is general for the study of protein carboxyl groups, which are often significantly implicated in protein function and structure; this study establishes the method's limits and problems . The Raman difference spectrum between wild-type ecDHFR and the Asp-27 to serine mutant (D27S) in the pH range 5.6-9.0 has been taken . No protonation of the carboxyl group was detected, implying that its pKa is probably less than 5.0 . We did, however, detect a pH dependence in the intensity of Raman bands in the difference spectrum with a pKa of 6.3, indicating that the apo enzyme undergoes a pH-dependent conformational change . Because the carboxyl group of Asp-27 at the active site is the only ionizable group in the binding site, other groups, away from the catalytic site, must be responsible for the pH behavior of ecDHFR.

Nucleic Acids Res, 1997 Feb 1, 25(3), 682 - 4
High efficiency of site-directed mutagenesis mediated by a single PCR product; Chen X et al.; We describe a highly efficient procedure for site-specific mutagenesis of double-stranded plasmids . The method relies on a single PCR primer which incorporates both the mutations at the selection site and the desired single base substitutions at the mutant site . This primer is annealed to the denatured plasmid and directs the synthesis of the mutant strand . After digestion with selection enzyme, the plasmid DNA is amplified into Escherichia coli strain BMH71-18 and subjected to a second digestion and amplification into the bacterial strain DH5alpha . A mutagenesis efficiency >80% was consistently achieved in the case of two unrelated plasmids.

Nucleic Acids Res, 1997 Feb 1, 25(3), 597 - 603
Replication bypass and mutagenic effect of alpha-deoxyadenosine site-specifically incorporated into single-stranded vectors; Shimizu H et al.; alpha-2'-Deoxyadenosine (alpha) is a major adenine lesion produced by gamma-ray irradiation of DNA under anoxic conditions . In this study, single-stranded recombinant M13 vectors containing alpha were constructed and transfected into Escherichia coli to assess lethal and mutagenic effects of this lesion . The data for alpha were further compared with those obtained with M13 vectors containing normal A or a model abasic site (F) at the same site . The transfection assay revealed that alpha constituted a moderate block to DNA replication . The in vivo replication capacity to pass through alpha was approximately 20% relative to normal A, but 20-fold higher than that of F constituting an almost absolute replication block . Similar data were obtained by in vitro replication of oligonucleotide templates containing alpha or F by E.coli DNA polymerase I . The mutagenic consequence of replicating M13 DNA containing alpha was analyzed by direct DNA sequencing of progeny phage . Mutagenesis was totally targeted at the site of alpha introduced into the vector . Mutation was exclusively a single nucleotide deletion and no base substitutions were detected . The deletion frequency associated alpha was dependent on the 3'-nearest neighbor base: with the 3'-nearest neighbor base T mutation (deletion) frequency was 26%, whereas 1% with the 3'-nearest neighbor base G . A possible mechanism of the single nucleotide deletion associated with alpha is discussed on the basis of the misinsertion-strand slippage model.

Nucleic Acids Res, 1997 Feb 1, 25(3), 537 - 45
Characterization of DbpA, an Escherichia coli DEAD box protein with ATP independent RNA unwinding activity; Boddeker N et al.; DbpA is a putative Escherichia coli ATP dependent RNA helicase belonging to the family of DEAD box proteins . It hydrolyzes ATP in the presence of 23S ribosomal RNA and 93 bases in the peptidyl transferase center of 23S rRNA are sufficient to trigger 100% of the ATPase activity of DbpA . In the present study we characterized the ATPase and RNA unwinding activities of DbpA in more detail . We report that-in contrast to eIF-4A, the prototype of the DEAD box protein family-the ATPase and the helicase activities of DbpA are not coupled . Moreover, the RNA unwinding activity of DbpA is not specific for 23S rRNA, since DbpA is also able to unwind 16S rRNA hybrids . Furthermore, we determined that the ATPase activity of DbpA is triggered to a significant extent not only by the 93 bases of the 23S rRNA previously reported but also by other regions of the 23S rRNA molecule . Since all these regions of 23S rRNA are either part of the 'functional core' of the 50S ribosomal subunit or involved in the 50S assembly, DbpA may play an important role in the ribosomal assembly process.

Nucleic Acids Res, 1997 Feb 1, 25(3), 491 - 6
Selective recognition of a cisplatin-DNA adduct by human mismatch repair proteins; Yamada M et al.; The antitumor agent cis-diamminedichloroplatinum(II) (cisplatin) introduces cytotoxic DNA damage predominantly in the form of intrastrand crosslinks between adjacent purines . Binding assays using a series of duplex oligonucleotides containing a single 1,2 diguanyl intrastrand crosslink indicate that human cell extracts contain factors that preferentially recognise this type of damage when the complementary strand contains T opposite the 3', and C opposite the 5'guanine in the crosslink . Under the conditions of the band-shift assay used, little binding is observed if the positions of the T and C are reversed in the complementary strand . Similarly, duplexes containing CC or TT opposite the crosslink are recognised relatively poorly . The binding activity is absent from extracts of the colorectal carcinoma cell lines LoVo and DLD-1 in which the hMutSalpha mismatch recognition complex is inactivated by mutation . Extensively purified human hMutSalpha exhibits the same substrate preference and binds to the mismatched platinated DNA at least as well as to an identical unplatinated duplex containing a single G.T mismatch . It is likely, therefore, that human mismatch repair may be triggered by 1,2 diguanyl intrastrand crosslinks that have undergone replicative bypass.

Nucleic Acids Res, 1997 Feb 1, 25(3), 480 - 91
Differential human nucleotide excision repair of paired and mispaired cisplatin-DNA adducts; Moggs JG et al.; In order to understand the action of the chemotherapeutic drug cisplatin, it is necessary to determine why some types of cisplatin-DNA intrastrand crosslinks are repaired better than others . Using cell extracts and circular duplex DNA, we compared nucleotide excision repair of uniquely placed 1,2-GG, 1,2-AG, and 1,3-GTG cisplatin-crosslinks, and a 2-acetylaminofluorene lesion . The 1,3 crosslink and the acetylaminofluorene lesion were repaired by normal cell extracts approximately 15-20 fold better than the 1,2 crosslinks . No evidence was found for selective shielding of 1,2 cisplatin crosslinks from repair by cellular proteins . Fractionation of cell extracts to remove putative shielding proteins did not improve repair of the 1,2-GG crosslink, and cell extracts did not selectively inhibit access of UvrABC incision nuclease to 1,2-GG crosslinks . The poorer repair of 1,2 crosslinks in comparison to the 1,3 crosslink is more likely a consequence of different structural alterations of the DNA helix . In support of this, a 1,2-GG-cisplatin crosslink was much better repaired when it was opposite one or two non-complementary thymines . Extracts from cells defective in the hMutSalpha mismatch binding activity also showed preferential repair of the 1,3 crosslink over the 1,2 crosslink, and increased repair of the 1,2 adduct when opposite thymines, showing that hMutSalphais not involved in the differential NER of these substrates in vitro . Mismatched cisplatin adducts could arise by translesion DNA synthesis, and improved repair of such adducts could promote cisplatin-induced mutagenesis in some cases.

Nucleic Acids Res, 1997 Feb 1, 25(3), 474 - 9
Kinetics of excision of purine lesions from DNA by Escherichia coli Fpg protein; Karakaya A et al.; The kinetics of excision of damaged purine bases from oxidatively damaged DNA by Escherichia coli Fpg protein were investigated . DNA substrates, prepared by treatment with H2O2/Fe(III)-EDTA or by gamma-irradiation under N2O or air, were incubated with Fpg protein, followed by precipitation of DNA . Precipitated DNA and supernatant fractions were analyzed by gas chromatography/isotope-dilution mass spectrometry . Kinetic studies revealed efficient excision of 8-hydroxyguanine (8-OH-Gua), 2,6-diamino-4-hydroxy-5-formamidopyrimidine (FapyGua) and 4, 6-diamino-5-formamidopyrimidine (FapyAde) . Thirteen other modified bases in the oxidized DNA substrates, including 5-hydroxycytosine and 5-hydroxyuracil, were not excised . Excision was measured as a function of enzyme concentration, substrate concentration, time and temperature . The rate of release of modified purine bases from the three damaged DNA substrates varied significantly even though each DNA substrate contained similar levels of oxidative damage . Specificity constants (kcat/KM) for the excision reaction indicated similar preferences of Fpg protein for excision of 8-OH-Gua, FapyGua and FapyAde from each DNA substrate . These findings suggest that, in addition to 8-OH-Gua, FapyGua and FapyAde may be primary substrates for this enzyme in cells.

Arch Biochem Biophys, 1997 Feb 1, 338(1), 104 - 10
Catalytic and structural importance of Gly-454, Tyr-455, and Leu-456 in the carboxy-terminal region of Escherichia coli F1-ATPase alpha subunit; Yabuki M et al.; Monoclonal antibody alpha110 recognizes Leu-456 in the alpha subunit of the Escherichia coli F1-ATPase . Binding of this antibody to the alpha subunit or mutation of this residue to Pro caused enhancement of the ATPase activity, suggesting that this residue is involved in the catalytic mechanism of this molecule (H . Kanazawa et al . (1995) Arch . Biochem . Biophys . 317, 348-356) . Leu-456 together with Gly-454 and Tyr-455 are the only residues in the carboxy-terminal 75 amino acids conserved among various species, suggesting that these three residues play important roles in catalysis by the ATPase . Here, we introduced site-directed mutations into these residues . Not only L456P but also G454L, Y455K, Y455L, and L456N mutations caused enhancement of the ATPase activity . Surprisingly, Y455V, L456H, and L456S caused assembly defects of F1 subunits on the membrane . Reconstitution of the alpha betagamma complex from the wild-type beta and gamma subunits with the mutant alpha subunit (L4gamma6P) exhibited enhanced ATPase activity . Addition of delta or epsilon fused to glutathione S-transferase which are functionally similar to the delta and epsilon subunits, respectively, to the reconstituted F1-ATPase did not cause significant enhancement of its activity . Decreased interaction between alpha and beta subunits with the L456P mutation was detected by the yeast two-hybrid system . According to the deduced three-dimensional structure of the bovine a subunit, Leu-456, Gly-454, and Tyr-455 are included in a small alpha helix . These results suggest that this alpha helix affects interaction of the alpha subunit with the beta subunit but not with delta or epsilon, which may be important for the catalytic mechanism and F1 assembly.

Arch Biochem Biophys, 1997 Feb 1, 338(1), 83 - 9
Reversal of the nucleotide specificity of ketol acid reductoisomerase by site-directed mutagenesis identifies the NADPH binding site; Rane MJ et al.; Analysis of the published amino acid sequences of the enzyme ketol acid reductoisomerase (KARI) from seven organisms identified three regions with highly conserved sequences . One of these regions is predicted to be the dinucleotide fold where NADPH binds . In order to confirm that this region did include the NADPH binding site, we used oligonucleotide-mediated site-directed mutagenesis to study the function of specific amino acids in this region in terms of their interactions with NADPH . Four positively charged amino acids, R68, K69, K75, and R76, were mutated singly, in different combinations, and finally as a quartet in order to evaluate electrostatic interactions with NADPH . Mutation of each of the arginines singly to glutamine results in a 60- to 100-fold reduction in k(cat)/K(m) for NADPH . Mutation of each of the lysines singly does not significantly alter the steady state kinetic parameters associated with NADPH . None of these mutations significantly alters the affinity of the enzyme for NADH . After looking at double mutations of these four amino acids, we constructed the quadruplet mutant R68DK69LK75VR76D . This mutant has K(m) and k(cat) values of 19.3 microM and 5.3 min(-1) for NADH, which compares to 207 microM and 0.11 min(-1) for the wild-type enzyme . For the quadruplet mutant the corresponding values for NADPH are >200 microM for K(m) and 2 min(-1) for k(cat) compared to 7.3 microM and 7.2 min for the wild-type enzyme . By altering these four amino acids, the specificity constants for NADH and NADPH are almost exactly reversed in the mutant relative to the wild type.

Arch Biochem Biophys, 1997 Feb 1, 338(1), 57 - 66
Mutation of conserved residues in the NADP(H)-binding domain of the proton translocating pyridine nucleotide transhydrogenase of Escherichia coli; Bragg PD et al.; Possible NADP(H)-binding sites of the beta subunit of the pyridine nucleotide transhydrogenase of Escherichia coli were examined by site-directed mutagenesis . The sequence of the beta subunit at positions 314-350 showed several features typical of NADP(H)-binding sites . Mutation of betaGly314, the first glycine residue of the GXGXXV motif, and of betaArg350, which probably interacts with the 2'-phosphate of the substrate NADP(H), resulted in drastic loss of enzyme activity . The loss of activity in the betaArg350 mutants was not due to loss of ability to bind NADP(H) . Several residues (betaVal319, betaGly337, betaHis345, and betaArg350) were mutated to make the sequence more similar to that of a NAD(H)-binding site . The introduction of multiple mutations resulted in improper assembly of the enzyme and decreased incorporation into the membrane . The GXGXXG motif, typical of beta alphabeta nucleotide-binding folds, in the sequence of the beta subunit at positions 274-279 was mutated without causing major changes in transhydrogenase activities . It is unlikely to be part of a nucleotide-binding domain . Deletion of the carboxy-terminal 32 amino acids of the beta subunit, a possible nucleotide-binding site, prevented assembly and incorporation of the truncated enzyme into the cytoplasmic membrane of E . coli.

Arch Biochem Biophys, 1997 Feb 1, 338(1), 35 - 42
Cytochrome P450 2C11: Escherichia coli expression, purification, functional characterization, and mechanism-based inactivation of the enzyme; Licad-Coles E et al.; The male-specific P450 enzyme CYP 2C11, whose expression is developmentally and hormonally regulated, is the major steroid 16alpha-hydroxylase of the untreated rat liver . The enzyme metabolizes a host of substrates, including mechanism-based inactivators, such as 3,5-dicarbethoxy-2,6-dimethyl-4-ethyl-1,4-dihydropyridine (DDEP) and spironolactone (SPL) . Structural and functional characterization of the specific mode of such inactivation, however, requires sufficient quantities of the fully purified enzyme . Although several laboratories including our own have isolated and purified the enzyme from male rats, the yields are typically low and of the order of 1% . For these reasons, we chose to heterologously express the enzyme in Escherichia coli . The full-length cDNA was excised from the yeast vector pD2M1 and cloned into the plasmid vector pCW after appropriate modifications for optimal expression in E . coli . The enzyme was isolated and purified from E . coli membranes in relatively high yields (approximately 60%) and relatively high specific content (19 nmol/mg protein) . The purified recombinant enzyme had spectral and functional characteristics comparable to those reported for the native rat liver enzyme, including mechanism-based inactivation by DDEP and SPL . Studies with 14C-heme-labeled enzyme indicated that the major mode of DDEP inactivation was via heme-N-ethylation . On the other hand, studies with radiolabeled SPL-SH (the proximal inactivating deacetylated metabolite of SPL) revealed that although both {22-14C}SPL-SH and SPL-35SH inactivated the enzyme, only SPL-35SH was found to irreversibly radiolabel the 2C11 protein . The latter findings thus suggest that during mechanism-based inactivation of 2C11, the thiol moiety of SPL-SH is oxidatively activated to a species that attacks the 2C11 protein during or after cleavage from the thiosteroid . Thus, these modes of mechanism-based 2C11 inactivation by DDEP and SPL-SH considerably differ from the corresponding modes of P450 3A inactivation by these agents, wherein heme modification of the protein predominates.

J Immunol, 1997 Feb 1, 158(3), 1293 - 301
A Ca2+-dependent autoregulation of lipopolysaccharide-induced IL-8 receptor expression in human polymorphonuclear neutrophils; Bhattacharya C et al.; IL-8, a potent neutrophil chemotactic agent, is known to be a key mediator in several inflammatory diseases . We found that 10 ng/ml of serum-activated LPS (Escherichia coli) efficiently up-regulated IL-8R on the surface of neutrophils within 30 min of LPS stimulation by 115 to 120% through de novo protein synthesis . After 30 min of LPS stimulation, reduction of IL-8R level was initiated and the normal level was restored within 2 h of LPS interaction . EDTA or EGTA and bestatin separately inhibited the receptor down-regulation by 98%, indicating the involvement of metalloprotease(s), more specifically an aminopeptidase in the process . Induction and subsequent reduction of IL-8 binding in serum-activated LPS-stimulated cells have been demonstrated in autoradiography . Intracellular Ca2+ level in these stimulated neutrophils was increased and decreased with alteration of IL-8R level . Although IL-8 binding was drastically reduced, the total IL-8R level, as detected by anti-IL-8R Ab measured by 125I-labeled anti-rabbit IgG, remained almost unaltered, indicating that minimal proteolysis occurred in IL-8R . Anti-IL-8R Ab and IL-8 itself could prevent this down-regulation significantly, suggesting that the susceptible epitope(s) might be in the IL-8 binding domain of the receptor . Under Ca2+-depleted conditions, the proteolysis was inhibited, which was accelerated upon addition of 1 mM of CaCl2 . The study demonstrates that LPS-induced up-regulation of IL-8R leads to amplified IL-8-mediated biologic responses of neutrophils that are restored to normal level by activation of a Ca2+-dependent aminopeptidase . This may be useful for understanding the regulation of LPS-mediated inflammatory responses of neutrophils during bacterial infection.

Insect Mol Biol, 1997 Feb, 6(1), 97 - 104
The nonvitellogenic female protein of Musca domestica is an adult-specific hexamerin; Capurro Mde L et al.; During Musca domestica vitellogenesis a protein is preferentially synthesized by the female fat body and accumulates in the haemolymph but not in the ovaries . This protein, designated nonvitellogenic female protein (NVFP), was purified and shown to be a hexamer with an M(r) = 430 kDa, and subunits of M(r) = 70 kDa . The hexamer dissociates into subunits when the pH is elevated from 7.0 to 9.0 . Two cDNA clones, F0 and F2, were isolated and analysed . The 2.2 kb F2 clone has an open reading frame that encodes a conceptual translation product that has similarity to the Drosophila melanogaster LSP-2 hexamerin . Recombinant protein from the F2-cDNA is recognized by a specific anti-NVFP serum . The temporal pattern of mRNA expression of the gene represented by the F2 clone follows that determined for the synthesis of NVFP . The data support the conclusion that NVFP is an hexamerin specific to the adult stage of Musca domestica.

Cancer Res, 1997 Feb 1, 57(3), 461 - 5
In vivo gene therapy for alpha-fetoprotein-producing hepatocellular carcinoma by adenovirus-mediated transfer of cytosine deaminase gene; Kanai F et al.; The alpha-fetoprotein (AFP) gene is normally expressed in fetal liver and is transcriptionally silent in adult liver but overexpressed in human hepatocellular carcinoma (HCC) . Here, we demonstrate that replication defective recombinant adenoviral vectors, containing the human AFP promoter/enhancer, can be used to express the Escherichia coli cytosine deaminase (CD) gene (AdAFPCD) and the beta-galactosidase gene (AdAF-PlacZ) in AFP-producing HCC cell lines . Expression of the CD gene by adenovirus from the AFP promoter/enhancer (AdAFPCD) induced cells sensitive to 5-fluorocytosine (5FC) in the AFP-producing cells but not in the AFP-nonproducing cells . Transduction by an adenoviral vector harboring an ubiquitous strong promoter and CD gene showed enzymatic activity and 5FC killing in all cell lines . When AdAFPlacZ was injected into the s.c . established hepatoma in vivo, expression of the beta-galactosidase gene was confined to AFP-producing HCC xenografts . Moreover, HCC xenografts regressed by transduction with AdAFPCD and subsequently with 5FC treatment in vivo . These findings suggest that utilization of the AFP promoter/enhancer in an adenoviral vector can confer selective expression of a heterologous suicide gene in hepatocellular carcinoma cells in vitro and in vivo.

Infect Immun, 1997 Feb, 65(2), 847 - 51
Adhesive factor/rabbit 2, a new fimbrial adhesin and a virulence factor from Escherichia coli O103, a serogroup enteropathogenic for rabbits; Fiederling F et al.; Enteropathogenic Escherichia coli-like E . coli strains belonging to serovar O103:K-:H2 and rhamnose-negative biotypes are highly pathogenic diarrhea-inducing strains for weaned European rabbits . We describe here the cloning and sequencing of the major subunit gene of a new fimbrial adhesin, adhesive factor/rabbit 2 (AF/R2), which confers on these strains the ability to attach to rabbit enterocytes and to HeLa cells in a diffuse manner and which is associated with in vivo virulence . The chromosomal operon that encodes functional AF/R2 has been cloned from strain B10 . The major subunit gene afr2G, as well as an adjacent open reading frame, afr2H, has been sequenced . The Afr2G protein shows homologies with FaeG and ClpG, which are the respective major subunits of fimbrial adhesin K88 (F4) and afimbrial adhesin CS31A . Plasmid carrying the operon transcomplements an AF/R2-negative TnphoA mutant for its ability to express AF/R2 . As a whole, AF/R2 is a new member of the E . coli K88 adhesin family which is associated with virulence and which may serve in the design of vaccines.

Infect Immun, 1997 Feb, 65(2), 531 - 6
Escherichia coli K5 capsule expression enhances colonization of the large intestine in the gnotobiotic rat; Herias MV et al.; The role of capsule expression in the capacity of Escherichia coli to colonize in the large intestinal environment was studied in a gnotobiotic rat model . The rats were given perorally a mixture of two mutant strains differing in K5 expression . After 2 weeks, the rats were sacrificed, and subsequently intestinal contents, intestinal mucosae, and mesenteric lymph nodes were homogenized and bacterial numbers were quantified . Two E . coli mutant pairs were used, the first pair (972-998) lacking the O-specific side chain and the second pair (973-997) carrying the O75 lipopolysaccharide . The K5+ mutants established themselves at a higher level than the K5- mutants (10(9) versus 10(6) CFU/g {P < 0.001} for the first pair and 10(9) versus 10(8) CFU/g {P < 0.01} for the second pair, respectively) . The results were confirmed by serology showing a K5+ phenotype for practically all isolates . The bacterial population associated with the mucosa was similar to that in the luminal contents with respect to the proportions of the respective mutants, and translocation occurred in numbers proportional to the intestinal population densities of the respective mutants . All mutants were able to express type 1 as well as P fimbriae . After colonization, the expression of P fimbriae remained high whereas only a minority of the isolates expressed type 1 fimbriae . The results suggest that capsule expression and P fimbriae enhance intestinal colonization by E . coli and that these virulence factors, by increasing bacterial densities in the intestine, secondarily increase translocation.

Infect Immun, 1997 Feb, 65(2), 507 - 13
A new putative fimbrial colonization factor, CS19, of human enterotoxigenic Escherichia coli; Grewal HM et al.; A gene probe derived from the colonization factor antigen I (CFA/I) operon cross-hybridized at very low stringency to plasmid DNA from coli surface antigen 17 (CS17)-producing enterotoxigenic Escherichia coli (ETEC) and from the ETEC strain F595C, which was negative for previously described CFAs, CSs, and putative colonization factors (PCFs) . A 16-kDa protein was identified in sodium dodecyl sulfate-polyacrylamide gel electrophoresis of heat extracts prepared after growth of strain F595C at 37 degrees C on CFA agar containing bile salts . Transmission electron microscopy revealed bile salt- and temperature-dependent expression of fimbriae with a diameter of 7 nm . After transformation with a recombinant plasmid harboring the cfaR gene, which encodes a positive regulator of several CFAs, PCFs, and CSs, the 16-kDa protein was hyperexpressed . Polyclonal antibodies raised against this protein bound to the fimbriae and inhibited the adhesion of F595C bacteria to tissue-cultured Caco-2 cells . Nucleotide sequence determination of the gene encoding the 16-kDa fimbrial subunit revealed a high degree of amino acid sequence homology to the CFA/I, CS1, CS2, CS4, CS14, and CS17 polypeptides . The term CS19 is proposed for the new fimbria.

Infect Immun, 1997 Feb, 65(2), 412 - 21
Molecular characterization of a 6.6-kilodalton Borrelia burgdorferi outer membrane-associated lipoprotein (lp6.6) which appears to be downregulated during mammalian infection; Lahdenne P et al.; Isolated outer membranes of Borrelia burgdorferi 297 were utilized to obtain partial amino acid sequence information for a low-molecular-weight, outer membrane-associated polypeptide . Degenerate oligonucleotide primers based upon this information were used to amplify a 100-bp probe for detection of the corresponding full-length gene within a B . burgdorferi total genomic library . The relevant open reading frame (ORF) encoded a polypeptide comprised of a 17-amino-acid putative signal peptide terminated by LFVAC, a probable consensus sequence for lipoprotein modification, and a mature protein of 51 amino acids (predicted molecular mass of 5.8 kDa) . The DNA sequences of the corresponding ORFs in B . burgdorferi 297 and B31 were identical; the corresponding ORF in strain N40 differed by only one nucleotide . Assuming conventional processing and acylation, the molecular weight of the lipoprotein, designated lp6.6, is about 6,600 . The lp6.6 gene, which was localized to the 49-kb linear plasmid of B . burgdorferi, subsequently was cloned and expressed in Escherichia coli as a fusion protein with glutathione S-transferase . Immunoblot analysis with monoclonal antibody 240.7 revealed that lp6.6 was identical to a low-molecular-weight, highly conserved B . burgdorferi lipoprotein reported previously (L . I . Katona, G . Beck, and G . S . Habicht, Infect . Immun . 60:4995-5003, 1992) . Results of indirect immunofluorescence assays, growth inhibition assays, passive immunizations, and active immunizations indicated that this outer membrane-associated antigen is not surface exposed in B . burgdorferi . Particularly interesting was the finding that mice and rhesus monkeys chronically infected with B . burgdorferi failed to develop antibodies against this antigen . We propose that high-level expression of lp6.6 is associated with the arthropod phase of the spirochetal life cycle and that expression of the gene is downregulated during mammalian infection.

Infect Immun, 1997 Feb, 65(2), 349 - 57
Identification of the gene family encoding the 160-kilodalton Trypanosoma cruzi complement regulatory protein; Norris KA et al.; Trypanosoma cruzi trypomastigotes are exquisitely resistant to the lytic effects of vertebrate complement, and this characteristic contributes to the survival of the parasites in the host bloodstream . Trypomastigotes avoid complement-mediated lysis by the production of a surface glycoprotein that inhibits the formation of the alternative and classical C3 convertase, thus preventing activation and amplification of the complement cascade at the parasite surface . We have developed a monoclonal antibody to the 160-kDa T . cruzi complement regulatory protein (CRP) and describe a one-step immunoaffinity purification procedure . The CRP was purified to homogeneity and subjected to amino-terminal peptide sequence analysis . Based on the protein sequence obtained, the CRP was identified as a member of a large family of trypomastigote-specific genes, and a complete cDNA was isolated and sequenced . The complete coding sequence was cloned in Escherichia coli, and antibodies raised against the full-length recombinant protein reacted specifically with a 160-kDa protein in trypomastigote membrane protein preparations as well as with native, purified CRP . Indirect immunofluorescence revealed that the protein is uniformly expressed at the cell surfaces of trypomastigotes.

J Bacteriol, 1997 Feb, 179(3), 968 - 71
Purification and in vitro phosphorylation of HupT, a regulatory protein controlling hydrogenase gene expression in Rhodobacter capsulatus; Elsen S et al.; The HupT protein of Rhodobacter capsulatus, involved in negative regulation of hydrogenase gene expression, is predicted to be a histidine kinase on the basis of sequence comparisons . The protein was overproduced in Escherichia coli, purified to homogeneity, and demonstrated to autophosphorylate in vitro in the presence of {gamma-32P}ATP . An H217N hupt mutant was constructed, and the mutant protein was shown to have lost kinase activity . This result, and the fact that the phosphoryl group in phosphorylated HupT appeared to be bound to an N atom, support the suggestion from sequence comparisons that HupT is a histidine kinase, which can autophosphorylate on the His217 residue.

J Bacteriol, 1997 Feb, 179(3), 959 - 63
Variation in RNA polymerase sigma subunit composition within different stocks of Escherichia coli W3110; Jishage M et al.; The composition of RNA polymerase sigma subunits was analyzed for stock strains of Escherichia coli K-12 W3110 in Japan . Heterogeneity was discovered with respect to two sigma subunits, sigma28 (sigmaF, the rpoF gene product) and sigma38 (sigmaS, the rpoS gene product) . Five different types of W3110 were identified: A-type lineages have both sigma subunits in intact forms; B-type lineages carry a truncated sigma38 subunit and an intact sigma28 subunit; C-type lineages carry an intact sigma28 subunit but lack a sigma38 subunit; D-type lineages have only a sigma38 subunit without a sigma28 subunit; and E-type stocks lack both sigma subunits . All the lineages examined, however, contain the intact forms of sigma70 (sigmaD, the rpoD gene product) and sigma54 (sigmaN, the rpoN gene product) . As expected from the lack of a sigma28 subunit, cells of D- and E-type lineages are nonmotile . The truncated form of the sigma38 subunit in B-type stocks carries two mutations near its N terminus and lacks C-terminal proximal region 4 due to an amber mutation . The failure of C- and E-type W3110 cells to express sigma38 and that of D- and E-type cells to express sigma28 were found to be due to defects in transcription even though the respective sigma subunit genes remain intact . These findings emphasize the importance of paying attention to possible variations in the genetic background between laboratory stocks originating from the same strain.

J Bacteriol, 1997 Feb, 179(3), 956 - 8
Isolation and characterization of a priB mutant of Escherichia coli influencing plasmid copy number of delta rop ColE1-type plasmids; Berges H et al.; The lethality induced by the overproduction in Escherichia coli of a heterologous protein was used to select bacterial mutants . In one of these, the mutation responsible was mapped to priB . We describe the isolation of this mutant, the sequencing of the mutated gene, and its in vivo effect on plasmid replication.

J Bacteriol, 1997 Feb, 179(3), 949 - 51
Dihydroneopterin triphosphate epimerase of Escherichia coli: purification, genetic cloning, and expression; Haussmann C et al.; The enzyme catalyzing the epimerization at position 2' of dihydroneopterin triphosphate was purified by a factor of about 10,000 from cell extract of Escherichia coli . The cognate gene was cloned, sequenced, expressed, and mapped to kb 2427 on the E . coli chromosome.

J Bacteriol, 1997 Feb, 179(3), 909 - 18
The Escherichia coli stpA gene is transiently expressed during growth in rich medium and is induced in minimal medium and by stress conditions; Free A et al.; The transcriptional regulation of the stpA gene, encoding the Escherichia coli H-NS-like protein StpA, has been studied as a function of a variety of environmental conditions, and its response to trans-acting factors has been characterized . Chromosomally located stpA is expressed primarily from a promoter immediately upstream of the gene which is severely repressed by the homologous nucleoid-associated protein H-NS . However, we show here that even in a strain containing functional H-NS, stpA is transiently induced during growth of a batch culture in rich medium . It can also be induced strongly by osmotic shock and, to a lesser extent, by an increase in growth temperature . Moreover, when cells are grown in minimal medium, we observe a more sustained induction of stpA which is dependent on the leucine-responsive regulatory protein (Lrp) . This enhanced level of stpA transcription is virtually abolished in an H-NS-independent manner when the culture undergoes carbon starvation . A sensitivity of the stpA promoter to DNA topology may contribute to some of these responses . Results reported here show that cloned fragments of the stpA promoter region can confer H-NS and Lrp responsiveness upon a lacZ reporter gene and suggest that several hundred base pairs of DNA upstream of the transcriptional start may be required for regulation by these two proteins.

J Bacteriol, 1997 Feb, 179(3), 880 - 8
Stability of linear DNA in recA mutant Escherichia coli cells reflects ongoing chromosomal DNA degradation; Kuzminov A et al.; To study the fate of linear DNA in Escherichia coli cells, we linearized plasmid DNA at a specific site in vivo and monitored its behavior in recA mutant cells deficient in recombinational repair . Earlier, we had found that in wild-type (WT) cells linearized DNA is degraded to completion by RecBCD nuclease . We had also found that in WT cells chi sites on linear DNA inhibit RecBCD degradation by turning off its nucleolytic activities . Now we report that chi sites do not work in the absence of the RecA protein, suggesting that RecA is required in vivo to turn off the degradative activities of the RecBCD enzyme . We also report that the degradation of linearized plasmid DNA, even devoid of chi sites, is never complete in recA cells . Investigation of this linear DNA stability indicates that a fraction of recA cells are recBC phenocopies due to ongoing chromosomal DNA degradation, which titrates RecBCD nuclease . A possible role for RecBCD-promoted DNA degradation in controlling chromosomal DNA replication in E . coli is discussed.

J Bacteriol, 1997 Feb, 179(3), 825 - 30
Genetic analysis of the catalytic domain of the chemotaxis-associated histidine kinase CheA; Ellefson DD et al.; Escherichia coli cells express two forms of CheA, the histidine kinase associated with chemotaxis . The long form, CheA(L), plays a critical role in chemotactic signal transduction by phosphorylating two chemotaxis-associated response regulators, CheY and CheB . CheA(L) first autophosphorylates amino acid His-48 before its phosphoryl group is transferred to these response regulators . The short form, CheA(S), lacks the amino-terminal 97 amino acids of CheA(L) and therefore does not possess the site of phosphorylation . The centrally located transmitter domain of both forms of CheA contains four regions, called N, G1, F, and G2, highly conserved among histidine kinases of the family of two-component signal transduction systems . On the basis of sequence similarity to highly conserved regions of certain eukaryotic kinases, the G1 and G2 regions are purported to be involved in the binding and hydrolysis of ATP . We report here that alleles mutated in the G1, G2, or F region synthesize CheA variants that cannot autophosphorylate in vitro and which cannot support chemotaxis in vivo . We also show that in vitro, the nonphosphorylatable CheA(S) protein mediates transphosphorylation of a CheA(L) variant defective in both G1 and G2 . In contrast, CheA(L) variants defective for either G1 or G2 mediate transphosphorylation of each other poorly, if at all . These results are consistent with a mechanism by which the G1 and G2 regions of one protomer of a CheA dimer form a unit that mediates transphosphorylation of the other protomer within that dimer.

J Bacteriol, 1997 Feb, 179(3), 784 - 93
Two polypeptide products of the Escherichia coli cell division gene ftsW and a possible role for FtsW in FtsZ function; Khattar MM et al.; Two new mutations in the cell division gene ftsW have been isolated and characterized . The ftsW263(Ts) mutation results in a block to division at the initiation stage, similar to that previously observed with the ftsW201(Ts) mutation . The ftsW1640(Ts) mutation, however, causes a block to division at a later stage . The ftsW201 and ftsW263 mutants were shown to be phenotypically sensitive to the genetic background and growth conditions and are possibly relA dependent . Immunofluorescence microscopy showed that the FtsZ protein can localize to presumptive division sites in strains carrying ftsW(Ts) mutations at the nonpermissive temperature, suggesting that FtsW is unlikely to be specifically required for the localization of FtsZ to the division site . Examination of the localization of FtsZ in an ftsW rodA double mutant (lemon-shaped cells) revealed several classes of cells ranging from a common class where an FtsZ ring structure is absent to a class where FtsZ forms a complete ring at the midpoint of a lemon-shaped cell, suggesting a role for FtsW in the establishment of a stable FtsZ-based septal structure . We further demonstrate that two FtsW peptides, FtsWL (large) and FtsWS (small), can be identified and that the expression of ftsWS is sufficient for complementation of ftsW(Ts) mutations.

J Bacteriol, 1997 Feb, 179(3), 742 - 53
Importance of structural differences between complementary RNA molecules to control of replication of an IncB plasmid; Wilson IW et al.; Replication of the IncB miniplasmid pMU720 is dependent on the expression of repA, the gene encoding replication initiator protein RepA . Binding of a small antisense RNA (RNAI) to its complementary target (stem-loop I {SLI}) in the RepA mRNA prevents the participation of SLI in the formation of a pseudoknot that is an enhancer of translation of this mRNA . Thus, RNAI regulates the frequency of replication of pMU720 by controlling the efficiency of translation of the RepA mRNA . Mutational analysis of the two seven-base complementary sequences involved in formation of the pseudoknot showed that only the five central bases of each were critical for the formation of the pseudoknot . Physical analysis of SLI showed that despite the complete complementarity of its sequence to that of RNAI, the structures of the two molecules are different . The most prominent difference between the two structures is the presence of a 4-base internal loop immediately below the hairpin loop of SLI but not that of RNAI . Closure of this internal loop in SLI resulted in a 40-fold reduction in repA expression and loss of sensitivity of the residual expression to inhibition by RNAI . By contrast, repA expression was largely unaffected by the closure of a lower internal loop whose presence in SLI and RNAI is essential for effective interaction between these two molecules . These results suggest that the interaction of SLI with the distal pseudoknot bases is fundamentally different from the RNAI-SLI binding interaction and that the differences in structure between RNAI and SLI are necessary to allow SLI to be able to efficiently bind RNAI and to participate in pseudoknot formation.

J Bacteriol, 1997 Feb, 179(3), 735 - 41
Role of conserved residues in hydrophilic loop 8-9 of the lactose permease; Pazdernik NJ et al.; A peptide motif, GXXX(D/E)(R/K)XG(R/K)(R/K), has been conserved in a large group of evolutionarily related membrane proteins that transport small molecules across the membrane . Within the superfamily, this motif is located in two cytoplasmic loops that connect transmembrane segments 2 and 3 and transmembrane segments 8 and 9 . In a previous study concerning the loop 2-3 motif of the lactose permease (A . E . Jessen-Marshall, N . J . Paul, and R . J . Brooker, J . Biol . Chem . 270:16251-16257, 1995), it was shown that the first-position glycine and the fifth-position aspartate are critical for transport activity since a variety of site-directed mutations greatly diminished the rate of transport . In the current study, a similar approach was used to investigate the functional significance of the conserved residues in the loop 8-9 motif . In the wild-type lactose permease, however, this motif has been evolutionarily modified so that the first-position glycine (an alpha-helix breaker) has been changed to proline (also a helix breaker); the fifth position has been changed to an asparagine; and one of the basic residues has been altered . In this investigation, we made a total of 28 single and 7 double mutants within the loop 8-9 motif to explore the functional importance of this loop . With regard to transport activity, amino acid substitutions within the loop 8-9 motif tend to be fairly well tolerated . Most substitutions produced permeases with normal or mildly defective transport activities . However, three substitutions at the first position (i.e., position 280) resulted in defective lactose transport . Kinetic analysis of position 280 mutants indicated that the defect decreased the Vmax for lactose uptake . Besides substitutions at position 280, a Gly-288-to-Thr mutant had the interesting property that the kinetic parameters for lactose uptake were normal yet the rates of lactose efflux and exchange were approximately 10-fold faster than wild-type rates . The results of this study suggest that loop 8-9 may facilitate conformational changes that translocate lactose.

J Bacteriol, 1997 Feb, 179(3), 730 - 4
Oxidative inactivation of glutamine synthetase from the cyanobacterium Anabaena variabilis; Martin G et al.; In crude extracts of the cyanobacterium Anabaena variabilis, glutamine synthetase (GS) could be effectively inactivated by the addition of NADH . GS inactivation was completed within 30 min . Both the inactivated GS and the active enzyme were isolated . No difference between the two enzyme forms was seen in sodium dodecyl sulfate-gels, and only minor differences were detectable by UV spectra, which excludes modification by a nucleotide . Mass spectrometry revealed that the molecular masses of active and inactive GS are equal . While the Km values of the substrates were unchanged, the Vmax values of the inactive GS were lower, reflecting the inactivation factor in the crude extract . This result indicates that the active site was affected . From the crude extract, a fraction mediating GS inactivation could be enriched by ammonium sulfate precipitation and gel filtration . GS inactivation by this fraction required the presence of NAD(P)H, Fe3+, and oxygen . In the absence of the GS-inactivating fraction, GS could be inactivated by Fe2+ and H2O2 . The GS-inactivating fraction produced Fe2+ and H2O2, using NADPH, Fe3+, and oxygen . Accordingly, the inactivating fraction was inhibited by catalase and EDTA . This GS-inactivating system of Anabaena is similar to that described for oxidative GS inactivation in Escherichia coli . We conclude that GS inactivation by NAD(P)H is caused by irreversible oxidative damage and is not due to a regulatory mechanism of nitrogen assimilation.

J Bacteriol, 1997 Feb, 179(3), 721 - 9
Nitrate- and nitrite-sensing protein NarX of Escherichia coli K-12: mutational analysis of the amino-terminal tail and first transmembrane segment; Williams SB et al.; Nitrate and nitrite control of anaerobic respiratory gene expression is mediated by dual two-component regulatory systems . The sensors NarX and NarQ each communicate nitrate and nitrite availability to the response regulators NarL and NarP . In the presence of nitrate, the NarX protein acts as a positive regulator ("kinase") of both NarL and NarP activity . In the presence of nitrite, the NarX protein acts primarily as a negative regulator ("phosphatase") of NarL activity but remains a positive regulator of NarP activity . In other topologically similar sensory proteins, such as the methyl-accepting chemotaxis proteins, the transmembrane regions are important for signal transduction . We therefore used localized mutagenesis of the amino-terminal coding region to isolate mutations in narX that confer an altered signaling phenotype . Five of the mutations studied alter residues in the amino-terminal cytoplasmic tail, and five alter residues in the first transmembrane segment . Based on patterns of target operon expression in various regulatory mutant strain backgrounds, most of the mutant NarX proteins appear to have alterations in negative control function . One mutant, with a change of residue Leu-11 to Pro in the cytoplasmic tail, exhibits strikingly altered patterns of NarL- and NarP-dependent gene expression . We conclude that the amino terminus of the NarX protein is important for the differential response to nitrate and nitrite.

J Bacteriol, 1997 Feb, 179(3), 620 - 6
Detection and comparison of specific hemin binding by Porphyromonas gingivalis and Prevotella intermedia; Tompkins GR et al.; A radioligand assay was designed to detect and compare specific hemin binding by the periodontal anaerobic black-pigmenting bacteria (BPB) Porphyromonas gingivalis and Prevotella intermedia . The assay included physiological concentrations of the hemin-binding protein rabbit serum albumin (RSA) to prevent self-aggregation and nonspecific interaction of hemin with cellular components . Under these conditions, heme-starved P . intermedia cells (two strains) expressed a single binding site species (4,100 to 4,600 sites/cell) with a dissociation constant (Kd) of 1.0 x 10(-9) M . Heme-starved P . gingivalis cells (two strains) expressed two binding site species; the higher-affinity site (1,000 to 1,500 sites/cell) displayed a Kd of between 3.6 x 10(-11) and 9.6 x 10(-11) M, whereas the estimated Kd of the lower-affinity site (1.9 x 10(5) to 6.3 x 10(5) sites/cell) ranged between 2.6 x 10(-7) and 6.5 x 10(-8) M . Specific binding was greatly diminished in heme-replete cells of either BPB species and was not displayed by iron-replete Escherichia coli cells, which bound as much hemin in the absence of RSA as did P . intermedia . Hemin binding by BPB was reduced following treatment with protein-modifying agents (heat, pronase, and N-bromosuccinimide) and was blocked by protoporphyrin IX and hemoglobin but not by Congo red . Hemopexin also inhibited bacterial hemin binding . These findings indicate that both P . gingivalis and P . intermedia express heme-repressible proteinaceous hemin-binding sites with affinities intermediate between those of serum albumin and hemopexin . P . gingivalis exhibited a 10-fold-greater specific binding affinity and greater heme storage capacity than did P . intermedia, suggesting that the former would be ecologically advantaged with respect to heme acquisition.

J Clin Microbiol, 1997 Feb, 35(2), 527 - 30
Distribution of colonization factor antigens among enterotoxigenic Escherichia coli strains isolated from patients with diarrhea in Nepal, Indonesia, Peru, and Thailand; Nirdnoy W et al.; Samples (1,318) of enterotoxigenic Escherichia coli (ETEC) isolated in 1994-1995 from children with diarrhea from Nepal, Indonesia, Peru, and Thailand were examined for colonization factor antigen (CFA) and coli surface (CS) antigens . Fifty-five percent of 361 heat-labile and heat-stable (LT-ST), 14% of 620 LT-only, and 48% of 337 ST-only ETEC had CFA/CS antigens . LT-ST ETEC strains were predominantly in the CFA II group, and ST only strains were in the CFA IV group . Additional studies are needed to identify ETEC strains that do not have CFA/CS antigens.

Mol Cell Biol, 1997 Feb, 17(2), 977 - 88
Cleavage of membrane-associated pref-1 generates a soluble inhibitor of adipocyte differentiation; Smas CM et al.; pref-1 is an epidermal growth factor-like repeat protein present on the surface of preadipocytes that functions in the maintenance of the preadipose state . pref-1 expression is completely abolished during 3T3-L1 adipocyte differentiation . Bypassing this downregulation by constitutive expression of full-length transmembrane pref-1 in preadipocytes drastically inhibits differentiation . For the first time, we show processing of cell-associated pref-1 to generate both a soluble pref-1 protein of approximately 50 kDa that corresponds to the ectodomain and also smaller products of 24 to 25 kDa and 31 kDa . Furthermore, while all four of the alternately spliced forms of pref-1 produce cell-associated protein, only the two largest of the four alternately spliced isoforms undergo cleavage in the juxtamembrane region to release the soluble 50-kDa ectodomain . We demonstrate that addition of Escherichia coli-expressed pref-1 ectodomain to 3T3-L1 preadipocytes blocks differentiation, thus overriding the adipogenic actions of dexamethasone and methylisobutylxanthine . The inhibitory effects of the pref-1 ectodomain are blocked by preincubation of the protein with pref-1 antibody . That the ectodomain alone is sufficient for inhibition demonstrates that transmembrane pref-1 can be processed to generate an inhibitory soluble form, thereby greatly extending its range of action . Furthermore, we present evidence that alternate splicing is the mechanism that governs the production of transmembrane versus soluble pref-1, thereby determining the mode of action, juxtacrine or paracrine, of the pref-1 protein.

Mol Cell Biol, 1997 Feb, 17(2), 571 - 83
The human Myt1 kinase preferentially phosphorylates Cdc2 on threonine 14 and localizes to the endoplasmic reticulum and Golgi complex; Liu F et al.; Entry into mitosis requires the activity of the Cdc2 kinase . Cdc2 associates with the B-type cyclins, and the Cdc2-cyclin B heterodimer is in turn regulated by phosphorylation . Phosphorylation of threonine 161 is required for the Cdc2-cyclin B complex to be catalytically active, whereas phosphorylation of threonine 14 and tyrosine 15 is inhibitory . Human kinases that catalyze the phosphorylation of threonine 161 and tyrosine 15 have been identified . Here we report the isolation of a novel human cDNA encoding a dual-specificity protein kinase (designated Myt1Hu) that preferentially phosphorylates Cdc2 on threonine 14 in a cyclin-dependent manner . Myt1Hu is 46% identical to Myt1Xe, a kinase recently characterized from Xenopus laevis . Myt1Hu localizes to the endoplasmic reticulum and Golgi complex in HeLa cells . A stretch of hydrophobic and uncharged amino acids located outside the catalytic domain of Myt1Hu is the likely membrane-targeting domain, as its deletion results in the localization of Myt1Hu primarily to the nucleus.

J Virol, 1997 Feb, 71(2), 1538 - 46
The barley stripe mosaic virus 58-kilodalton beta(b) protein is a multifunctional RNA binding protein; Donald RG et al.; The barley stripe mosaic virus (BSMV) beta(b) gene product is the major viral nonstructural protein synthesized during early stages of the infection cycle and is required for systemic movement of the virus . To examine the biochemical properties of beta(b), a histidine tag was engineered at the amino terminus and the protein was purified from BSMV-infected barley tissue by metal affinity chromatography . The beta(b) protein bound ATPs in vitro, with a preference for ATP over dATP, and also exhibited ATPase activity . In addition, beta(b) bound RNA without detectable sequence specificity . However, binding was selective, as the beta(b) protein had a strong affinity for both single-stranded (ss) and double-stranded (ds) RNAs but not for tRNA or DNA substrates . Mutational analyses of beta(b) purified from Escherichia coli indicated that the protein has multiple RNA binding sites . These sites appear to contribute differently, because mutants that were altered in their binding affinities for ss and ds RNA substrates were recovered.

J Chromatogr A, 1997 Jan 31, 760(2), 165 - 71
Role of polyethyleneimine in the purification of recombinant human tumour necrosis factor beta; Loh KC et al.; The chromatographic behaviour of recombinant human tumour necrosis factor beta (rhTNF-beta) (pI approximately 9.0) during cation-exchange chromatography at pH 7.5 is investigated . Without prior treatment of the Escherichia coli cell extract with polyethyleneimine (PEI), very little rhTNF-beta was bound to the column . However, upon addition of 5% PEI (100 microliters ml-1) to the cell lysate, rhTNF-beta was shown to bind to cation-exchange columns normally . TNF-beta was readily precipitated from the clarified cell extract by 20% ammonium sulphate, but ony ca . 25% of this precipitate could be re-solubilized for further purification . However, when 5% PEI was included in the solubilization buffer, the balance of the rhTNF-beta could be recovered . It is proposed that charge interaction between rhTNF-beta and nucleic acids in the cell extract is responsible for both of these anomalous phenomena, and that PEI (a cationic polyelectrolyte) was able to disrupt this interaction by displacing rhTNF-beta from the charge complex.

Mutat Res, 1997 Jan 31, 383(1), 31 - 7
Different repair of O6-methylguanine occurring in DNA modified by MMS in vivo or in vitro; Sledziewska-Gojska E et al.; Lack of the adaptive response effect on the level of GC-->AT transitions induced by methyl methanesulfonate (MMS) in E . coli {Sledziewska-Gojska, E . (1993) The level of GC-->AT transitions induced by MMS is not affected by adaptive response of Escherichia coli K12 . Mutation Res., 294, 1-8.} can be explained by MMS inactivation of the ada encoded O6-methylguanine-DNA methyltransferase {Takahashi, K.Y., Kawazoe, K., Sakumi, Nakabeppu Y . and M . Sekiguchi (1988) Activation of Ada protein as a transcriptional regulator by direct alkylation with methylating agents, J . Biol . Chem., 263, 13490-13492; Sledziewska-Gojska, E . (1995) Inactivation of O6-methylguanine-DNA methyltransferase in vivo by SN2 alkylating agents, Mutation Res., 336, 61-67} . To evaluate this explanation and clarify the origin of MMS-induced GC-->AT transitions, we compared the repair of DNA treated by MMS in vivo or in vitro . Replication forms of lacZ mutants of E . coli phage M13mp18 were used to analyse the effect of the adaptive response on the frequency of GC-->AT transitions occurring in control and mismatch repair deficient strains . It was shown that DNA lesions, leading to GC-->AT transitions, induced by MMS in vivo are not repaired in adapted E . coli cells . In contrast, induction of the adaptive response causes efficient repair of these DNA lesions induced by MMS in vitro . This repair is consistent with the assumption that GC-->AT transitions induced by MMS are originated by O6-methylguanine and that MMS treatment of the cells during in vivo mutagenesis interfere with the adaptation mediated repair of the lesion . In agreement with this we have shown that treatment of the adapted cultures with 5 mM MMS completely blocks repair of in vitro modified DNA . Increased level of GC-->AT transitions induced by MMS occurs in mutS- strains . These mutations are avoided in adapted mutS- cells, when induced by MMS in vitro . This confirms that mismatch repair system of E . coli recognises mismatches formed in DNA by O6-methylguanine.

Mutat Res, 1997 Jan 31, 383(1), 21 - 30
Rejoining of DNA double-strand breaks after the introduction of chromosome 11 into a radiosensitive bladder carcinoma cell line; Hofseth LJ et al.; Insertion of a normal chromosome 11 into tumour cell lines can protect against a sensitivity to irradiation and oxidative stress . A possible mechanism underlying this effect is that there is a correction of a defect in the rejoining of double-strand breaks (dsb) by the chromosome insertion . In order to explore this hypothesis, three cell lines were evaluated for their ability to rejoin dsb: (1) a bladder carcinoma cell line ('parent') previously shown to be sensitive to irradiation and radical generating species; (2) a derivative of this cell line into which a normal chromosome 11 had been inserted by microcell fusion ('hybrid') showing corrected radiosensitivity; and (3) a 'revertant' cell line that had spontaneously lost the insert and reverted to the radiosensitive phenotype . Nuclear extracts from the 3 lines were isolated and evaluated for their capacity to rejoin plasmid (pUC18) DNA broken at defined restriction sites (SalI, EcoRI, KpnI, SmaI) in the lacZ gene . The extent of rejoining was determined by gel electrophoresis and the fidelity of rejoining determined by expression of the lacZ gene in E . coli DH5 alpha bacteria . Results suggest there is no difference between the 'parent', 'hybrid' and 'revertant' nuclear extracts in the fidelity and the total extent of rejoining, regardless of the type of break . However, there is an alteration in the distribution of rejoined products . Nuclear extracts from 'hybrid' cells tend to rejoin linear DNA into circular monomers with a greater efficiency than extracts from both 'parent' and 'revertant' cells . This alteration in distribution is observed when 3'- or 5'-protruding ends are rejoined but not in the rejoining of blunt ends . The results suggest that loci on chromosome 11 are involved in the rejoining of dsb, affecting the relative amount of the different rejoined products . Whether this alteration plays a role in the 'parent' cell's radiosensitivity is yet to be determined.

J Mol Biol, 1997 Jan 31, 265(4), 385 - 93
Role of the spacer boxA of Escherichia coli ribosomal RNA operons in efficient 23 S rRNA synthesis in vivo; Pfeiffer T et al.; A boxA sequence, known to be important for transcriptional antitermination, is found in both the leader region and in the spacer between the 16 S and 23 S genes of Escherichia coli ribosomal RNA operons . We have shown that a functional leader boxA is important for efficient completion of 16 S rRNA transcription . In this study, point mutations were introduced into the 16S-23S spacer boxA of a plasmid-encoded E . coli rrnB operon in order to study the contribution of this conserved sequence element to ribosomal RNA synthesis in vivo . The rrnB mutant constructs contained an additional point mutation in each of the 16 S and 23 S genes, which were used to distinguish rRNA derived from plasmid and chromosomal rrn operons by primer extension analysis . Mutations in the spacer boxA reduced the proportion of plasmid-derived 23 S rRNA without affecting synthesis of plasmid-derived 16 S rRNA or spacer boxA RNA, indicating that premature termination of transcription occurred during 23 S rRNA synthesis . Reductions in plasmid-derived 23 S rRNA were very similar for total cellular RNA, 50 S subunits and 70 S ribosomes, suggesting that plasmid-derived rRNAs from mutant operons were functional in ribosome biogenesis . In the presence of a wild-type leader boxA, single nucleotide exchanges in the spacer boxA reduced the proportion of plasmid-derived 23 S rRNA from 70% to about 55% under conditions of exponential growth in rich medium . This proportion further decreased to 20 to 25% with an additional point mutation in the leader boxA . We conclude that modification of RNA polymerase into a termination-resistant form has to be renewed at the spacer boxA in order to ensure the faithful completion of full-length 23 S rRNA.

Gene, 1997 Jan 31, 185(1), 111 - 7
A simple screening for mutant DNA binding proteins: application to murine transcription factor PEBP2alpha subunit, a founding member of the Runt domain protein family; Akamatsu Y et al.; Mouse transcription factor PEBP2 (polyomavirus enhancer-binding protein (2) is composed of two distinct subunits alpha and beta . The alpha subunit has an ability to bind the specific DNA sequences, which is enhanced by formation of a heterodimer with the beta subunit . The DNA binding and heterodimerization activities of the alpha subunit are both localized within a 128-amino-acid (aa) region termed as the Runt domain for its homology to the Drosophila segmentation gene runt . To characterize the molecular determinants for these activities, the Runt domain was randomly mutagenized and produced in E . coli as a secreted form . Using E . coli culture supernatant, the DNA binding and heterodimerization of mutant Runt domains were analyzed by gel retardation assay . Nine randomly picked single-aa substitution mutants showed various functional alterations in DNA binding and heterodimerization either separately or simultaneously . This observation suggests that the structure of Runt domain is highly ordered and is quite sensitive to modulations in its primary structure . The method presented here provides a simple and quick method to characterize a large number of mutant DNA binding proteins.

Gene, 1997 Jan 31, 185(1), 105 - 9
Cloning and expression of AatII restriction-modification system in Escherichia coli; Nwankwo DO et al.; The genes encoding the AatII restriction endonuclease and methylase from Acetobacter aceti have been cloned and expressed in Escherichia coli . The nucleotide sequences of aatIIM and aatIIR genes were determined . The aatIIM and aatIIR genes are 996 bp and 1038 bp, respectively, encoding the 331-aa methylase with a predicted molecular mass of 36.9 kDa, and the 345-aa AatII restriction endonuclease with a predicted molecular mass of 38.9 kDa . The two genes overlap by 4 base pairs and are transcribed in the same orientation . The aatIIRM genes are located next to a putative gene for plasmid mobilization . A stable overproducing strain was constructed, in which the aatIIM gene was expressed from a pSC101-derived plasmid . The aatIIR gene was inserted into a modified T7 expression vector that carries transcription terminators upstream from the T7 promoter . The recombinant AatII restriction endonuclease was purified to near homogeneity by chromatography through DEAE Sepharose, Heparin Sepharose, and phosphocellulose columns.

J Biol Chem, 1997 Jan 31, 272(5), 2834 - 40
Overexpression, purification, and characterization of the catalase-peroxidase KatG from Mycobacterium tuberculosis; Johnsson K et al.; Wild-type catalase-peroxidase KatG from Mycobacterium tuberculosis as well as a specific mutant (R463L) frequently found in isoniazid-resistant strains have been overexpressed in Escherichia coli, allowing purification of sufficient quantities of enzyme for physical and kinetic characterization . Optical absorption and EPR spectroscopies indicate that KatG is similar to a growing class of bacterial catalase-peroxidases . Optical and EPR spectra of KatG in the presence of either a strong field or weak field ligand suggest that, like horseradish peroxidase and metmyoglobin, KatG is likely to have a histidine as a proximal ligand . The wild-type enzyme functions as a highly active catalase as well as a broad specificity peroxidase . Wild-type KatG and the R463L mutant of KatG exhibit identical spectroscopic and kinetic properties . Furthermore, both enzymes are equally capable of metabolizing the important antituberculosis drug isoniazid.

J Biol Chem, 1997 Jan 31, 272(5), 2744 - 52
Siroheme biosynthesis in higher plants . Analysis of an S-adenosyl-L-methionine-dependent uroporphyrinogen III methyltransferase from Arabidopsis thaliana; Leustek T et al.; Siroheme, the prosthetic group for both nitrite and sulfite reductases, is a methylated, iron-containing modified tetrapyrrole . Here we report the first molecular characterization of the branch point enzyme in higher plants, which directs intermediates toward siroheme synthesis . A cDNA was cloned from Arabidopsis thaliana (UPM1) that functionally complements an Escherichia coli cysG mutant, a strain that is unable to catalyze the conversion of uroporphyrinogen III (Uro'gen-III) to siroheme . UPM1 is 1484 base pairs and encodes a 369-amino acid, 39.9-kDa protein . The UPM1 product contains two regions that are identical to consensus sequences found in bacterial Uro'gen-III and precorrin methyltransferases . Recombinant UPM1 protein was found to catalyze S-adenosyl-L-methionine-dependent transmethylation by UPM1 in a multistep process involving the formation of a covalently linked complex with S-adenosyl-L-methionine . The UPM1 product has a sequence at the amino terminus that resembles a transit peptide for localization to mitochondria or plastids . The protein produced by in vitro expression is able to enter isolated intact chloroplasts but not mitochondria . Genomic blot analysis showed that UPM1 is encoded in the A . thaliana genome . The genomic DNA corresponding to UPM1 was cloned and sequenced and found to contain at least five introns.

J Biol Chem, 1997 Jan 31, 272(5), 2714 - 21
Real time conformational changes in the retinal phosphodiesterase gamma subunit monitored by resonance energy transfer; Berger AL et al.; The gamma subunit of the retinal cGMP phosphodiesterase (gammaPDE) acts as an inhibitor of phosphodiesterase (PDE) catalytic activity and mediates enzyme regulation by the alpha subunit of the GTP-binding protein transducin (alphaT) . In order to characterize conformational changes in the 87-amino acid gammaPDE subunit that may accompany the activation of the holoenzyme, gammaPDE was labeled with the fluorescent probes 5-iodoacetamidofluorescein and eosin-5-isothiocyanate for use in resonance energy transfer measurements . 5-Iodoacetamidofluorescein specifically labeled a cysteine residue at position 68 and served as a resonance energy transfer donor . The site of modification of eosin-5-isothiocyanate, which served as the resonance energy transfer acceptor, was determined to be within the first seven residues of the amino terminus of gammaPDE . Energy transfer between the labeled sites on free, unbound gammaPDE indicated that they were separated by a distance of 63 A, consistent with a random conformation . Upon binding the catalytic alphabeta subunits of the PDE, the distance between the two probes on gammaPDE increased to 77 A . Binding of the labeled gammaPDE by alphaT.guanosine 5'-3-O-(thio)triphosphate did not affect the distance between the probes under conditions where the PDE was activated . These data are consistent with the view that the binding of activated alphaT to gammaPDE, which is essential for the stimulation of PDE activity, does not impart significant alterations in the tertiary structure of the gammaPDE molecule . They also support a model for PDE activation that places active alphaT in a complex with the holoenzyme.

J Biol Chem, 1997 Jan 31, 272(5), 2682 - 7
Biosynthesis of the escherichia coli K4 capsule polysaccharide . A parallel system for studies of glycosyltransferases in chondroitin formation; Lidholt K et al.; Escherichia coli K4 bacteria synthesize a capsule polysaccharide (GalNAc-GlcA(fructose))n with the carbohydrate backbone identical to chondroitin . GlcA- and GalNAc-transferase activities from the bacterial membrane were assayed with acceptors derived from the capsule polysaccharide and radiolabeled UDP-{14C}GlcA and UDP-{3H}GalNAc, respectively . It was shown that defructosylated oligosaccharides (chondroitin) could serve as substrates for both the GlcA- and the GalNAc-transferases . The radiolabeled products were completely degraded with chondroitinase AC; the {14C}GlcA unit could be removed by beta-D-glucuronidase, and the {3H}GalNAc could be removed by beta-N-acetylhexosaminidase . A fructosylated oligosaccharide acceptor tested for GlcA-transferase activity was found to be inactive . These results indicate that the chain elongation reaction of the K4 polysaccharide proceeds in the same way as the polymerization of the chondroitin chain, by the addition of the monosaccharide units one by one to the nonreducing end of the polymer . This makes the biosynthesis of the K4 polysaccharide an interesting parallel system for studies of chondroitin sulfate biosynthesis . In the biosynthesis of capsule polysaccharides from E . coli, a similar mechanism has earlier been demonstrated for polysialic acid (NeuNAc)n (Rohr, T . E., and Troy, F . A . (1980) J . Biol . Chem . 255, 2332-2342) and for the K5 polysaccharide (GlcAbeta1-4GlcNAcalpha1-4)n (Lidholt, K., Fjelstad, M., Jann, K., and Lindahl, U . (1994) Carbohydr . Res . 255, 87-101) . In contrast, chain elongation of hyaluronan (GlcAbeta1-3GlcNAcbeta1-4)n is claimed to occur at the reducing end (Prehm, P . (1983) Biochem . J . 211, 181-189).

Nature, 1997 Jan 30, 385(6615), 442 - 6
Crosstalk between G proteins and protein kinase C mediated by the calcium channel alpha1 subunit; Zamponi GW et al.; The modulation of voltage-dependent Ca2+ channels at presynaptic nerve terminals is an important factor in the control of neurotransmitter release and synaptic efficacy . Some terminals contain multiple Ca2(+)-channel subtypes (N and P/Q), which are differentially regulated by G-protein activation and by protein kinase C (PKC)-dependent phosphorylation . Regulation of channel activity by crosstalk between second messenger pathways has been reported although the molecular mechanisms underlying crosstalk have not been described . Here we show that crosstalk occurs at the level of the presynaptic Ca2(+)-channel complex . The alpha1 subunit domain I-II linker, which connects the first and second transmembrane domains, contributes to the PKC-dependent upregulation of channel activity, while G-protein-dependent inhibition occurs through binding of Gbetagamma to two sites in the I-II linker . Crosstalk results from the PKC-dependent phosphorylation of one of the Gbetagamma binding sites which antagonizes Gbetagamma-induced inhibition . The results provide a mechanism for the highly regulated and dynamic control of neurotransmitter release that depends on the integration of multiple presynaptic signals.

Biochemistry, 1997 Jan 28, 36(4), 932 - 40
Equilibrium and kinetic analyses of unfolding and refolding for the conserved proline mutants of tryptophan synthase alpha subunit; Ogasahara K et al.; To elucidate the role of conserved proline residues of the tryptophan synthase alpha subunit from Escherichia coli in stability and folding, equilibrium and kinetic studies of the unfolding-refolding induced by guanidine hydrochloride for six mutant alpha subunits (Pro-->Ala) were carried out by peptidyl circular dichroism and aromatic fluorescence measurements at pH 7 and 25 degrees C . These results were analyzed assuming the presence of one intermediate (I) state in the denaturation process . (I) For all mutant and wild-type proteins, the Gibbs energy change (delta Gni(H2O)) in water between the native (N) and I states coincided with the difference (delta G++u(H2O)-delta G++r(H2O)) between the activation Gibbs energy changes in water for the unfolding (delta G++u(H2O) and refolding (delta G++r(H2O) reactions . This means that the early folding intermediate of the alpha subunit corresponds to the equilibrium intermediate . Delta Gni(H2O) values of all mutant proteins decreased compared with that of the wild-type protein . Gibbs energy change (delta Gid(H2O) in water between I and the denatured (D) states was not substantially affected by the substitutions . Delta G++u(H2O) and delta G++r(H2O) decreased and increased, respectively, for all mutant proteins . (2) Six conserved prolines played roles in stability and folding of the alpha subunit in a different manner: prolines 28 and 96 by stabilizing the N state and prolines 28, 96, 132, and 207 by destabilizing the I state . The contributions of prolines 57 and 62 to the stability were marginal . (3) Cis proline 28 was not the origin of the slow phase in the refolding kinetics assumed to arise from the cis-trans isomerization reaction of proline.

Biochemistry, 1997 Jan 28, 36(4), 894 - 902
Uncompetitive substrate inhibition and noncompetitive inhibition by 5-n-undecyl-6-hydroxy-4,7-dioxobenzothiazole (UHDBT) and 2-n-nonyl-4-hydroxyquinoline-N-oxide (NQNO) is observed for the cytochrome bo3 complex: implications for a Q(H2)-loop proton translocation mechanism; Musser SM et al.; The cytochrome bo3 ubiquinol oxidase complex from Escherichia coli contains two binding sites for ubiquinone(ol) (UQ(H2)) . One of these binding sites, the ubiquinol oxidation site, is clearly in dynamic equilibrium with the UQ(H2) pool in the membrane . The second site has a high affinity for ubiquinone (UQ), stabilizes a semiquinone species, and is located physically close to the low-spin heme b component of the enzyme . The UQ molecule in this site has been proposed to remain strongly bound to the enzyme during enzyme turnover and to act as a cofactor facilitating the transfer of electrons from the substrate ubiquinol to heme b {Sato-Watanabe et al . (1994) J . Biol . Chem . 269, 28908-28912} . In this paper, the steady-state turnover of the enzyme is examined in the presence and absence of inhibitors (UHDBT and NQNO) that appear to be recognized as ubisemiquinone analogs . It is found that the kinetics are accounted for best by a noncompetitive inhibitor binding model . Furthermore, at high concentrations, the substrates ubiquinol-1 and ubiquinol-2 inhibit turnover in an uncompetitive fashion . Together, these observations strongly suggest that there must be at least two UQ(H2) binding sites that are in rapid equilibrium with the UQ(H2) pool under turnover conditions . Although these data do not rule out the possibility that a strongly bound UQ molecule functions to facilitate electron transfer to heme b, they are more consistent with the behavior expected if the two UQ(H2) binding sites were to function in a Q(H2)-loop mechanism (similar to that of the cytochrome bc1 complex) as originally proposed by Musser and co-workers {(1993) FEBS Lett . 327, 131-136} . In this model, ubiquinol is oxidized at one site and ubiquinone is reduced at the second site . While the structural similarities of the heme-copper ubiquinol and cytochrome c oxidase complexes suggest the possibility that these two families of enzymes translocate protons by similar mechanisms, the current observations indicate that the Q(H2)-loop proton translocation mechanism for the heme-copper ubiquinol oxidase complexes should be further investigated and experimentally tested.

Biochemistry, 1997 Jan 28, 36(4), 812 - 22
Spectroscopic properties of Escherichia coli UDP-N-acetylenolpyruvylglucosamine reductase; Axley MJ et al.; Purified uridine diphosphate N-acetylenolpyruvylglucosamine reductase (E.C . 1.1.1.158) was analyzed by circular dichroism (CD) and UV-visible spectroscopy to establish the spectral properties of its tightly bound flavin adenine dinucleotide (FAD) cofactor . The polypeptide backbone displayed a single circular dichroic minimum at 208 nm and a single maximum at 193 nm . The CD spectrum of bound flavin exhibited a single major negative Cotton peak at 364 nm and two minor negative Cotton peaks at 464 and 495 nm . The protein was reversibly unfolded in 9.8 M urea and refolded in buffer in the presence of excess FAD . The refolded enzyme incorporated FAD and catalyzed full activity . The bound FAD displayed an absorption maximum at 464 nm with an extinction coefficient of epsilon 464 = 11700 M-1 cm-1 . Anaerobic reduction with dithionite was complete at 1 equiv . Anaerobic reduction with nicotinamide adenine dinucleotide phosphate, reduced form (NADPH), also was essentially complete at 1 equiv and produced a long-wavelength absorbance band characteristic of an FAD-pyridine nucleotide charge transfer complex . Photochemical bleaching in the presence of ethylenediaminetetraacetic acid (EDTA) followed exponential kinetics . None of the anaerobic reductive titrations produced a spectral intermediate characteristic of a flavin semiquinone, and all reduced enzyme species could be fully reoxidized by oxygen, with full recovery of catalytic activity . Photochemically reduced enzyme was reoxidized by titration with either NADP+ or uridine diphospho N-acetylglucosamine enolpyruvate (UNAGEP) . Reoxidation by NADP+ reached a chemical equilibrium, whereas reoxidation by UNAGEP was stoichiometric . Binding of NADP+ or UNAGEP to the oxidized form of the enzyme produced a dead-end complex that could be titrated by following a 10-nm red shift in the absorption spectrum of the bound FAD . The Kd of NADP+ for oxidized enzyme was 0.7 +/- 0.3 microM and the Kd of UNAGEP was 2.7 +/- 0.3 microM . Solvent deuterium isotope effects on binding were observed for both NADP+ and UNAGEP, depending on the pH . At pH 8.5, the HKd/DKd was 2.2 for NADP+ and 3.9 for UNAGEP . No spectral changes were observed in the presence of a 40-fold excess of uridine diphospho N-acetylmuramic acid (UNAM) either aerobically or anaerobically . These studies have identified spectral signals for five steps in the kinetic mechanism, have indicated that product formation is essentially irreversible, and have indicated that hydrogen bonding or protonation contributes significantly to ground-state complex formation with the physiological substrate.

Biochemistry, 1997 Jan 28, 36(4), 699 - 710
Phosphotransfer site of the chemotaxis-specific protein kinase CheA as revealed by NMR; Zhou H et al.; Bacterial chemotaxis involves autophosphorylation of a histidine kinase and transfer of the phosphoryl group to response regulators to control flagellar rotation and receptor adaptation . The phosphotransfer domain, CheA1-134, of the chemotaxis-specific histidine autokinase CheA from Escherichia coli contains the site of phosphorylation, His48, and two other histidine residues, His26 and His67 . Two-dimensional 1H-15N NMR techniques were applied to characterize the protonation states of these histidine residues and to evaluate the structural changes in the domain that occur upon phosphorylation of His48 . The pKa of His48 was determined to be 7.8 (in 50 mM NaPO4 buffer at 30 degrees C) . At high pH, its imidazole ring exists primarily as the normally unfavored N delta 1H tautomer, suggesting hydrogen bond formation to the ring nitrogen atom(s) to stabilize this state . The pKa values and predominant tautomeric states of the imidazole rings of His26 (pKa approximately 7.1, N epsilon 2H tautomer) and His67 (pKa approximately 6.5, N delta 1H tautomer) were also determined . His48 of CheA1-134 and CheA1-233 was phosphorylated by full-length CheA . The phosphorylation site was confirmed to be the N epsilon 2 position in the imidazole ring . Phosphorylation of His48 only results in small changes in the amide 1H and 15N chemical shifts of a few residues from helices B and C, suggesting that only very small changes in structure are associated with phosphorylation of the phosphotransfer domain of CheA . These residues occupy a small surface area of the helix bundle and form the active site of the protein . At the active site, in addition to His48, residues Gly52, His67, and Glu70 are conserved in the CheA homologous phosphotransfer domains from 10 different organisms . Sequence comparison of these CheA homologs suggest that the phosphotransfer domains likely fold in a similar helix-bundle structure and the structural features at the active site are well-conserved.

Mol Gen Genet, 1997 Jan 27, 253(4), 515 - 9
Functional relationship between Escherichia coli RNase E and the CafA protein; Wachi M et al.; We analyzed the functional relationship between the Escherichia coli RNase E and the CafA protein, which show extensive sequence similarity . The temperature-sensitive growth of the RNase E mutant strain ams1 was partially suppressed by multicopy plasmids bearing the cafA gene . Introduction of a cafA::cat mutation enhanced the temperature sensitivity of the ams1 mutant . These results suggest that there is a functional homology between these two proteins.

FEBS Lett, 1997 Jan 27, 402(1), 62 - 6
Cloning of the fabF gene in an expression vector and in vitro characterization of recombinant fabF and fabB encoded enzymes from Escherichia coli; Edwards P et al.; Analysis of the beta-ketoacyl-ACP synthase (KAS) encoded by the fabF gene of Escherichia coli has been hampered by a reported instability of the cloned gene . Here we describe biochemical characterization of purified, active protein from the recombinant fabF gene . This enzyme has the properties ascribed to KAS II and not those of a putative KAS IV reported to be encoded by fabJ, a genomic clone with DNA sequence identical to that of fabF . We also characterize active protein from a recombinant fabB gene and suggest that this method may have a general utility for analysis of KAS enzymes.

J Mol Biol, 1997 Jan 24, 265(3), 302 - 9
Leading versus lagging strand mutagenesis induced by 7,8-dihydro-8-oxo-2'-deoxyguanosine in Escherichia coli; Wagner J et al.; We have previously shown that a single N-2-acetylaminofluorene (AAF) adduct bound to the C-8 position of a guanine residue located within plasmids containing the unidirectional ColE1 origin of replication induces a 20-fold higher mutation frequency when the adduct is located in the lagging strand as compared to the leading strand . In this study, single 7,8-dihydro-8-oxo-2'-deoxyguanosine (8-oxodG) lesions have been introduced in the leading and lagging strand orientation within the same sequence context as for the AAF adducts . The induced frequency of guanine to thymine transversions has been measured, using a specific PCR-based quantitative assay, in strains deficient in the repair of the oxidative lesion . The potential involvement of the UvrABC excision repair system in the removal of 8-oxodG has also been investigated and ruled out . Concerning the mutation frequency asymmetry, in contrast to AAF adducts, 8-oxodG adducts induce the same mutation frequency, irrespective of their location in the leading or lagging strands . This striking difference between 8-oxodG and dGuo-C8-AAF adducts is discussed in terms of their differential capacity to block DNA replication.

Cell, 1997 Jan 24, 88(2), 277 - 85
Endostatin: an endogenous inhibitor of angiogenesis and tumor growth; O'Reilly MS et al.; We previously identified the angiogenesis inhibitor angiostatin . Using a similar strategy, we have identified endostatin, an angiogenesis inhibitor produced by hemangioendothelioma . Endostatin is a 20 kDa C-terminal fragment of collagen XVIII . Endostatin specifically inhibits endothelial proliferation and potently inhibits angiogenesis and tumor growth . By a novel method of sustained release, E . coli-derived endostatin was administered as a nonrefolded suspension . Primary tumors were regressed to dormant microscopic lesions . Immunohistochemistry revealed blocked angiogenesis accompanied by high proliferation balanced by apoptosis in tumor cells . There was no toxicity . Together with angiostatin data, these findings validate a strategy for identifying endogenous angiogenesis inhibitors, suggest a theme of fragments of proteins as angiogenesis inhibitors, and demonstrate dormancy therapy.

Cell, 1997 Jan 24, 88(2), 235 - 42
The solution structure of the S1 RNA binding domain: a member of an ancient nucleic acid-binding fold; Bycroft M et al.; The S1 domain, originally identified in ribosomal protein S1, is found in a large number of RNA-associated proteins . The structure of the S1 RNA-binding domain from the E . coli polynucleotide phosphorylase has been determined using NMR methods and consists of a five-stranded antiparallel beta barrel . Conserved residues on one face of the barrel and adjacent loops form the putative RNA-binding site . The structure of the S1 domain is very similar to that of cold shock protein, suggesting that they are both derived from an ancient nucleic acid-binding protein . Enhanced sequence searches reveal hitherto unidentified S1 domains in RNase E, RNase II, NusA, EMB-5, and other proteins.

J Biol Chem, 1997 Jan 24, 272(4), 2559 - 69
Abasic translesion synthesis by DNA polymerase beta violates the "A-rule" . Novel types of nucleotide incorporation by human DNA polymerase beta at an abasic lesion in different sequence contexts; Efrati E et al.; The "A-rule" reflects the preferred incorporation of dAMP opposite abasic lesions in Escherichia coli in vivo . DNA polymerases (pol) from procaryotic and eucaryotic organisms incorporate nucleotides opposite abasic lesions in accordance with the A-rule . However, recent in vivo data demonstrate that A is not preferentially incorporated opposite abasic lesions in eucaryotes . Purified human DNA polymerases beta and alpha are used to measure the specificity of nucleotide incorporation at a site-directed tetrahydrofuran abasic lesion, in 8-sequence contexts, varying upstream and downstream bases adjacent to the lesion . Extension past the lesion is measured in 4 sequence contexts, varying the downstream template base . Pol alpha strongly favors incorporation of dAMP directly opposite the lesion . In marked contrast, pol beta violates the A-rule for incorporation directly opposite the lesion . In addition to incorporation taking place directly opposite the lesion, we also analyze misalignment incorporation directed by a template base downstream from the lesion . Lesion bypass by pol beta occurs predominantly by "skipping over" the lesion, by insertion of a nucleotide complementary to an adjacent downstream template site . Misalignment incorporation for pol beta occurs by a novel "dNTP-stabilized" mechanism resulting in both deletion and base substitution errors . In contrast, pol alpha shows no propensity for this type of synthesis . The misaligned DNA structures generated during dNTP-stabilized lesion bypass do not conform to misaligned structures reported previously.

J Biol Chem, 1997 Jan 24, 272(4), 2429 - 36
Dissection of glutathionylspermidine synthetase/amidase from Escherichia coli into autonomously folding and functional synthetase and amidase domains; Kwon DS et al.; The bifunctional glutathionylspermidine synthetase/amidase from Escherichia coli catalyzes both the ATP-dependent formation of an amide bond between N1 of spermidine (N-(3-amino)propyl-1, 4-diaminobutane) and the glycine carboxylate of glutathione (gamma-Glu-Cys-Gly) and the opposing hydrolysis of this amide bond (Bollinger, J . M., Jr., Kwon, D . S., Huisman, G . W., Kolter, R., and Walsh, C . T . (1995) J . Biol . Chem . 270, 14031-14041) . In our previous work describing its initial characterization, we proposed that the 619-amino acid (70 kDa) protein might possess separate amidase (N-terminal) and synthetase (C-terminal) domains . In the present study, we have confirmed this hypothesis by expression of independently folding and functional amidase and synthetase modules . A fragment containing the C-terminal 431 amino acids (50 kDa) has synthetase activity only, with steady-state kinetic parameters similar to the full-length protein . A fragment containing the N-terminal 225 amino acids (25 kDa) has amidase activity only and is significantly activated relative to the full-length protein for hydrolysis of glutathionylspermidine analogs . This observation suggests that the amidase activity in the full-length protein is negatively autoregulated . The amidase active site catalyzes hydrolysis of amide and ester derivatives of glutathione (e.g . glutathione ethyl ester and glutathione amide) but lacks activity toward acetylspermidine (N1 and N8) and acetylspermine (N1), indicating that glutathione provides the primary recognition determinants for glutathionylspermidine amide bond cleavage . No metal ion is required for the amidase activity . A tetrahedral phosphonate analogue of glutathionylspermidine, designed as a mimic of the proposed tetrahedral intermediate for either reaction, inhibits the synthetase activity (Ki approximately 10 microM) but does not inhibit the amidase activity.

J Biol Chem, 1997 Jan 24, 272(4), 2259 - 67
Influence of Mg2+ and temperature on formation of the transcription bubble; Zaychikov E et al.; The transcription bubble formed in the binding complex of T7A1 promoter upon Escherichia coli RNA polymerase was analyzed by chemical probes, namely by single-strand specific reagents, to map the unpaired bases in the bubble, and by FeEDTA, to analyze the accessibility of the DNA backbone . The latter probe could also be used as a local hydroxyl radical probe placed close to the Mg2+-binding site in the active center . The data show that the transcription bubble consists of two parts, an Mg2+-dependent part and an Mg2+-independent part, both having individual transition temperatures . The data further suggest that formation of a transcription active open complex is preceded by a transition state complex having enhanced affinity for those Mg2+ ions presumably participating in the formation of the catalytic site . Our data also suggests that the three catalytically active Mg2+ ions in RNA polymerase are functionally not equivalent . One/two of the three Mg2+ ions are responsible for the polymerization, the other two/one for enlargement of the transcription bubble.

Biochem Biophys Res Commun, 1997 Jan 23, 230(3), 582 - 6
Escherichia coli rnpB promoter mutants altered in stringent response; Jung YH et al.; The promoter of the rnpB gene (encoding the RNA component of Escherichia coli RNase P) shares a consensus discriminator sequence, located between the -10 hexamer sequence and the transcription start site, with other promoters whose activities are repressed upon stringent condition . Under stringent conditions induced by seryl-tRNA starvation the transcription of the rnpB gene was repressed in wild type E . coli but not in a relaxed strain carrying a relA- mutation . Site-directed mutagenesis was carried out to examine sequences of the rnpB promoter necessary for stringent control . The results indicate that the discriminator region is responsible for the transcription repression of the rnpB gene during the stringent response and that both the content and position of GC pairs in the region determine the strength of negative stringent signals.

Nature, 1997 Jan 23, 385(6614), 365 - 8
Crystal structure of the NG domain from the signal-recognition particle receptor FtsY; Montoya G et al.; Newly synthesized proteins destined either for secretion or incorporation into membranes are targeted to the membrane translocation machinery by a ubiquitous system consisting of a signal-recognition particle (SRP) and its receptor . Both the SRP receptor and the protein within the SRP that binds the signal sequence contain GTPases . These two proteins, together with the RNA component of the SRP, form a complex and thereby regulate each other's GTPase activity . Here we report the structure of the GTPase-containing portion of FtsY, the functional homologue of the SRP receptor of Escherichia coli, at 2.2 A resolution without bound nucleotide . This so-called NG domain displays similarities to the Ras-related GTPases, as well as features unique to the SRP-type GTPases, such as a separate amino-terminal domain, an insertion within the p21ras (Ras) effector domain, and a wide-open GTP-binding region . The structure explains the low affinity of FtsY for GTP, and suggests rearrangements that may occur on nucleotide binding . It also identifies regions potentially involved in the transmission of signals between domains and in interactions with regulatory proteins.

Nature, 1997 Jan 23, 385(6614), 361 - 4
Structure of the conserved GTPase domain of the signal recognition particle; Freymann DM et al.; The signal-recognition particle (SRP) and its receptor (SR) function in the co-translational targeting of nascent protein-ribosome complexes to the membrane translocation apparatus . The SRP protein subunit (termed Ffh in bacteria) that recognizes the signal sequence of nascent polypeptides is a GTPase, as is the SR-alpha subunit (termed FtsY) . Ffh and FtsY interact directly, each stimulating the GTP hydrolysis activity of the other . The sequence of Ffh suggests three domains: an amino-terminal N domain of unknown function, a central GTPase G domain, and a methionine-rich M domain that binds both SRP RNA and signal peptides . Sequence conservation suggests that structurally similar N and G domains are present in FtsY . Here we report the structure of the nucleotide-free form of the NG fragment of Ffh . Consistent with a role for apo Ffh in protein targeting, the side chains of the empty active-site pocket form a tight network of interactions which may stabilize the nucleotide-free protein . The structural relationship between the two domains suggests that the N domain senses or controls the nucleotide occupancy of the GTPase domain . A structural subdomain unique to these evolutionarily conserved GTPases constitutes them as a distinct subfamily in the GTPase superfamily.

Nature, 1997 Jan 23, 385(6614), 353 - 7
Bcl-x(L) forms an ion channel in synthetic lipid membranes; Minn AJ et al.; Bcl-2-related proteins are critical regulators of cell survival that are localized to the outer mitochondrial, outer nuclear and endoplasmic reticulum membranes . Despite their physiological importance, the biochemical function of Bcl-2-related proteins has remained elusive . The three-dimensional structure of Bcl-xL, an inhibitor of apoptosis, was recently shown to be similar to the structures of the pore-forming domains of bacterial toxins . A key feature of these pore-forming domains is the ability to form ion channels in biological membranes . Here we demonstrate that Bcl-xL shares this functional feature . Like the bacterial toxins, Bcl-xL can insert into either synthetic lipid vesicles or planar lipid bilayers and form an ion-conducting channel . This channel is pH-sensitive and becomes cation-selective at physiological pH . The ion-conducting channel(s) formed by Bcl-xL display multiple conductance states that have identical ion selectivity . Together, these data suggest that Bcl-xL may maintain cell survival by regulating the permeability of the intracellular membranes to which it is distributed.

Biochim Biophys Acta, 1997 Jan 21, 1344(2), 139 - 52
Characterization of human apolipoprotein A-I expressed in Escherichia coli; Bergeron J et al.; Human apolipoprotein A-I (apoA-I), with an additional N-terminal extension (Met-Arg-Gly-Ser-(His)6-Met) (His-apoA-I), has been produced in Escherichia coli with a final yield after purification of 10 mg protein/1 of culture medium . We have characterized the conformation and structural properties of His-apoA-I in lipid-free form, and in reconstituted lipoproteins containing two apoA-I per particle (Lp2A-I) by both immunochemical and physicochemical techniques . The lipid-free forms of the two proteins present very similar secondary structure and stability, and have also very similar kinetics of association with dimyristoyl phosphatidylcholine . His-apoA-I and native apoA-I can be complexed with 1-palmitoyl-2-oleoyl phosphatidylcholine (POPC) to form similar, stable, either discoidal or spherical (sonicated) Lp2A-I particles . Lipid-bound native apoA-I and His-apoA-I showed very similar alpha-helical content (69% and 66%, respectively in discoidal Lp2A-I and 54% and 51%, respectively in spherical Lp2A-I) . The conformation of His-apoA-I in lipid-free form and in discoidal or spherical Lp2A-I has also been shown to be similar to native apoA-I by immunochemical measurements using 13 monoclonal antibodies recognizing distinct apoA-I epitopes . In the free protein and in reconstituted Lp2A-I, the N-terminal has no effect on the affinity of any of the monoclonal antibodies and minimal effect on immunoreactivity values . Small differences in the exposure of some apoA-I epitopes are evident on discoidal particles, while no difference is apparent in the expression of any epitope of apoA-I on spherical Lp2A-I . The presence of the N-terminal extension also has no effect on the reaction of LCAT with the discoidal Lp2A-I or on the ability of complexes to promote cholesterol efflux from fibroblasts in culture . In conclusion, we show that His-apoA-I expressed in E . coli exhibits similar physicochemical properties to native apoA-I and is also identical to the native protein in its ability to interact with phospholipids and to promote cholesterol esterification and cellular cholesterol efflux.

Proc Natl Acad Sci U S A, 1997 Jan 21, 94(2), 463 - 8
Activities of human recombination protein Rad51; Gupta RC et al.; Homologous pairing and strand exchange, which are catalyzed by Escherichia coli RecA protein, are central to homologous recombination . Homologs of this protein are found in eukaryotes; however, little has been reported on the recombinase activities of the mammalian homologs, including the human protein, denoted HsRad51 . For the studies described here, we purified HsRad51 form E . coli . Although the activities of HsRad51 and RecA were qualitatively similar in the presence of ATP, there were also striking differences . The stoichiometry of binding to DNA and the rate of renaturation of complementary strands were similar for the two proteins, but rates of ATP hydrolysis, homologous pairing, and subsequent strand exchange promoted by HsRad51 were less than 1/10 those of RecA . In addition, HsRad51 bound gamma-thio-ATP and formed stable presynaptic complexes that promoted renaturation as rapidly as RecA, but the recombinant human protein catalyzed neither strand exchange nor homologous pairing of a single strand with duplex DNA in the presence of the ATP analog . By contrast, RecA promoted both of the latter reactions in control experiments . These observations suggest that among RecA-like proteins, HsRad51 may be a variant in which homologous pairing and strand exchange are more closely linked to the hydrolysis of ATP.

Proc Natl Acad Sci U S A, 1997 Jan 21, 94(2), 391 - 6
Discrimination of a single base change in a ribozyme using the gene for dihydrofolate reductase as a selective marker in Escherichia coli; Fujita S et al.; For use of ribozymes in vivo, it is desirable to select functional ribozymes in the cellular environment (in the presence of inhibitory factors and limited concentrations of mandatory Mg2+ ions, etc.) . As a first step toward this goal, we developed a new screening system for detection in vivo of an active ribozyme from pools of active and inactive ribozymes using the gene for dihydrofolate reductase (DHFR) as a selective marker . In our DHFR expression vector, the sequence encoding either the active or the inactive ribozyme was connected to the DHFR gene . The plasmid was designed such that, when the ribozyme was active, the rate of production of DHFR was high enough to endow resistance to trimethoprim (TMP) . We demonstrated that the active ribozyme did indeed cleave the primary transcript in vivo, whereas the inactive ribozyme had no cleavage activity . Cells that harbored the active-ribozyme-coding plasmid grew faster in the presence of a fixed concentration of TMP than the corresponding cells that harbored the inactive-ribozyme-coding plasmid . Consequently, when cells were transformed by a mixture that consisted of active- and inactive-ribozyme-coding plasmids at a ratio of 1:1, (i) mainly those cells that harbored active ribozymes survived in the presence of TMP and (ii) both active- and inactive-ribozyme-harboring cells grew at an identical rate in the absence of TMP, a demonstration of a positive selection system in vivo . If the background "noise" can be removed completely in the future, the selection system might usefully complement existing selection systems in vitro.

Biochemistry, 1997 Jan 21, 36(3), 615 - 25
Use of 1H-15N heteronuclear multiple-quantum coherence NMR spectroscopy to study the active site of aspartate aminotransferase; Mollova ET et al.; Aspartate aminotransferase from Escherichia coli, an 88 kDa enzyme, was uniformly and selectively enriched with 15N and was studied by heteronuclear multiple-quantum coherence NMR spectroscopy in H2O . Good resolution was obtained for the downfield region (above 9.5 ppm chemical shift in the 1H dimension) for NH protons in the amide, indole, imidazole, and guanidinium group regions and several resonances were tentatively assigned . Two downfield resonances, at 12.6 and 11.36 ppm, appear to belong to oxygen- or sulfur-bound protons . The most downfield amide resonance at 11.78 ppm was assigned to the active site cysteine 192 whose peptide proton is 2.9 A away from the negatively charged carboxyl group of aspartate 199 . Large downfield shifts (up to 1.15 ppm) of the indole NH resonance of the active site tryptophan 140 were observed upon binding of dicarboxylic inhibitors to the pyridoxal 5'-phosphate (PLP) form and of inorganic dianions to the pyridoxamine 5'-phosphate (PMP) form of the enzyme . We discuss these striking differences in the light of the available crystallographic data . Active sites of proteins, as well as specific inhibitory molecules, often contain negatively charged groups . These may be able to form hydrogen-bonds to NH groups and to shift the NH resonances downfield into a less crowded and therefore more readily observable region for many large proteins . Our approach, which makes use of both HMQC spectroscopy and NOE observations, should be widely applicable.

Biochemistry, 1997 Jan 21, 36(3), 604 - 14
Escherichia coli dimethylallyl diphosphate:tRNA dimethylallyltransferase: a binding mechanism for recombinant enzyme; Moore JA et al.; Escherichia coli dimethylallyl diphosphate:tRNA dimethylallyltransferase (DMAPP-tRNA transferase) catalyzes the first step in the biosynthesis of the hypermodified A37 residue in tRNAs that read codons beginning with uridine . The enzyme, encoded by the miaA gene, was overproduced and purified to apparent homogeneity in three steps by ion-exchange (DE52 and Mono-Q) and size exclusion chromatography . Affinity-tagged DMAPP-tRNA transferase containing a C-terminal tripeptide alpha-tubulin epitope also was overproduced and purified to apparent homogeneity in two steps by ion-exchange and immunoaffinity chromatography . Addition of the C-terminal tripeptide alpha-tubulin epitope to DMAPP-tRNA transferase did not affect the activity of the enzyme . Undermodified tRNA(Phe) used as substrate in the DMAPP-tRNA transferase-catalyzed reaction was isolated and purified from an overexpressing clone in a miaA deficient strain of E . coli . Active recombinant E . coli DMAPP-tRNA transferase is monomeric . The enzyme transferred the dimethylallyl moiety of DMAPP to A37, located adjacent to the anticodon in undermodified tRNA(Phe) . The enzyme required Mg2+ for activity and exhibited a broad pH optimum . Michaelis constants for tRNA(Phe) and DMAPP are 96 +/- 11 nM and 3.2 +/- 0.5 microM, respectively, and Vmax = 0.83 +/- 0.02 micromol min-1 mg-1 . DMAPP-tRNA transferase bound tRNA(Phe) with a dissociation constant of 5.2 +/- 1.2 nM . In contrast, DMAPP did not bind to the enzyme in the absence of tRNA . However, DMAPP was bound with a dissociation constant of 3.4 +/- 0.6 microM in the presence of a minihelix analogue of the anticodon stem-loop of tRNA(Phe) where the base corresponding to A37 was replaced by inosine . These results suggest an ordered sequential mechanism for substrate binding.

Biochemistry, 1997 Jan 21, 36(3), 461 - 8
Escherichia coli RNA polymerase activity observed using atomic force microscopy; Kasas S et al.; Fluid tapping-mode atomic force microscopy (AFM) was used to observe Escherichia coli RNA polymerase (RNAP) transcribing two different linear double-stranded (ds) DNA templates . The transcription process was detected by observing the translocation of the DNA template by RNAP on addition of ribonucleoside 5'-triphosphates (NTPs) in sequential AFM images . Stalled ternary complexes of RNAP, dsDNA and nascent RNA were adsorbed onto a mica surface and imaged under continuously flowing buffer . On introduction of all four NTPs, we observed some DNA molecules being pulled through the RNAP, some dissociating from the RNAP and others which did not move relative to the RNAP . The transcription rates were observed to be approximately 0.5-2 bases/s at our NTP concentrations, approximately 5 microM . The RNA transcripts were not unambiguously imaged in fluid . However, in experiments using a small single-stranded (ss) circular DNA template, known as a rolling circle, transcripts up to 1 or 2 microns long could be observed with tapping mode AFM once the samples were dried and imaged in air . This confirmed our observations of the transcriptional activity of RNAP adsorbed onto mica . This work illustrates that the development of tapping-mode in fluid has made it possible to use AFM to follow biological processes at the molecular level and get new insights about the variability of activity of individual molecules bound to a surface.

Circulation, 1997 Jan 21, 95(2), 455 - 62
Recombinant staphylokinase variants with altered immunoreactivity . III: Species variability of antibody binding patterns; Collen D et al.; BACKGROUND: The "charged cluster-to-alanine" substitution variants SakSTAR(K35A,E38A,K74A,E75A,R77A) and SakSTAR(K74A,E75A,R77A,E80A,D82A), previously identified as SakSTAR.M38 and SakSTAR.M89, respectively, induce less antibody formation in patients than wild-type recombinant staphylokinase (SakSTAR), but their specific activities are reduced by 50% . Therefore, the effect of the reversal of one or more of these substituted amino acids on the ratio of activity to antigenicity was studied . METHODS AND RESULTS: Fourteen mutants with one to four "alanine-to-wild-type" reversals were expressed in Escherichia coli and highly purified (> 95%) . In rabbits immunized with wild-type SakSTAR, the combined K35,E38,K74,E75,R77 or K74,E75,R77,E80,E82 epitope accounted for only 30% of antibody absorption from plasma, and no clear immunodominant residue could be identified . In baboons immunized with SakSTAR, the K35,E38 and K74,E75,R77 epitopes or the K74,E75,R77 and E80,D82 epitopes contributed equally to account for 50% of total antibody binding, but no immunodominant residues were apparent . In pooled plasma from patients with peripheral arterial occlusion treated with wild-type SakSTAR, about 40% of the antibodies depended on K74 of epitope K74,E75,R77 for binding, whereas epitopes K35,E38 and E80,D82 had a negligible contribution toward antibody recognition . CONCLUSIONS: The recognition pattern by SakSTAR variants of antibodies induced with wild-type SakSTAR differs markedly among species . This implies that a systematic evaluation of reduced antigen recognition and antibody induction in humans will require the development of human or humanized systems . Surprisingly, SakSTAR(K74), with a single substitution of Lys74 with Ala, had an intact specific activity but did not absorb 40% of the antibodies induced in patients by treatment with wild-type SakSTAR.

Virology, 1997 Jan 20, 227(2), 474 - 83
Sequence close to the N-terminus of L2 protein is displayed on the surface of bovine papillomavirus type 1 virions; Liu WJ et al.; The bovine papillomavirus type 1 (BPV1) L2 protein purified from Escherichia coli was used as an antigen to produce monoclonal antibodies (MAbs) . A total of 26 individual clones which recognized the BPV1 L2 protein were obtained . Using infectious BPV1 virus particles, 3 of the MAbs were found to interact with BPV1 virus particles . Binding of the MAbs to BPV1 was confirmed by immunoelectron microscopy . A set of 92 13-mer peptides overlapping by 8 amino acids spanning the entire BPV1 L2 protein was synthesized on a membrane and used to map the epitopes recognized by these antibodies . Seventeen linear epitopes were identified . Our results revealed that a sequence toward the N-terminus of the L2 protein (aa 61-123) is displayed on the virus surface, while the remaining L2 sequences are hidden inside the virus capsid . Although the polyclonal antisera raised against BPV1 L2 neutralized the BPV1 virus, we failed to detect any neutralizing activity for the 3 L2-specific monoclonal antibodies which bound to the BPV1 particles . This suggests that extra binding sites may be needed for neutralization . This study prompted us to propose a model about how L1 and L2 proteins may interact during infectious papillomavirus assembly.

Virology, 1997 Jan 20, 227(2), 370 - 7
Genetic analysis of the phi X174 DNA binding protein; Jennings B et al.; The phi X174 J protein is 37 amino acids in length and contains 12 basic residues . There are no acidic amino acids in the protein . The basic residues are concentrated in two clusters in the N-terminus which are separated by a proline-rich region . To investigate the morphogenetic functions of the J protein and possible mechanisms by which it may bind DNA, a genetic analysis was conducted . Lysine --> leucine and arginine --> leucine substitutions were generated within the basic amino acid clusters . At least three substitutions were required to eliminate viability in vivo . Lethal mutants with three or four substitutions exhibit dominant lethal phenotypes, indicating that the mutant proteins retain enough function to interfere with productive assembly . In cells infected with a dominant lethal mutant, noninfectious packaged particles were produced . Infectivity can be restored by second-site suppressors in the viral coat protein which disrupt polar interactions atop the threefold axis of symmetry in the capsid . The viability of strains containing compensating frameshift mutations within the proline-rich region suggests that only the proline residues in this segment are critical for efficient function.

Virology, 1997 Jan 20, 227(2), 281 - 94
Analysis of the contributions of the equine herpesvirus 1 glycoprotein gB homolog to virus entry and direct cell-to-cell spread; Neubauer A et al.; Experiments to analyze the functions of the equine herpesvirus 1 (EHV-1) glycoprotein gB were performed . Cell lines which stably expressed either the full-length EHV-1 gB or only the extracellular portion of gB (amino acids 1 to 844) were constructed and were termed TCgBf and TCgB delta, respectively . Using the cell line TCgBf, a gB-negative viral mutant, L11delta gB, was generated by replacing a 2.1-kb BglII-NruI fragment in the EHV-1 strain RacL11 gB with the Escherichia coli LacZ gene . EHV-1 strain RacL11, the modified live vaccine strain RacH, and L11delta gB were used for functional studies . It was shown that: (i) EHV-1 gB is essential for virus growth in vitro since gB-negative L11delta gB exhibited titers of <10 PFU/ml when grown and titrated on noncomplementing cells . (ii) The cell line expressing truncated gB (TCgB delta) did not complement for the growth of L11delta gB, but the RacH virus grew to titers comparable to those of RacL11 in all cell lines tested . Since RacH had amino acids 944-980 of gB replaced by 7 missense amino acids as determined by nucleotide sequence analysis, the extreme carboxyterminus but not a domain between amino acid residues 845 and 943, probably the transmembrane domain, of EHV-1 gB is dispensable for virus growth in cultured cells . (iii) Single infected cells but no plaque formation were observed after infection of noncomplementing cells with L11delta gB, demonstrating the requirement of EHV-1 gB for direct cell-to-cell spread of infection . (iv) The attachment of gB-negative L11delta gB virions to target cells was similar to both phenotypically complemented L11delta gB and parent RacL11 virus . (v) L11delta gB viral titers could be enhanced by using the fusogen polyethylene glycol (PEG) . The increase of L11delta gB titers by PEG treatment, however, was considerably lower compared to gB-negative pseudorabies virus, suggesting that EHV-1 gB might not be as stringently required for virus penetration as are its homologs in other Alphaherpesvirinae.

Hum Gene Ther, 1997 Jan 20, 8(2), 195 - 204
Modulation of erythropoietin delivery from engineered muscles in mice; Bohl D et al.; In most relevant diseases, the permanent systemic delivery of a therapeutic protein from engineered cells might be proposed only if secretion levels can be regulated . The tetracycline resistance operon of Escherichia coli provides a transcriptional regulatory system effective in mammalian cells, which could be used for that purpose . A chimeric transactivator (tTA) consisting of the tetracycline repressor fused to the transactivation domain of the herpes simplex virus VP16 protein stimulates transcription by binding a minimal cytomegalovirus (CMV) promoter containing repeats of the tetracycline operator (tetO-CMV) . Binding is abolished by tetracycline, thus impairing promoter activation . We have transduced C2.7 myoblasts with two U3-deleted retroviral vectors containing these regulatory elements . The tetP-Epo vector expressed the murine erythropoietin (Epo) cDNA under the control of the tetO-CMV promoter . The D-De-tTA vector expressed tTA under the control of the muscle-specific human desmin enhancer-promoter . Northern blot analysis showed background Epo mRNA expression in myoblasts . Myotubes differentiation induced tTA expression, leading to a 28-fold increase of Epo mRNAs, which was suppressed by tetracycline . Basal Epo secretion in myoblasts increased 23- to 41-fold during the formation of multinucleated myotubes, and turned back close to myoblast level when tetracycline was added . Myoblasts transduced with both vectors and treated with mitomycin with the aim to prevent tumor formation were engrafted in skeletal muscles of syngeneic C3H mice . Hematocrit levels were significantly higher in animals bearing cells transduced with both vectors than in control animals grafted with cells transduced with the Epo vector only . This difference was abolished when tetracycline was given to mice . These data indicate that the tetracycline regulatory elements can modulate transcription in the context of retroviral vector genomes, and that this system can be used to control the in vivo delivery of a therapeutic protein from genetically modified muscles.

J Exp Med, 1997 Jan 20, 185(2), 367 - 71
HLA class I binding motifs derived from random peptide libraries differ at the COOH terminus from those of eluted peptides; Davenport MP et al.; Recombinant HLA-A2, HLA-B8, or HLA-B53 heavy chain produced in Escherichia coli was combined with recombinant beta2-microglobulin (beta2m) and a pool of randomly synthesised nonamer peptides . This mixture was allowed to refold to form stable major histocompatability complex (MHC) class I complexes, which were then purified by gel filtration chromatography . The peptides bound to the MHC class I molecules were subsequently eluted and sequenced as a pool . Peptide binding motifs for these three MHC class I molecules were derived and compared with previously described motifs derived from analysis of naturally processed peptides eluted from the surface of cells . This comparison indicated that the peptides bound by the recombinant MHC class I molecules showed a similar motif to naturally processed and presented peptides, with the exception of the peptide COOH terminus . Whereas the motifs derived from naturally processed peptides eluted from HLA-A2 and HLA-B8 indicated a strong preference for hydrophobic amino acids at the COOH terminus, this preference was not observed in our studies . We propose that this difference reflects the effects of processing or transport on the peptide repertoire available for binding to MHC class I molecules in vivo.

FEBS Lett, 1997 Jan 20, 401(2-3), 243 - 6
Identification of a C-terminal binding site for G-protein betagamma-subunits in phosducin-like protein; Schroder S et al.; Phosducin-like protein (PhLP) has recently been identified as a ubiquitous inhibitor of G-protein betagamma-subunit (G betagamma)-mediated signaling, with an affinity about 5-fold lower than that of phosducin . The G betagamma binding site of phosducin has been suggested to be contained in its N-terminus . A region corresponding to this N-terminus is lacking in PhLP, suggesting that PhLP must utilize a different mode of G betagamma binding . To map the G betagamma binding site in PhLP, a series of deletion mutants were constructed, expressed in E . coli as glutathione S-transferase (GST) fusion proteins, and the purified fusion proteins were examined for their ability to attenuate G(o) GTPase activity . Progressive N-terminal truncations of PhLP caused only minor reductions in potency, whereas the complementary N-terminal PhLP fragments turned out to be inactive . We further identified a short C-terminal segment comprising residues 168 to 195 that inhibited G0 GTPase activity similar in efficacy and potency to full-length PhLP . This C-terminal fragment was also capable of antagonizing a second G betagamma-mediated function, the enhancement of rhodopsin phosphorylation by the beta-adrenergic receptor kinase . Taken together, these data indicate that PhLP interacts with G betagamma via a short C-terminal binding site which is distinct from that identified previously in phosducin.

FEBS Lett, 1997 Jan 20, 401(2-3), 175 - 9
Acetylation of ribosomal protein S5 affected by defects in the central pseudoknot in 16S ribosomal RNA?
Poot RA, Jeeninga RE, Pleij CW, van Duin J.
We have analyzed the ribosomal protein profile of Escherichia coli 30S subunits with the mutation C18A in the central pseudoknot of their 16S ribosomal RNA . This mutation was shown to inhibit translational activity in vivo and to affect ribosome stability in vitro . The majority of the mutant 30S particles were present as free subunits in which a reproducible decrease in amount of proteins S1, S2, S18 and S21 was observed . The protein gels also showed the appearance of a satellite band next to S5 . This band reacted with anti-S5 antibodies and had a slightly increased positive charge . The simplest interpretation of these findings, also considering published data, is that the satellite band is S5 with a non-acetylated N-terminal alanine . Underacetylation of S5 due to mutations in the 16S rRNA implies that the modification is performed on the ribosome.

FEBS Lett, 1997 Jan 20, 401(2-3), 171 - 4
Identification of a conserved phosphorylation site modulating nuclear lamin polymerization; Stuurman N; Mitotic lamin disassembly results from phosphorylation at specific sites . In vitro, lamins can form head-to-tail polymers that disassemble upon phosphorylation by cdc2 kinase . A co-immunoprecipitation assay, employing Drosophila nuclear lamin Dm0 fragments was used to study the effect of phosphorylation on head-to-tail binding . Phosphorylation of serine-50 by cAMP-dependent kinase inhibited head-to-tail binding in the same manner as phosphorylation of serine-42 by cdc2 kinase . Results suggest that multiple pathways may be employed to disassemble nuclear lamins in vivo.

FEBS Lett, 1997 Jan 20, 401(2-3), 127 - 32
NMR structural characterization of the CDK inhibitor p19INK4d; Kalus W et al.; p19INK4d is a 165 amino acid protein that belongs to the INK4 family of CDK4 and CDK6 inhibitors . Assignments of 1H, 15N and 13C resonances have enabled the determination of the secondary structure of the protein which is largely alpha-helical (residues 14-18, 21-29, 54-62, 77-83, 87-95, 110-116, 120-128, 142-148 and 152-160) . The protein comprises five 32-amino acid ankyrin-like repeats; each ankyrin repeat contains a helix-beta-turn-helix core . The exception is the second ankyrin repeat, which lacks the first helix . All beta-turns have a central glycine residue flanked by two residues in beta-conformations . There is also a high conservation of Ala at position 8 in the first helix and Leu-Leu(Val) at positions 17-18 of the second helix in all ankyrin repeats of p19 . The location of the helix-turn-helix segments found in p19 should be general for all other members of the INK4 family, including, for example, a homologous tumor suppressor p16INK4a . 1H-15N heteronuclear steady-state NOE measurements on p19 indicate that most of the backbone of p19INK4d exists in a well defined structure of limited conformational flexibility on the nano- to picosecond time scale.

FEBS Lett, 1997 Jan 20, 401(2-3), 109 - 12
Isolation and characterization of a cDNA encoding a Translin-like protein, TRAX; Aoki K et al.; Translin is a DNA binding protein which specifically binds to consensus sequences at breakpoint junctions of chromosomal translocations in many cases of lymphoid malignancies . To investigate its functional significance at such recombination hotspots, we examined whether Translin interacts with other proteins using a yeast two-hybrid system and identified an associated 33 kd protein partner, TRAX, with extensive amino acid homology . The TRAX protein was established to contain bipartite nuclear targeting sequences in its N-terminal region, suggesting a possible role in the selective nuclear transport of Translin protein lacking any nuclear targeting motifs.

Carbohydr Res, 1997 Jan 17, 297(3), 297 - 9
Structure determination of the O-antigenic polysaccharide from the enterotoxigenic Escherichia coli (ETEC) O101; Staaf M et al.; The O-antigenic polysaccharide of the lipopolysaccharide from the enterotoxigenic Escherichia coli O101 has been investigated . The composition and sequence of the repeating units was established by sugar and methylation analysis together with 1H and 13C NMR spectroscopy . The sequence was corroborated using the computer program CASPER . The structure of the repeating unit of the polysaccharide from E . coli O101 is as follows: -->6)-alpha-D-GlcpNAc-1-->4-alpha-D-GalpNAc-(1-->.

J Mol Biol, 1997 Jan 17, 265(2), 161 - 72
A natural antibody missing a cysteine in VH: consequences for thermodynamic stability and folding; Proba K et al.; While the disulfide bridge is highly conserved within the immunoglobulin fold, a few antibody variable domains lack one of the essential cysteine residues . In the levan binding antibody ABPC48 one of the essential cysteine residues (Cys H92) of the heavy chain variable domain is replaced by tyrosine . We expressed scFv fragments with the ABPC48 sequence and a mutant in which the VH disulfide bond has been restored in Escherichia coli, purified both proteins by antigen affinity chromatography and characterized them by equilibrium denaturation . While the ABPC48 protein was found to be significantly less stable than an average scFv molecule, the restored disulfide increased its stability above that of other, unrelated scFv fragments, explaining why it tolerates the disulfide loss . Surprisingly, we observed that under some refolding conditions, the unpaired cysteine residue of functional scFv of ABPC48 is derivatized by glutathione . It is easily accessible to other reagents and thus appears to be solvent-exposed, in contrast to the deeply buried disulfide of ordinary variable domains . This implies a very unusual conformation of stand b containing the unpaired Cys H22, which might be stabilized by interactions with the tyrosine residue in position H92.

J Mol Biol, 1997 Jan 17, 265(2), 142 - 52
Identification of elements on GeneX-secA RNA of Escherichia coli required for SecA binding and secA auto-regulation; Salavati R et al.; The protein translocation ATPase of Escherichia coli, SecA protein, auto-regulates its translation by binding to its translation initiation region in geneX-secA mRNA . To analyze this regulation further the secondary structure of this portion of geneX-secA RNA was investigated utilizing structure-specific nucleases and chemical probing approaches . The results of this analysis were consistent with the existence of two adjacent helices, helix I and the lower portion of helix II, whose function in secA activation and repression, respectively, has been demonstrated . Binding of SecA protein to geneX-secA RNA or various mutant derivatives of this RNA was studied by measurement of affinity constants, RNA footprint analysis, and quantitation of auto-repression in vivo . This analysis showed that the SecA-binding site in geneX-secA RNA was remarkably large spanning a region of 96 nucleotides including a 3' portion of helix II, the secA translation initiation region and distal sequences . From the size of the SecA-binding site and the plasticity of its response to mutational alteration, it is suggested that SecA protein contains two distinct RNA-binding sites . Finally, it was shown that SecA binding was not sufficient to promote auto-regulation and that sequences both upstream (helix I) and within the binding site can contribute to auto-regulation without affecting SecA-binding affinity.

J Mol Biol, 1997 Jan 17, 265(2), 128 - 41
Dual regulation of Escherichia coli secA translation by distinct upstream elements; McNicholas P et al.; The regulation of the Escherichia coli secA gene, whose translation is auto-repressed except when protein secretion becomes limiting, was investigated using a combination of genetic and biochemical approaches . Oligonucleotide-directed deletion and point mutagenesis was used to show that only the last quarter of the upstream gene, geneX, and the geneX-secA intergenic are essential for proper regulation . This region previously shown to contain a secretion-responsive element contains two predicted helices, helix I and II, the latter of which would occlude the secA Shine-Dalgarno sequence . Mutations that destabilized the lower portion of helix II increased secA basal expression, reduced auto-repression by SecA protein, but retained a normal pattern of derepression of secA expression during a protein export block . The introduction of compensatory mutations into helix II that were predicted to restore base-pairing restored secA regulation to wild-type levels or nearly so, suggesting that this helix does play a role in secA auto-regulation in vivo . In contrast, mutations in the lower portion of helix I decreased secA basal expression, reduced auto-repression by SecA protein, and abolished the responsiveness of secA expression to a protein export block . In this latter case introduction of compensatory mutations into helix I that were predicted to restore base-pairing did not restore proper secA regulation, indicating that specific nucleotides in this region are required for normal secA regulation . Primer-extension inhibition (toeprint) analysis with 30 S ribosoma subunits, tRNAMet, and a model segment of geneX-secA RNA carrying the relevant mutations was used to show that mutations that destabilized helix II increased ribosome binding at the secA translation initiation site, while mutations that perturbed helix I decreased ribosome binding at this site . Our results suggest strongly that there is a system of dual regulation of secA translation, whereby helix I serves as an activator element while helix II serves as a repressor element.

J Biol Chem, 1997 Jan 17, 272(3), 1950 - 5
The role of cysteine residues in the rearrangement of uridine to pseudouridine catalyzed by pseudouridine synthase I; Zhao X et al.; Escherichia coli tRNA pseudouridine synthase I (PSUI) catalyzes the conversion of uridine residues to pseudouridine in positions 38, 39, and 40 of various tRNA molecules . In previous biochemical studies with this enzyme (Kammen, H . O., Marvel, C . C., Hardy, L., and Penhoet, E . E . (1988) J . Biol . Chem . 263, 2255-2263) it was reported that cysteine residues are important in maintaining the active structure of the enzyme and are possibly involved in the catalytic reaction mechanism via a covalent cysteine intermediate . In order to further investigate the biochemical properties of PSUI, a high level expression and purification system for the enzyme and its corresponding mutants was developed . PSUI has three cysteine residues among 270 amino acids . In the present investigation, each cysteine residue was individually changed to serine and alanine . In addition, a triple mutant was prepared wherein all three cysteine residues were replaced by alanine . Surprisingly, while two of the three cysteine to serine mutants were inactive, all alanine mutants exhibited near wild-type levels of activity, including the triple mutant . These results provide the first direct and unambiguous chemical evidence against a covalent cysteine intermediate in the rearrangement mechanism of uridine to pseudouridine.

J Biol Chem, 1997 Jan 17, 272(3), 1910 - 9
Protein phosphorylation affects binding of the Escherichia coli transcription activator UhpA to the uhpT promoter; Dahl JL et al.; Expression of the Escherichia coli sugar phosphate transporter UhpT is induced by extracellular glucose 6-phosphate through a transmembrane signaling process dependent on the sensor kinase UhpB and the UhpT homolog, UhpC . These proteins are thought to regulate the phosphorylation of the transcription activator, UhpA . To examine the effect of protein phosphorylation on the binding of UhpA to target sequences in the uhpT promoter region, the UhpA protein was overexpressed and purified . Purified UhpA was phosphorylated by acetyl phosphate in a reaction that was dependent on Mg2+ and on the presence of aspartate 54, the site of phosphorylation in homologous response regulators . Gel electrophoretic mobility shift and DNase I and hydroxyl radical protection assays showed that UhpA bound specifically to the region of the uhpT promoter extending from -80 to -50 bp, relative to the transcription start site . At higher concentrations of UhpA, binding was extended to the -32 region . Binding to the -64 element exhibited positive cooperativity and was stimulated severalfold by phosphorylation of UhpA, whereas extension to the downstream region was more strongly affected by phosphorylation . The consensus sequences for the high affinity UhpA-binding sites in the -64 element and for the downstream, low affinity sites are proposed . The pattern of in vitro binding by UhpA agreed with the in vivo observations that phosphorylation-independent assembly of the transcription initiation complex can occur at elevated concentrations of UhpA.

J Biol Chem, 1997 Jan 17, 272(3), 1891 - 5
Effect of the amino acid attached to Escherichia coli initiator tRNA on its affinity for the initiation factor IF2 and on the IF2 dependence of its binding to the ribosome; Wu XQ et al.; We show that the nature of the amino acid in the formylaminoacyl-tRNA influences initiation factor (IF) 2 dependence of its ribosome binding and that this IF2 dependence reflects the relative affinity of the formylaminoacyl-tRNA for the initiation factor IF2 . We compared the template-dependent ribosome binding activities, in the presence of initiation factors, of wild type and anticodon sequence mutants of Escherichia coli initiator tRNAs that carry formylmethionine (fMet), formylglutamine (fGln), or formylvaline (fVal) . The fGln-tRNA bound less well than fMet-tRNA whereas the fVal-tRNA bound as well as fMet-tRNA . The rate and extent of binding of fGln-tRNA to the ribosome was significantly increased by further addition of purified initiation factor IF2 . In contrast, the binding of fVal-tRNA or fMet-tRNA was not affected much by the addition of IF2 . Using gel mobility shift assay, we have measured the apparent Kd values of the IF2.formylaminoacyl-tRNA binary complexes . These are 1.8, 3.5, and 10.5 microM for fMet-tRNA, fVal-tRNA, and fGln-tRNA, respectively.

J Biol Chem, 1997 Jan 17, 272(3), 1761 - 8
Histidine 225, a residue of the NhaA-Na+/H+ antiporter of Escherichia coli is exposed and faces the cell exterior; Olami Y et al.; Cysteine residues were found nonessential in the mechanism of the NhaA antiporter activity of Escherichia coli . The functional C-less NhaA has provided the groundwork to study further histidine 225 of NhaA which has previously been suggested to play an important role in the activation of NhaA at alkaline pH (Rimon, A., Gerchman, Y., Olami, Y., Schuldiner, S . and Padan, E . (1995) J . Biol . Chem . 270, 26813-26817) . C-less H225C was constructed and shown to possess an antiporter activity 60% of that of C-less antiporter and a pH profile similar to that of both the C-less or wild-type antiporters . Remarkably, whereas neither the wild-type nor the C-less antiporters were affected by N-ethylmaleimide, C-less H225C was inhibited by this reagent . To determine the degree of alkylation of the antiporter protein by N-ethylmaleimide, antiporter derivatives tagged at their C termini with six histidines residues were constructed . Alkylation of C-less H225C was measured by labeling of everted membrane vesicles with {14C}N-ethylmaleimide, affinity purification of the His-tagged antiporter, and determination of the radioactivity of the purified protein . This assay showed that H225C is alkylated to a much higher level than any of the native cysteinyl residues of NhaA reaching saturation at alkyl/NhaA stoichiometry of 1 . The wild-type derivative showed at least 10-fold less alkylation even at higher concentrations, suggesting that H225C resides in a domain that is much more exposed to N-ethylmaleimide than the native cysteinyl residues of NhaA . Since H225C residues both in right-side out and inside-out membrane vesicles were quantitatively alkylated by N-ethylmaleimide, this assay was used to determine the accessibility of H225C to other SH reagents by titrating the H225C left free to react with N-ethylmaleimide, following exposure of the membranes to the reagents . Furthermore, since membrane-impermeant probes can react with residues in membrane-embedded protein only if accessible to the medium containing the reagent, the assay was used to determine the membrane topology of H225C . As expected for a membrane-permeant probe, p-chloromercuribenzoate reacted with H225C as efficiently as N-ethylmaleimide in both membrane orientations . Similar results were obtained with methanethiosulfonate ethylammonium supporting the recent observations that this probe is membrane-permeant . On the other hand, both membrane-impermeant reagents p-chloromercuribenzosulfonate and methanethiosulfonate ethyl-trimethyl ammonium bromide reacted with H225C 10-fold more in right-side out than in inside-out vesicles, and p-chloromercuribenzosulfonate also blocked completely the H225C in intact cells . These results strongly suggest that H225C is exposed at the periplasmic face of the membrane.

J Biol Chem, 1997 Jan 17, 272(3), 1473 - 9
Roles of DNA topoisomerases in the regulation of R-loop formation in vitro; Phoenix P et al.; We have recently found that stable R-loop formation occurs in vivo and in vitro when a portion of the Escherichia coli rrnB operon is transcribed preferentially in its physiological orientation . Our results also suggested that the formation of such structures was more frequent in topA mutants and was sensitive to the template DNA supercoiling level . In the present report we investigated in greater detail the involvement of DNA topoisomerases in this process . By using an in vitro transcription system with phage RNA polymerases, we found that hypernegative supercoiling of plasmid DNAs in the presence of DNA gyrase is totally abolished by RNase H, suggesting that extensive R-looping occurs during transcription in the presence of DNA gyrase . When RNase A is present, significant hypernegative supercoiling occurs only when the 567-base pair rrnB HindIII fragment is transcribed in its physiological orientation . This result suggests that more stable R-loops are being produced in this orientation . Our results also suggest that DNA gyrase can participate in the process of R-loop elongation . The strong transcription-induced relaxing activity of E . coli DNA topoisomerase I is shown to efficiently counteract the effect of DNA gyrase and thus inhibit extensive R-looping . In addition, we found that an R-looped plasmid DNA is a better substrate for relaxation by E . coli DNA topoisomerase I as compared with a non-R-looped substrate.

J Biol Chem, 1997 Jan 17, 272(3), 1421 - 4
Spodoptera frugiperda caspase-1, a novel insect death protease that cleaves the nuclear immunophilin FKBP46, is the target of the baculovirus antiapoptotic protein p35; Ahmad M et al.; Employing the degenerate primer-dependent polymerase chain reaction approach used recently to clone human Mch2, we have identified and cloned the insect Spodoptera frugiperda target of the baculovirus antiapoptotic protein p35 . This protein named Sf caspase-1 belongs to the family of caspases and is highly related to human Mch3 and CPP32 in sequence and specific activity . The proenzyme of Sf caspase-1 is 299 amino acids in length and can undergo autocatalytic processing in Escherichia coli to an active enzyme heterocomplex . Autoprocessing occurs at Asp-28, Asp-184, and Asp-195 to generate the large p19/p18 and small p12 subunits . Sf caspase-1 is able to induce apoptosis in Sf9 cells and is capable of cleaving p35 to similar sized fragments as observed with extracts from p35 null mutant baculovirus-infected Sf9 cells . Sf caspase-1 activity is potently inhibited by p35, suggesting that it is an important target of this antiapoptotic protein . Finally, the Sf9 nuclear immunophilin FKBP46 was identified as a death-associated substrate for Sf caspase-1.

Science, 1997 Jan 17, 275(5298), 377 - 80
Kinetic measurement of the step size of DNA unwinding by Escherichia coli UvrD helicase; Ali JA et al.; The kinetic mechanism by which the DNA repair helicase UvrD of Escherichia coli unwinds duplex DNA was examined with the use of a series of oligodeoxynucleotides with duplex regions ranging from 10 to 40 base pairs . Single-turnover unwinding experiments showed distinct lag phases that increased with duplex length because partially unwound DNA intermediate states are highly populated during unwinding . Analysis of these kinetics indicates that UvrD unwinds duplex DNA in discrete steps, with an average "step size" of 4 to 5 base pairs (approximately one-half turn of the DNA helix) . This suggests an unwinding mechanism in which alternating subunits of the dimeric helicase interact directly with duplex DNA.

Biochim Biophys Acta, 1997 Jan 16, 1318(1-2), 225 - 34
High cyclic transhydrogenase activity catalyzed by expressed and reconstituted nucleotide-binding domains of Rhodospirillum rubrum transhydrogenase; Yamaguchi M et al.; The hydrophilic, extramembranous domains I (alpha 1 subunit) and III of the Rhodospirillum rubrum nicotinamide nucleotide transhydrogenase were expressed in Escherichia coli and purified therefrom as soluble proteins . These domains bind NAD(H) and NADP(H) . respectively, and together they form the enzyme's catalytic site . We have demonstrated recently that the isolated domains I and III of the bovine transhydrogenase (or domain I of R . rubrum plus domain III of the bovine enzyme) reconstitute to catalyze transhydrogenation in the absence of the membrane-intercalated domain II, which carries the enzyme's proton channel . Here we show that the expressed domains I and III of the R . rubrum transhydrogenase catalyze a very high NADP(H)-dependent cyclic transhydrogenation from NADH to AcPyAD (3-acetylpyridine adenine dinucleotide) with a Vmax of 214 mumol AcPyAD reduced (min x mg of domain I)-1 . The reaction mechanism is 'ping-pong' with respect to NADH and AcPyAD, as these nucleotides bind interchangeably to domain I, and the stereospecificity of hydride ion transfer is from the 4A position of NADH to the 4A position of AcPyAD . The expressed domain I is dimeric, like the native alpha 1 subunit of the enzyme, but the expressed domain III is monomeric and contains 0.94 mol NADP(H) per mol.

Nature, 1997 Jan 16, 385(6613), 275 - 8
Synergistic effects of substrate-induced conformational changes in phosphoglycerate kinase activation; Bernstein BE et al.; Phosphoglycerate kinase (PGK), a key enzyme in glycolysis, catalyses the transfer of a phosphoryl-group from 1,3-bisphosphoglycerate to ADP to form 3-phosphoglycerate and ATP . Despite extensive kinetic and structural investigations over more than two decades, the conformation assumed by this enzyme during catalysis remained unknown . Here we present the 2.8 A crystal structure of a ternary complex of PGK from Trypanosoma brucei, the causative agent of sleeping sickness . This structure determination relied on a procedure in which fragments containing less than 10% of the scattering mass were successively positioned in the unit cell to obtain phases . The PGK ternary complex exhibits a dramatic closing of the large cleft between the two domains seen in all previous studies, thereby bringing the two ligands, 3-phosphoglycerate and ADP into close proximity . Our results demonstrate that PGK is a hinge-bending enzyme, reveal a novel mechanism in which substrate-induced effects combine synergistically to induce major conformational changes and, to our knowledge, afford the first observation of the PGK active site in a catalytic conformation.

Eur J Biochem, 1997 Jan 15, 243(1-2), 482 - 92
Effects of minor and major groove-binding drugs and intercalators on the DNA association of minor groove-binding proteins RecA and deoxyribonuclease I detected by flow linear dichroism; Tuite E et al.; Linear and circular dichroic spectroscopies have been employed to investigate the effects of small DNA ligands on the interactions of two proteins which bind to the minor groove of DNA, viz . RecA protein from Escherichia coli and deoxyribonuclease I (bovine pancreas) . Ligands representing three specific non-covalent binding modes were investigated: 4',6-diamidino-2-phenylindole and distamycin A (minor groove binders), methyl green (major groove binder), and methylene blue, ethidium bromide and ethidium dimer (intercalators) . Linear dichroism was demonstrated to be an excellent detector, in real time, of DNA double-strand cleavage by deoxyribonuclease I . Ligands bound in all three modes interfered with the deoxyribonuclease I digestion of dsDNA, although the level of interference varied in a manner which could be related to the ligand binding site, the ligand charge appearing to be less important . In particular, the retardation of deoxyribonuclease I cleavage by the major groove binder methyl green demonstrates that accessibility to the minor groove can be affected by occupancy of the opposite groove . Binding of all three types of ligand also had marked effects on the interaction of RecA with dsDNA in the presence of non-hydrolyzable cofactor adenosine 5'-O-3-thiotriphosphate, decreasing the association rate to varying extents but with the strongest effects from ligands having some minor groove occupancy . Finally, each ligand was displaced from its DNA binding site upon completion of RecA association, again demonstrating that modification of either groove can affect the properties and behaviour of the other . The conclusions are discussed against the background of previous work on the use of small DNA ligands to probe DNA-protein interactions.

Eur J Biochem, 1997 Jan 15, 243(1-2), 408 - 14
Nicotinamide riboside, an unusual, non-typical, substrate of purified purine-nucleoside phosphorylases; Wielgus-Kutrowska B et al.; Nicotinamide 1-beta-D-riboside (Nir), the cationic, reducible moiety of the coenzyme NAD+, has been confirmed as an unusual substrate for purified purine-nucleoside phosphorylase (PNP) from a mammalian source (calf spleen) . It is also a substrate of the enzyme from Escherichia coli . The Km values at pH 7, 1.48 mM and 0.62 mM, respectively, were 1-2 orders of magnitude higher than for the natural substrate inosine, but the Vmax values were comparable, 96% and 35% that for Ino . The pseudo first-order rate constants, Vmax/Km, were 1.1% and 2.5% for the calf spleen and E . coli enzymes . The aglycon, nicotinamide, was neither a substrate nor an inhibitor of PNP . Nir was a weak inhibitor of inosine phosphorolysis catalyzed by both enzymes, with Ki values close to the Km for its phosphorolysis, consistent with simple competitive inhibition; this was further confirmed by Dixon plots . Phosphorolysis of the fluorescent positively charged substrate 7-methylguanosine was also inhibited in a competitive manner by both Ino and Nir . Phosphorolysis of Nir by both enzymes was inhibited competitively by several specific inhibitors of calf spleen and E . coli PNP, with Ki values similar to those for inhibition of other natural substrates . The pH dependence of the kinetic constants for the phosphorolysis of Nir and of a variety of other substrates, was extensively investigated, particularly in the alkaline pH range, where Nir exhibited abnormally high substrate activity relative to the reduced reaction rates of both enzymes towards other anionic or neutral substrates . The overall results are discussed in relation to present concepts regarding binding and phosphorolysis of substrates by PNP based on crystallographic data of enzyme-inhibitor complexes, and current studies on enzymatic and nonenzymatic mechanisms of the cleavage of the Nir glycosidic bond.

Eur J Biochem, 1997 Jan 15, 243(1-2), 384 - 92
Characterisation of the isolated Che Y C-terminal fragment (79-129)--Exploring the structure/stability/folding relationship of the alpha/beta parallel protein Che Y; Bruix M et al.; To gain insight into how the three-dimensional structure, stability and folding of the protein Che Y are related to one another, we have performed a conformational analysis of a long fragment of this protein, encompassing its C-terminal 51 residues (79-129) . This fragment consists of residues in the beta-strands 4 and 5 and alpha-helices 4 and 5 of native Che Y . The study has been performed by two-dimensional NMR and far-ultraviolet circular dichroism in aqueous solution and in 30% (by vol.) trifluoroethanol/ water at 273 K and 298 K . We observe little structure for this fragment in aqueous solution which could be due to low helical populations in the regions corresponding to helices 4 and 5 . Within the limits of the residual helical structure experimentally detected, helix 4 appears to extend beyond the N-terminus observed in the native structure by over four residues belonging to the preceding loop . In 30% trifluoroethanol the helical content of both helices increase and helix 4 extends further to include the preceding beta-strand 4 . None of the long-range NOEs present in native Che Y are observed under the explored experimental conditions . The conformational shifts of the H(alpha) protons within the alpha-helices of fragment 79-129 are identical to those of shorter synthetic peptides corresponding to the isolated alpha-helices . Thus, the fragment 79-129 appears to behave as an open chain with low local helical populations . The very low intrinsic ability for structure formation displayed by this region of Che Y at pH 2.5 suggests that in the folded protein this region could be mainly stabilised by interactions with the N-terminal Che Y region . This is in accordance with the contact map of Che Y, which shows that the strongest non-local contacts of C-terminal residues are with residues of the N-terminal region, while those within the C-terminal region are very weak . More importantly, the relationship appears to be possibly extended to the folding properties of the protein, since the C-terminal region is not structurally formed in the folding transition state of Che Y but in the final steps of the folding.

Eur J Biochem, 1997 Jan 15, 243(1-2), 336 - 43
Turnover number of Escherichia coli F0F1 ATP synthase for ATP synthesis in membrane vesicles; Etzold C et al.; The rate of ATP synthesized by the ATP synthase (F0F1-ATPase) is limited by the rate of energy production via the respiratory chain, when measured in everted membrane vesicles of an Escherichia coli atp wild-type strain . After energization of the membranes with NADH, fractional inactivation of F0F1 by the covalent inhibitor N,N'-dicyclohexylcarbodiimide allowed the rate of ATP synthesis/mol remaining active ATP synthase complexes to increase; the active ATP synthase complexes were calculated using ATP hydrolysis rates as the defining parameter . In addition, variation of the assay temperature revealed an increase of the ATP synthesis rate up to a temperature of 37 degrees C, the optimal growth temperature of E . coli . In parallel, the amount of F0F1 complexes present in membrane vesicles was determined by immunoquantitation to be 3.3 +/- 0.3% of the membrane protein for cells grown in rich medium and 6.6 +/- 0.3% for cells grown in minimal medium with glycerol as sole carbon and energy source . Based on these data, a turnover number for ATP synthesis of 270 +/- 40 s(-1) could be determined in the presence of 5% active F0F1 complexes . Therefore, these studies demonstrate that the ATP synthase complex of E . coli has, with respect to maximum rates, the same capacity as the corresponding enzymes of eukaryotic organells.

Eur J Biochem, 1997 Jan 15, 243(1-2), 268 - 73
Mitochondrial asparaginyl-tRNA synthetase is encoded by the yeast nuclear gene YCR24c; Landrieu I et al.; One of the open reading frames located on yeast Saccharomyces cerevisiae chromosome III, YCR24c, appeared to code for a protein of unknown function, but the predicted sequence showed similarity with asparaginyl-tRNA synthetase from Escherichia coli, with 38% amino acid identity . There is a putative mitochondrial targeting signal at the N-terminus of the YCR24c product . Northern blot analysis of total RNA from a wild-type strain sigma1278b confirmed that YCR24c was transcribed . Disruption of the chromosomal copy of YCR24c in a respiratory-competent haploid cell induced a petite phenotype, but did not affect cell viability . This respiratory-defective phenotype is typical for a mutation in a nuclear gene that induces a non-functional mitochondrial protein synthesis system . The protein encoded by YCR24c was expressed in Escherichia coli in a histidine-tagged form and isolated . The enzyme aminoacylated unfractionated Escherichia coli tRNA with asparagine . These results identified YCR24c as the structural gene for yeast mitochondrial asparaginyl-tRNA synthetase.

Eur J Biochem, 1997 Jan 15, 243(1-2), 202 - 8
Processing of thionin precursors in barley leaves by a vacuolar proteinase; Romero A et al.; Thionins are synthesized as precursors with a signal peptide and a long C-terminal acidic peptide that is post-translationally processed . A fusion protein including the maltose-binding protein from Escherichia coli (MalE), thionin DG3 from barley leaves, and its acidic C-terminal peptide has been used to obtain antibodies that recognize both domains of the precursor . In barley leaf sections, mature thionins accumulated in the vacuolar content, while the acidic peptide was not detected in any cell fraction . Brefeldin A and monensin inhibited processing of the precursor but its export from the microsomal fraction was not inhibited . Both purified vacuoles and an acid (pH 5.5) extract from leaves processed the fusion protein into a MalE-thionin and an acidic peptide fragment . A 70-kDa proteinase that effected this cleavage was purified from the acid extract . Processing of the fusion protein by both lysed vacuoles and the purified proteinase was inhibited by Zn2+ and by Cu2+, but not by inhibitors of the previously described vacuolar processing thiol or aspartic proteinases . In vivo processing of the thionin precursor in leaf sections was also inhibited by Zn2+ and Cu2+ . Variants of the fusion protein with altered processing sites that represented those of thionin precursors from different taxa were readily processed by the proteinase, whereas changing the polarity of either the C-terminal or N-terminal residues of the processing site prevented cleavage by the proteinase.

EMBO J, 1997 Jan 15, 16(2), 430 - 8
DNA double-strand breaks caused by replication arrest; Michel B et al.; We report here that DNA double-strand breaks (DSBs) form in Escherichia coli upon arrest of replication forks due to a defect in, or the inhibition of, replicative DNA helicases . The formation of DSBs was assessed by the appearance of linear DNA detected by pulse-field gel electrophoresis . Processing of DSBs by recombination repair or linear DNA degradation was abolished by mutations in recBCD genes . Two E . coli replicative helicases were tested, Rep, which is essential in recBC mutants, and DnaB . The proportion of linear DNA increased up to 50% upon shift of rep recBTS recCTS cells to restrictive temperature . No increase in linear DNA was observed in the absence of replicating chromosomes, indicating that the formation of DSBs in rep strains requires replication . Inhibition of the DnaB helicase either by a strong replication terminator or by a dnaBTS mutation led to the formation of linear DNA, showing that blocked replication forks are prone to DSB formation . In wild-type E . coli, linear DNA was detected in the absence of RecBC or of both RecA and RecD . This reveals the existence of a significant amount of spontaneous DSBs . We propose that some of them may also result from the impairment of replication fork progression.

EMBO J, 1997 Jan 15, 16(2), 260 - 6
A protein-arginine methyltransferase binds to the intracytoplasmic domain of the IFNAR1 chain in the type I interferon receptor; Abramovich C et al.; The intracytoplasmic domain (IC) of cytokine receptors provides docking sites for proteins which mediate signal transduction . Thus, in interferon-alpha,beta receptors (IFNAR1 and 2), the IC region binds protein-tyrosine and -serine/threonine kinases which phosphorylate the receptor and the associated Stat transcription factors . A two-hybrid screening was carried out to identify additional proteins which could interact with the IC domain of the IFNAR1 chain of the IFN-alpha,beta receptor . Several positive clones representing a protein sequence designated IR1B4 were recovered from a human cDNA library . IR1B4 was identified as the human homolog of PRMT1, a protein-arginine methyltransferase from rat cells . Flag-IR1B4 fusion proteins bind to the isolated IFNAR1 intracytoplasmic domain produced in Escherichia coli, as well as to the intact IFNAR1 chain extracted by detergent from human U266 cell membranes . S-Adenosylmethionine-dependent methyltransferase activity was precipitated by anti-IFNAR1 antibodies from untreated human cells . IR1B4/PRMT1 is involved in IFN action since U266 cells rendered deficient in this methyltransferase by antisense oligonucleotides become more resistant to growth inhibition by IFN . Methylation of proteins by enzymes which can attach to the IC domains of receptors may be a signaling mechanism complementing protein phosphorylation . Among substrates methylated by PRMT1 are RNA-binding heterogeneous nuclear ribonucleoproteins (hnRNPs) which are involved in mRNA processing, splicing and transport into the cytoplasm.

EMBO J, 1997 Jan 15, 16(2), 211 - 20
Molecular structure of the sarcomeric M band: mapping of titin and myosin binding domains in myomesin and the identification of a potential regulatory phosphorylation site in myomesin; Obermann WM et al.; The M band of sarcomeric muscle is a highly complex structure which contributes to the maintenance of the regular lattice of thick filaments . We propose that the spatial coordination of this assembly is regulated by specific interactions of myosin filaments, the M band protein myomesin and the large carboxy-terminal region of titin . Corresponding binding sites between these proteins were identified . Myomesin binds myosin in the central region of light meromyosin (LMM, myosin residues 1506-1674) by its unique amino-terminal domain My1 . A single titin immunoglobulin domain, m4, interacts with a myomesin fragment spanning domains My4-My6 . This interaction is regulated by phosphorylation of Ser482 in the linker between myomesin domains My4 and My5 . Myomesin phosphorylation at this site by cAMP-dependent kinase and similar or identical activities in muscle extracts block the association with titin . We propose that this demonstration of a phosphorylation-controlled interaction in the sarcomeric cytoskeleton is of potential relevance for sarcomere formation and/or turnover . It also reveals how binding affinities of modular proteins can be regulated by modifications of inter-domain linkers.

Mutat Res, 1997 Jan 15, 388(1), 45 - 57
Evaluation of potential genotoxicity of pulsed electric and electromagnetic fields used for bone growth stimulation; Jacobson-Kram D et al.; Medical devices emitting pulsed electric and electromagnetic fields have been found to be effective for a number of clinical applications including stimulation of bone and tissue growth . To determine whether pulsed fields of the type used in these clinical applications present a mutagenic hazard, electric and electromagnetic fields at two exposure levels were tested in the Ames test, CHO cell chromosomal aberration assay, BALB/3T3 cell transformation assay and unscheduled DNA synthesis assay in primary rat hepatocytes . For both field types, initial and independent repeat studies were performed for each assay at both clinical and supra clinical doses . In all assays, the results show a lack of cytotoxic, transforming and mutagenic activity . The data suggest that pulsed electric and electromagnetic fields of the type and dose levels used in bone growth stimulation lack mutagenic and transforming activity.

Arch Biochem Biophys, 1997 Jan 15, 337(2), 338 - 44
Fungal P450nor: expression in Escherichia coli and site-directed mutageneses at the putative distal region; Okamoto N et al.; P450nor, nitric oxide reductase from Fusarium oxysporum, was expressed in the soluble fraction of Escherichia coli cells by modifying the N-terminal codons and utilizing the pCW vector . The modified P450nor purified to electrophoretic homogeneity had spectral and enzymatic properties identical to those of native P450nor obtained from the fungi . Residues 239 to 247 of the modified P450nor were replaced with Lys by site-directed mutagenesis . Thr-243 to His- and Arg-mutants were also created . Among 11 mutants, only the Thr-243 to Lys-mutant exhibited an absorption spectrum characteristic of a nitrogenous ligand-bound form of P450 at pH 8.0 in the ferric state, but the spectrum was altered to that of the wild-type P450nor as the pH was lowered . Other mutants had spectra typical of the low- and high-spin mixed form of P450 in the ferric state . In the ferrous state, all mutants showed the same spectrum as the wild-type P450nor . Nitric oxide reductase activity was considerably decreased by the replacement of Thr-243 with Lys, His, or Arg or Ala-239 with Lys . These findings indicate that Thr-243 is located more closely to the heme iron than other residues in the putative distal helix of P450nor and plays an important role in the catalytic activity, but a specific difference in the structure of the heme pocket from other P450s is suggested.

Arch Biochem Biophys, 1997 Jan 15, 337(2), 232 - 8
Immunological analysis of two calpain-like Ca2+-dependent proteinases from lobster striated muscles: relationship to mammalian and Drosophila calpains; Beyette JR et al.; Lobster skeletal muscles contain four Ca2+-dependent cysteine proteinases (CDPs I, IIa, IIb, and III) that degrade myofibrillar proteins . Lobster CDPs share many properties with calpains from vertebrate tissues, but differ in native mass and subunit composition . Recently, cDNAs encoding a calpain-like protein (Dm-calpain; 91.5 or 94 kDa) have been isolated from fruit fly, Drosophila melanogaster . To further clarify the relationship between invertebrate CDPs and mammalian calpains, antibodies specific for mu-, m-, p94 (nCL-1), and Dm-calpains and lobster CDP IIb (native M(r) 195,000, subunit M(r) 95,000) were used in immunoblots to test for antigenic cross-reactivity . No common epitopes were found between CDP IIb and vertebrate calpains . However, polyclonal antibodies to CDP IIb cross-reacted strongly with a C-terminal 70-kDa portion of Dm-calpain expressed in Escherichia coli . Conversely, polyclonal antibodies to Dm-calpain recognized CDP IIb . A second CDP, CDP IIa (native M(r) 125,000), was partially purified from lobster muscle; enzyme activity coeluted with a 60-kDa polypeptide using anion-exchange chromatography . The 60-kDa protein reacted with a polyclonal antibody raised against a 20-amino acid peptide sequence found around the catalytic cysteine residue of mu- and m-calpains, but not with antibodies raised against other regions of mu- or m-calpain or with the anti-CDP IIb antibody . These results suggest that (1) the CDP IIb is the homolog of Drosophila calpain in crustaceans and (2) the active site regions of CDP IIa and mu- and m-calpains are similar.

Arch Biochem Biophys, 1997 Jan 15, 337(2), 202 - 8
Substrate specificity modification of the stromal glycerol-3-phosphate acyltransferase; Ferri SR et al.; The stromal glycerol-3-phosphate acyltransferases (GPATs; EC 2.3.1.15) from spinach (Spinacia oleracea) and squash (Cucurbita moschata) were expressed in Escherichia coli and their activities with palmitoyl-CoA and oleoyl-CoA compared . The GPAT from squash, a chilling-sensitive plant, was found to have the greatest difference in activities between the two substrates, using palmitoyl-CoA over three times faster than oleoyl-CoA . In contrast, the enzyme from spinach, a chilling-tolerant plant, preferred oleoyl-CoA over palmitoyl-CoA . By using conserved restriction endonuclease sites each of the two genes was divided into three fragments of roughly equal size and recombined to create six different chimeras . All chimeras retained a large portion of their original activity but in most cases the specificity was greatly altered . The central third of the protein was found to contain the structural features which determine substrate specificity of the wild-type GPATs . Two of the chimeras, which have a spinach-derived central region and a squash-derived carboxyl region, were found to have greatly enhanced specificities for 18:1 acyl chains, potentially making them ideal for decreasing the level of saturation of plant membrane lipids through genetic engineering.

Structure, 1997 Jan 15, 5(1), 7 - 11
Role REVersal: understanding how RRE RNA binds its peptide ligand; Grate D et al.; The structure of a complex between the HIV Revresponsive element (RRE) RNA and a fragment of the Rev protein has recently been determined by NMR . Together with previous studies of the Tat-TAR complex, these results show how RNA elements with considerable tertiary structure are able to play a more active role in directing binding to elements of protein secondary structure.

Structure, 1997 Jan 15, 5(1), 109 - 24
The DNA-binding domain of OmpR: crystal structures of a winged helix transcription factor; Martinez-Hackert E et al.; BACKGROUND: The differential expression of the ompF and ompC genes is regulated by two proteins that belong to the two component family of signal transduction proteins: the histidine kinase, EnvZ, and the response regulator, OmpR . OmpR belongs to a subfamily of at least 50 response regulators with homologous C-terminal DNA-binding domains of approximately 98 amino acids . Sequence homology with DNA-binding proteins of known structure cannot be detected, and the lack of structural information has prevented understanding of many of this familys functional properties . RESULTS: We have determined the crystal structure of the Escherichia coli OmpR C-terminal domain at 1.95 A resolution . The structure consists of three alpha helices packed against two antiparallel beta sheets . Two helices, alpha2 and alpha3, and the ten residue loop connecting them constitute a variation of the helix-turn-helix (HTH) motif . Helix alpha3 and the loop connecting the two C-terminal beta strands, beta6 and beta7, are probable DNA-recognition sites . Previous mutagenesis studies indicate that the large loop connecting helices alpha2 and alpha3 is the site of interaction with the alpha subunit of RNA polymerase . CONCLUSIONS: OmpRc belongs to the family of 'winged helix-turn-helix' DNA-binding proteins . This relationship, and the results from numerous published mutagenesis studies, have helped us to interpret the functions of most of the structural elements present in this protein domain . The structure of OmpRc could be useful in helping to define the positioning of the alpha subunit of RNA polymerase in relation to transcriptional activators that are bound to DNA.

Structure, 1997 Jan 15, 5(1), 1 - 5
A tale of two terminators: crystal structures sharpen the debate on DNA replication fork arrest mechanisms; Wake R et al.; The structure of the Tus-Ter DNA replication fork arrest complex of Escherichia coli reveals a novel architecture for the bound Tus protein and a new type of DNA-binding motif . The structure of the complex may explain how Tus can block movement of a replication fork approaching from one direction and not the other.

Nucleic Acids Res, 1997 Jan 15, 25(2), 410 - 6
Characterization of the autoantigen La (SS-B) as a dsRNA unwinding enzyme; Huhn P et al.; During the analysis of the La (SS-B) autoantigen for catalytic activities an ATP-dependent double-stranded RNA unwinding activity was detected . Both native and recombinant La proteins from different species displayed this activity, which could be inhibited by monospecific anti-La antibodies . La protein was able to melt dsRNA substrates with either two 3'-overhangs or a single 3'- and a 5'-overhang . Double-stranded RNAs with two 5'-overhangs were not unwound, indicating that at least one 3'-overhang is required for unwinding . Sequence elements of the La protein that might be involved in dsRNA unwinding, such as an evolutionarily conserved putative ATP-binding motif and an element that is homologous to the double-stranded RNA binding protein kinase PKR, are discussed.

Nucleic Acids Res, 1997 Jan 15, 25(2), 388 - 94
Binding site of the M-domain of human protein SRP54 determined by systematic site-directed mutagenesis of signal recognition particle RNA; Gowda K et al.; The interaction of protein SRP54M from the human signal recognition particle with SRP RNA was studied by systematic site-directed mutagenesis of the RNA molecule . Protein binding sites were identified by the analysis of mutations that removed individual SRP RNA helices or disrupted helical sections in the large SRP domain . The strongest effects on the binding activity of a purified polypeptide that corresponds to the methionine-rich domain of SRP54 (SRP54M) were caused by changes in helix 8 of the SRP RNA . Binding of protein SRP19 was diminished significantly by mutations in helix 6 and was stringently required for SRP54M to associate . Unexpectedly, mutant RNA molecules that resembled bacterial SRP RNAs were incapable of interaction with SRP54M, showing that protein SRP19 has an essential and direct role in the formation of the ternary complex with SRP54 and SRP RNA . Our findings provide an example for how, in eukaryotes, an RNA function has become protein dependent.

Nucleic Acids Res, 1997 Jan 15, 25(2), 326 - 32
Transcription activation at class II CRP-dependent promoters: the role of different activating regions; Rhodius VA et al.; Transcription activation by the Escherichia coli cyclic AMP receptor protein (CRP) at Class II promoters is dependent on direct interactions between two surface-exposed activating regions (AR1 and AR2) and two contact sites in RNA polymerase . The effects on transcription activation of disrupting either AR1 or AR2 have been measured at different Class II promoters . AR2 but not AR1 is essential for activation at all the Class II promoters that were tested . The effects of single positive control substitutions in AR1 and AR2 vary from one promoter to another: the effects of the different substitutions are contingent on the -35 hexamer sequence . Abortive initiation assays have been used to quantify the effects of positive control substitutions in each activating region on the kinetics of transcription initiation at the Class II CRP- dependent promoter pmelRcon . At this promoter, the HL159 substitution in AR1 results in a defect in the initial binding of RNA polymerase whilst the KE101 substitution in AR2 reduces the rate of isomerization from the closed to the open complex.

Nucleic Acids Res, 1997 Jan 15, 25(2), 304 - 11
Substitution and deletion mutations induced by 2-hydroxyadenine in Escherichia coli: effects of sequence contexts in leading and lagging strands; Kamiya H et al.; To evaluate the mutation frequency and the mutation spectrum of 2-hydroxyadenine (2-OH-Ade), an oxidative DNA lesion, the modified base was site-specifically incorporated into a unique restriction enzyme site (SalI, GTCGA*C or AflII, CTTA*AG where A* represents 2-OH-Ade) in single- and double-stranded vectors . The 2-OH-Ade residues were introduced into (+)- and (-)-strands of the double-stranded vectors and into the (+)-strand of single-stranded vectors . When the vectors were transfected intoEscherichia coli, the modified base showed little to no cytotoxicity . The mutation frequencies of 2-OH-Ade in the SalI and AflII sites were approximately 0.8 and 0.07%, respectively, with double-stranded (+)-vectors . An increase in the mutation frequencies was not observed with single-stranded vectors . When incorporated into the (-)-strand, the mutation frequencies of 2-OH-Ade in the SalI and AflII sites were approximately 0.3 and 0.1%, respectively . The mutations observed most frequently were -1 deletions at both positions, in the case of the (+)-strand . On the other hand, we observed that 2-OH-Ade in the (-)-strand induced A-->G and A-->T substitutions . These results indicate that 2-OH-Ade residues in DNA induce substitution and deletion mutations without blocking replication inE.coli.

FEMS Microbiol Lett, 1997 Jan 15, 146(2), 241 - 5
A unique DNA sequence of human enterotoxigenic Escherichia coli enterotoxin encoded by chromosomal DNA; Imamura S et al.; We detected Ent plasmids in 300 strains of human enterotoxigenic Escherichia coli, but one strain, E . coli 240-3, had neither a small nor a large plasmid and encoded the heat-labile enterotoxin (LTh(240-3)) gene on its chromosome . DNA sequences showed that LTh(240-3) differed by 12 and 14 base pairs from LT (LTh) and LT (LTp) from human H10407 and porcine EWD299 strains, respectively . In deduced precursor toxins, LTh(240-3), LTh and LTp differed from LTh, LTp and LTh(240-3) at nine, eight and eleven positions, respectively . These data suggest that although LTh(240-3) encoded in the chromosome is antigenically similar to LTh, it cannot be grouped with LTh due to differences in its DNA and amino acids sequences.

FEMS Microbiol Lett, 1997 Jan 15, 146(2), 191 - 8
Identification of the gene (aphA) encoding the class B acid phosphatase/phosphotransferase of Escherichia coli MG1655 and characterization of its product; Thaller MC et al.; An open reading frame located in the tyrB-uvrA intergenic region of the Escherichia coli MG1655 chromosome was identified as encoding the class B acid phosphatase of this species on the basis of cloning and expression experiments . A protocol for purification of the enzyme (named AphA) was developed, and its properties were analyzed . The enzyme is a 100-kDa homotetrameric protein which apparently requires a metal co-factor for activity . Similarly to other bacterial class B acid phosphatases, it is able to dephosphorylate several organic phosphomonoesters as well as to catalyze the transfer of low-energy phosphate groups from phosphomonoesters to hydroxyl groups of various organic compounds.

J Immunol Methods, 1997 Jan 15, 200(1-2), 89 - 97
A sensitive fluorometric assay for quantitatively measuring specific peptide binding to HLA class I and class II molecules; Ottenhoff TH et al.; A sensitive, highly reproducible assay was developed for measuring binding of peptides to various HLA class I and II alleles . The assay is based on competition for binding to HLA between a peptide of interest and a fluorescent labelled standard peptide . This mixture is incubated with HLA to obtain equilibrium binding, and subsequently separated on an HPLC size-exclusion column in (i) a protein fraction containing HLA and bound peptide and (ii) a free peptide fraction . Each assay uses only 100 fmol labelled peptide and approximately 10 pmol of HLA . The analytical system contains an autosampler that samples from 96-well microtiter plates . Injections and data recording/evaluation is fully automated . Typical analysis time is 10-12 min per sample . The fluorescence in the HLA-bound peptide and free peptide containing fractions is measured on-line . The ratios of fluorescence signal in protein and peptide fractions at various concentrations of the peptide of interest are determined . IC50 values are calculated from the binding curve as obtained by curve fitting of the data . Here we show results for peptide binding to HLA-DR1 and -DR17 molecules purified from detergent solubilized cell lysates . and for recombinant HLA-A*0201 and HLA-A*0301 expressed in E . coli . The assay reported is sensitive and reproducible . It is non-radioactive and is non-labor intensive due to the high degree of automation.

J Immunol Methods, 1997 Jan 15, 200(1-2), 69 - 77
High level production of soluble single chain antibodies in small-scale Escherichia coli cultures; Kipriyanov SM et al.; We have investigated the effect of growth and induction conditions on the production of soluble single-chain Fv antibody fragments in Escherichia coli under the control of wt lac promoter . The scFv was directed into the periplasmic space by a pelB leader sequence . Addition of sucrose to the medium gave a 15-25-fold increase in the yield of soluble scFv-phOx (3.0 mg/l) for bacterial shake-tube cultures and an increase of 80-150-fold (16.5 mg/l) for shake-flask cultures . Using flask culture in the presence of 0.4 M sucrose, a significant amount of scFv was released into the medium . We found that the scFv could be made to accumulate in the periplasm or be secreted into the medium by simply changing the incubation conditions and the concentration of the inducer . The ratio between soluble antibody fragments and insoluble scFv aggregates proved to be dependent on the strength of the promoter . Lowering the incubation temperature below 20 degrees C had no effect on the yield of soluble antibody fragments in the periplasm, but they were no longer secreted into the medium . An example of high level production in shake-flask cultures and one-step purification by immobilized metal affinity chromatography (IMAC) is described for a soluble scFv specific for the T cell surface antigen CD3 . The biological activity of the purified anti-CD3 scFv was demonstrated by flow cytometry . This method should be especially useful for the functional screening of a large number of clones in small-scale cultures.

J Immunol Methods, 1997 Jan 15, 200(1-2), 27 - 37
Interest of immunomodulation as a mean to improve the preparation of polyclonal and monoclonal antibody reagents; Bouige P et al.; Immunomodulation by monoclonal antibodies (mAbs) was investigated in mice in order to improve the preparation of antibody reagents . Three different types of representative immunogens were chosen: a human soluble protein (secretory immunoglobulin A, SIgA), a bacterial polysaccharide from E . coli K1 and an envelope protein from the hepatitis B virus . These Ag are all of importance for diagnosis and exhibit different levels of immunogenicity . Antibody-mediated enhancement was observed against restricted and defined regions of each immunogen i.e.: the Fab epitopes of SIgA, the preS1 domain of the HBV envelope and associated cell wall components of the capsular PS . The epitopes which were enhanced appeared to be different from those recognized by the modulating mAb . Negative modulations were also observed . Moreover, new epitopes seemed to be generated . In both cases the level and direction of the modulation were irrespective of isotypy and affinity of the mAbs . Interestingly the positive modulatory effect was found to be correlated with an in vitro assay based on the binding of immune complex to antigen-presenting cells.

J Immunol Methods, 1997 Jan 15, 200(1-2), 1 - 16
Implications for the assay and biological activity of interleukin-8: results of a WHO international collaborative study; Mire-Sluis AR et al.; Eight ampouled preparations of interleukin-8 (IL-8) have been evaluated for their suitability to serve as an international standard for IL-8 by 30 laboratories in 12 countries in an international collaborative study . The preparations were assayed in a wide range of in vitro bioassays and immunoassays . It is clear from the study that different recombinant preparations of IL-8 can have different biological specific activities, even though all were produced using E . coli . It is of interest that the intra-laboratory variability of estimates provided by several neutrophil degranulation bioassays was less than that of the immunoassays, suggesting that these bioassays can be as precise, if not more so, than immunoassays . In addition, immunoassay estimates of IL-8 preparations differed from those of bioassays, illustrating the fact that immunoassays do not necessarily measure biologically active cytokine . The large reduction in the inter-laboratory variability of estimates in terms of a common reference preparation clearly illustrates the need for a standard for IL-8 . On the basis of the results reported here, with the agreement of the participants of the study and with the authorisation of the Expert Committee on Biological Standardization (ECBS) of the World Health Organization (WHO) the preparation of IL-8 (89/520) was established as the International Standard for IL-8 with an assigned unitage of 1000 IU/ampoule.

Biochim Biophys Acta, 1997 Jan 14, 1323(1), 29 - 44
Interaction of surfactants with DNA . Role of hydrophobicity and surface charge on intercalation and DNA melting; Bhattacharya S et al.; A probe, 9-(anthrylmethyl)trimethylammonium chloride, 1 . was prepared, 1 binds to caF-thymus DNA or Escherichia coli genomic DNA with high affinity, as evidenced from the absorption titration . Strong hypochromism, spectral broadening and red-shifts in the absorption spectra were observed . Half-reciprocal plot constructed from this experiment gave binding constant of 5 +/- 0.5 x 10(4) M-1 in base molarity . We employed this anthryl probe-DNA complex for studying the effects of addition of various surfactant to DNA . Surfactants of different charge types and chain lengths were used in this study and the effects of surfactant addition to such probe-DNA complex were compared with that of small organic cations or salts . Addition of either salts or cationic surfactants led to structural changes in DNA and under these conditions, the probe from the DNA-bound complex appeared to get released . However, the cationic surfactants could induce such release of the probe from the probe-DNA complex at a much lower concentration than that of the small organic cations or salts . In contrast the anionic surfactants failed to promote any destabilization of such probe-DNA complexes . The effects of additives on the probe-DNA complexes were also examined by using a different technique (fluorescence spectroscopy) using a different probe ethidium bromide . The association complexes formed between the cationic surfactants and the plasmid DNA pTZ19R, were further examined under agarose gel electrophoresis and could not be visualized by ethidium bromide staining presumably due to cationic surfactant-induced condensation of DNA . Most of the DNA from such association complexes can be recovered by extraction of surfactants with phenol-chloroform . Inclusion of surfactants and other additives into the DNA generally enhanced the DNA melting temperatures by a few degrees C and at high {surfactant}, the corresponding melting profiles got broadened.

Biochemistry, 1997 Jan 14, 36(2), 432 - 41
Dinuclear center of ferritin: studies of iron binding and oxidation show differences in the two iron sites; Treffry A et al.; The ferroxidase activity of human ferritin has previously been associated with a diiron site situated centrally within the four-helix bundle of H-type chains (HuHF) . However, direct information about the site of Fe(II) binding has been lacking, and events between Fe(II) binding and its oxidation have not previously been studied . A sequential stopped-flow assay has now been developed to enable the dissection of binding and oxidation . It depends on the ability of 1,10-phenanthroline to complex protein-bound Fe(II) and to distinguish it from the more immediately available free Fe(II) . This approach, aided by the use of site-directed variants, indicates that in HuHF and the non-heme ferritin of Escherichia coli the first 48 Fe(II) atoms/molecule added are bound and oxidized at the dinuclear centers . At a constant iron concentration, the rate of Fe(II) oxidation was maximal for additions of 2 Fe(II) atoms/subunit, consistent with a two-electron oxidation of the Fe(II) pair . Although, at low Fe(II)/protein ratios, no cooperativity in Fe(II) binding was observed; a preferred order of binding was deduced {Fe(II) binding first at site A and then at site B} . Binding of Fe(II) at both sites was essential for fast oxidation . Modification of site A ligands resulted in slow iron binding and slow oxidation . Modification of site B did not prevent Fe(II) binding at site A but greatly reduced its oxidation rate . These differences may mean that dioxygen is initially bound to Fe(II) at site B.

Biochemistry, 1997 Jan 14, 36(2), 390 - 8
Binding affinity and specificity of Escherichia coli RNase H1: impact on the kinetics of catalysis of antisense oligonucleotide-RNA hybrids; Lima WF et al.; In this study we report for the first time the binding affinity of RNase H1 for oligonucleotide duplexes . We used a previously described 17-mer antisense sequence {Monia, B . P., Johnston, J . F., Ecker, D . J., Zounes, M . A., Lima, W . F., & Freier, S . M . (1992) J . Biol . Chem . 267, 19954-19962} hybridized to a complementary oligoribonucleotide to evaluate both the binding affinity and the catalytic rate of RNase H1 . The dissociation constants (Kd) of RNase H1 for the various substrates tested were determined by inhibition analysis using chemically modified noncleavable oligonucleotide heteroduplexes . Catalytic rates were determined using heteroduplex substrates containing chimeric antisense oligonucleotides composed of a five-base deoxynucleotide sequence flanked on either side by chemically modified nucleotides . We find that the enzyme preferentially binds A-form duplexes: RNase H bound A-form duplexes (RNA:RNA and DNA:RNA) approximately 60-fold tighter than B-form duplexes (DNA:DNA) and approximately 300-fold tighter than single-strand oligonucleotides . The enzyme exhibited equal affinity for both the wild type (RNA:DNA) oligonucleotide substrate and heteroduplexes containing various 2'-sugar modifications, while the cleavage rates for these chemically modified substrates were without exception slower than for the wild type substrate . The introduction of a single positively charged 2'-propoxyamine modification into the chimeric antisense oligonucleotide portion of the heteroduplex substrate resulted in both decreased binding affinity and a slower rate of catalysis by RNase H . The cleavage rates for heteroduplexes containing single-base mismatch sequences within the chimeric oligonucleotide portion varied depending on the position of the mismatch but had no effect on the binding affinity of the enzyme . These results offer further insights into the physical binding properties of the RNase H-substrate interaction as well as the design of effective antisense oligonucleotides.

Biochemistry, 1997 Jan 14, 36(2), 301 - 6
Hepatocyte nuclear factor 4 inhibits the activity of site A from the rat apolipoprotein AI gene; Murao K et al.; The pivotal role of apolipoprotein AI (Apo AI) in mediating reverse cholesterol transport has lead us to the study of transcription factors that influence the expression of this gene . Previous studies show that rat HNF-4 enhances the activity of a cis-acting site C in the rat Apo AI promoter . Since sites C and A share 80% homology, we have examined whether HNF-4 binds to and modulates the transcriptional activity of the A-motif . Results show that HNF-4 binds to site A . The transcriptional activity of site A in a human hepatoma cell line, HuH-7, increases 2-2.5-fold in the presence of antisense HNF-4, but the sense construct has no effect on the activity of the reporter template . The lack of an effect of HNF-4 on site A activity may be due to high endogenous levels of the factor in HuH-7 cells . However, in BHK cells HNF-4 clearly inhibits the transcriptional activity of site A . Together these findings suggest that in contrast to the enhancing effects of HNF-4 on site C, the same factor inhibits site A activity . Since hepatocytes normally contain the T3 receptor and this nuclear factor increases site A action, cotransfection of T3 receptor along with antisense HNF-4 further augments the activity of p5'A.CAT . In summary, rat HNF-4 binds to site A from rat Apo AI DNA, and this factor suppresses site A activity . HNF-4 interferes with the enhancer role of the T3 receptor and thus contributes negatively to the net expression of the Apo AI gene.

Biochemistry, 1997 Jan 14, 36(2), 288 - 300
Truncation of the amino terminus of human apolipoprotein A-I substantially alters only the lipid-free conformation; Rogers DP et al.; An amino-terminal deletion mutant (residues 1-43) of human apolipoprotein A-I (apo hA-I) has been produced from a bacterial expression system to explore the structural and functional role of these amino acids, encoded by exon 3, in apo hA-I . Lipid binding of apo delta (1-43)A-I and lipid binding of apo hA-I are very similar as assessed by surface activity, lipid association with palmitoyloleoylphosphatidylcholine (POPC) vesicles, and lipid association with plasma lipoproteins . Preliminary kinetic measurements appear to show that the reactivity of lecithin:cholesterol acyltransferase (LCAT) with the mutant is slightly decreased compared to wild-type apo hA-I . Collectively, these results indicate that the N-terminal region is not necessary for lipid binding or activation of LCAT . In contrast, there are significant structural differences between lipid-free apo delta (1-43)A-I and apo hA-I, as judged by denaturant-induced unfolding, binding of the fluorescent probe 1-anilinonaphthalene-8-sulfonate, surface balance measurements, and far- and near-ultraviolet circular dichroic spectroscopy . All spectral and physical measurements indicate apo delta (1-43)A-I has a folded, tertiary structure, although it is significantly less stable than that of apo hA-I . It is concluded that the N-terminal 43 residues are an important structural element of the lipid-free conformational state of apo hA-I, the absence of which induces a fundamentally different fold for the remaining carboxy-terminal residues, compared to those in native apo hA-I.

Biochem Biophys Res Commun, 1997 Jan 13, 230(2), 442 - 7
Domain organization of murine mdr1b P-glycoprotein: the cytoplasmic linker region is a potential dimerization domain; Juvvadi SR et al.; P-glycoprotein is an integral membrane protein that functions as a cytotoxic drug-efflux pump . Studies suggest that the transporter exists in the membrane as a dimer and possibly higher order structures . We report the bacterial expression of the linker region (amino acids 621-688) of murine mdr1b P-glycoprotein and demonstrate that this region, which serves to link the two homologous halves of the transporter, has the potential to serve as a dimerization domain . The recombinant peptide (8742 daltons) eluted from a gel filtration column at a position corresponding to a dimer (i.e . 17,500 daltons) . A dimer:monomer equilibrium, as a function of peptide concentration, was confirmed by large zone gel filtration chromatography . The dissociation constant (KD) for the dimer:monomer equilibrium was 350 nM . It was possible to dissociate the dimer by low pH (3.0) or high ionic strength (2.5 M NaCl) . Dimerization was not affected by an alkaline pH of 10 or 5 mM EDTA . Studies with a truncated linker peptide indicated that the N-terminal 48 amino acids were sufficient for dimerization.

Biochem Biophys Res Commun, 1997 Jan 13, 230(2), 293 - 6
Purification of the human RARgamma ligand-binding domain and crystallization of its complex with all-trans retinoic acid; Rochel N et al.; A 28-kDa fragment (residues 178-423) of the human retinoic acid receptor gamma, hRARgamma D3E, encompassing the ligand-binding domain (LBD) was overproduced in Escherichia coli and purified as a monomer to more than 95% purity and homogeneity . The Kd for all-trans retinoic acid binding was 0.6 +/- 0.1 nM . Crystals of the LBD complexed with all-trans retinoic acid were grown at pH 7 from sodium acetate in the presence of detergents using the vapor diffusion method . They diffract to 2.0 A using a synchrotron radiation (lambda=0.91 A) and belong to the tetragonal space group P4(1)2(1)2 with unit cell parameters a=b=60.6 A and c=155.3 A, one monomer per asymmetric unit, a solvent content of ca . 33%, and a Vm value of approximately 2 A3/dalton.

FEBS Lett, 1997 Jan 13, 401(1), 15 - 9
Paramagnetic NMR spectroscopy of native and cobalt substituted manganese superoxide dismutase from Escherichia coli; Renault JP et al.; Manganese containing superoxide dismutase from E . coli has been investigated through paramagnetic NMR spectroscopy . The spectrum of the native form was rationalized using a tau(s) = 3x10(-11) s for the Mn(III) ion, consistent with previous estimates from NMRD measurements . Mn(III) has been replaced by a Co(II) ion and a tentative assignment of the NMR spectrum of the Co(II)-substituted derivative has been proposed, based on T1, chemical shifts and 1D-NOE data . The metal coordination geometry is provided by three histidines and a carboxylate group . The presence of a solvent molecule as a loosely bound fifth ligand is also proposed . The NMR data of the Co(II)-substituted derivative of E . coli MnSOD differs from those of Co(II)SOD from other bacterial sources . This suggests that Co(II) substitution is an efficient method to address the problem of metal ion selectivity in superoxide dismutase.

Pediatrics . 1997 Dec;100(6):E2.
Breastfeeding and the risk of life-threatening enterotoxigenic Escherichia coli diarrhea in Bangladeshi infants and children; Clemens JD et al.; OBJECTIVE: To assess the relationship between breastfeeding and the risk of life-threatening enterotoxigenic Escherichia coli (ETEC) diarrhea among Bangladeshi infants and young children <36 months of age . DESIGN: Case-control study . SETTING: A rural Bangladesh community . PARTICIPANTS: A total of 168 cases with clinically severe ETEC diarrhea detected in a treatment center-based surveillance system during 1985 to 1986 and 3679 controls selected in three surveys of the same community during the same calendar interval . OUTCOMES: Cases and controls were compared for the frequency of antecedent breastfeeding patterns . RESULTS: Compared with other feeding modes, exclusive breastfeeding of infants was associated with significant protection against severe ETEC diarrhea (relative risk {RR} = 0.51; 95% confidence interval {CI}: 0.28,0.96) . However, during the second and third years of life, the risk of this outcome was similar in both breastfed and nonbreastfed children (RR = 0.98; 95% CI: 0.45,2.12), and no significant overall protective association between breastfeeding and severe ETEC diarrhea was evident during the first 3 years of life (RR = 0.86; 95% CI: 0.43,1 . 74) . CONCLUSIONS: Exclusive breastfeeding appeared to protect infants against severe ETEC diarrhea, but breastfeeding was not associated with protection after infancy, nor was it associated with a major overall reduction of severe ETEC disease during the first 3 years of life . Although not diminishing the importance of breastfeeding, our findings suggest that other interventions, such as immunization and education about proper food hygiene, may also be required in efforts to prevent this major pediatric disease.

Cell, 1997 Jan 10, 88(1), 19 - 28
Arrangement of tRNAs in pre- and posttranslocational ribosomes revealed by electron cryomicroscopy; Stark H et al.; The three-dimensional structure of the translating 70S E . coli ribosome is presented in its two main conformations: the pretranslocational and the posttranslocational states . Using electron cryomicroscopy and angular reconstitution, structures at 20 A resolution were obtained, which, when compared with our earlier reconstruction of "empty" ribosomes, showed densities corresponding to tRNA molecules--at the P and E sites for posttranslocational ribosomes and at the A and P sites for pretranslocational ribosomes . The P-site tRNA lies directly above the bridge connecting the two ribosomal subunits, with the A-site tRNA fitted snugly against it at an angle of approximately 50 degrees, toward the L7/L12 side of the ribosome . The E-site tRNA appears to lie between the side lobe of the 30S subunit and the L1 protuberance.

Cell, 1997 Jan 10, 88(1), 121 - 9
Redox signal transduction: mutations shifting {2Fe-2S} centers of the SoxR sensor-regulator to the oxidized form; Hidalgo E et al.; SoxR is a {2Fe-2S} transcription factor triggered by oxidative stress and activated in vitro by one-electron oxidation or assembly of the iron-sulfur centers . To distinguish which mechanism operates in cells, we studied constitutively active SoxR (SoxRc) proteins . Three SoxRc proteins contained {2Fe-2S} centers required for in vitro transcription and, like wild-type SoxR, were inactivated by chemical reduction . However, in vivo spectroscopy showed that even without oxidative stress, the three SoxRc proteins failed to accumulate with reduced {2Fe-2S} (< or = 4% compared to > or = 40% for wild type) . One SoxRc protein had a redox potential 65 mV lower than wild type, consistent with its accumulation in the oxidized (activated) form in vivo . These results link in vitro and in vivo approaches showing novel redox regulation that couples an iron-sulfur oxidation state to promoter activation.

J Mol Biol, 1997 Jan 10, 265(1), 56 - 67
Differential binding of S-adenosylmethionine S-adenosylhomocysteine and Sinefungin to the adenine-specific DNA methyltransferase M.TaqI; Schluckebier G et al.; The crystal structures of the binary complexes of the DNA methyltransferase M.TaqI with the inhibitor Sinefungin and the reaction product S-adenosyl-L-homocysteine were determined, both at 2.6 A resolution . Structural comparison of these binary complexes with the complex formed by M.TaqI and the cofactor S-adenosyl-L-methionine suggests that the key element for molecular recognition of these ligands is the binding of their adenosine part in a pocket, and discrimination between cofactor, reaction product and inhibitor is mediated by different conformations of these molecules; the methionine part of S-adenosyl-L-methionine is located in the binding cleft, whereas the amino acid moieties of Sinefungin and S-adenosyl-L-homocysteine are in a different orientation and interact with the active site amino acid residues 105NPPY108 . Dissociation constants for the complexes of M.TaqI with the three ligands were determined spectrofluorometrically . Sinefungin binds more strongly than S-adenosyl-L-homocysteine or S-adenosyl-L-methionine, with KD=0.34 microM, 2.4 microM and 2.0 microM, respectively.

J Mol Biol, 1997 Jan 10, 265(1), 30 - 9
Binding and cleavage of nicked substrates by site-specific recombinases XerC and XerD; Blakely GW et al.; In Xer site-specific recombination two related recombinases, XerC and XerD, catalyse strand cleavage and rejoining reactions at a site, dif, in order to ensure normal chromosome segregation during cell division in Escherichia coli . We have used nicked suicide substrates to trap reaction intermediates and show that XerC cleaves the top strand efficiently while XerD is less efficient at cleaving the bottom strand of dif . Recombinase-mediated cleavage positions are separated by six base pairs and occur at either end of the dif central region adjacent to the recombinase binding sites . XerC can cleave the top strand of dif inefficiently in the absence of its partner recombinase during a reaction that does not require intermolecular synapsis . The presence of a nick in the bottom strand of dif allows cooperative interactions between two XerC protomers bound to adjacent binding sites, suggesting that a conserved interaction domain is present in both XerC and XerD . Cooperativity between two identical recombinase protomers does not occur on un-nicked linear DNA . Ethylation interference footprinting of two XerD catalytic mutant proteins suggests that the conserved domain II arginine from the integrase family RHRY tetrad may make direct contact with the scissile phosphate.

J Mol Biol, 1997 Jan 10, 265(1), 20 - 9
The bifunctional protein DCoH modulates interactions of the homeodomain transcription factor HNF1 with nucleic acids; Rhee KH et al.; The hepatocyte nuclear factor-1 (HNF1) is a homeodomain transcription factor that binds DNA as a dimer . HNF1 dimers associate with two molecules of DCoH, a bifunctional protein that also has an enzymatic function in the tetrahydrobiopterin regeneration, to form stable heterotetramers also capable of DNA binding . Employing purified, recombinant HNF1, HNF1/DCoH heterotetramers and DCoH homotetramers we investigated whether DCoH affects interactions of HNF1 with nucleic acids . Although we detected no direct binding of DCoH to DNA or RNA, DCoH stabilized HNF1/DNA complexes and promoted interactions with sub-optimal DNA target sequences such as the human alpha1-antitrypsin TATA box region . Importantly, we also observed interactions of HNF1 with RNA, but these interactions were completely abolished when HNF1 was complexed with DCoH . Interestingly, DCoH retains its enzymatic activity while complexed with HNF1 . Our results document intermolecular regulation of HNF1 binding to nucleic acids by DCoH.

J Mol Biol, 1997 Jan 10, 265(1), 8 - 19
A model for the mechanism of polymerase translocation; Guajardo R et al.; A general mechanism for polymerase translocation is elaborated . The central feature of this mechanism is that a rapid translocational equilibrium is established after each cycle of nucleoside monophosphate incorporation such that the polymerase distributes itself by diffusional sliding between all accessible positions on the template with relative occupancy determined by relative free energy . While alternative models for translocation have not been fully developed, much of the language currently used to describe this step suggests an active mechanism coupled to conformational transitions in the polymerase . For example, a recent study of force generation by Escherichia coli RNA polymerase during transcription suggests that it is a mechanoenzyme analogous to kinesin of myosin motor proteins . While the proposed mechanism does not rule out conformational transitions during polymerase translocation, it suggests that they may be unnecessary and that translocation can be explained in terms of the affinity of the active site for nucleoside triphosphate and the relative free energies of the polymerase bound at different positions on the template . This mechanism makes specific predictions which are borne out experimentally with polymerases as distinct as E . coli DNAP I, phage T7 RNAP, and E . coli RNAP.

J Mol Biol, 1997 Jan 10, 265(1), 1 - 7
Escherichia coli lac repressor-lac operator interaction and the influence of allosteric effectors; Horton N et al.; The wild type E . coli lac operator is embedded in a 35 base-pair DNA sequence containing extensive 2-fold symmetry, suggesting a symmetric repressor operator complex . However, deviations from strict 2-fold symmetry occur at the central base-pair and at three additional base-pairs . Using an operator fragment binding analysis we have determined: (a) a relative contribution each pair provides to the lac repressor-lac operator DNA complex, (b) the operator DNA length necessary for maximum binding to lac repressor; and (c) the contribution of the several non-symmetric base in the wild-type operator to the binding affinity . Since lac repressor-lac operator DNA interaction is reduced upon binding of the gratuitous inducer, isopropyl-beta-D-galactoside (IPTG), the same DNA fragment binding analysis was performed with the low affinity form of lac repressor . In the presence of inducer, the affinity for the left half site of the wild-type lac operator is reduced without significant reduction on the right half of the operator . Conversely, the anti-inducer orthonitrophenylfucoside (ONPF) which stabilizes the lac repressor-lac operator complex increases the binding affinity, particularly to the right half of the operator.

J Biol Chem, 1997 Jan 10, 272(2), 1302 - 7
Substrate binding by human apurinic/apyrimidinic endonuclease indicates a Briggs-Haldane mechanism; Strauss PR et al.; Apurinic/apyrimidinic endonuclease (AP endo) makes a single nick 5' to a DNA abasic site . We have characterized this reaction by steady-state and transient-state kinetics with purified human AP endo, which had been expressed in Escherichia coli . The substrate was a 49-base pair oligonucleotide with an abasic site at position 21 . This substrate was generated by treating a 49-mer duplex oligonucleotide with a single G/U located at position 21 with uracil-DNA glycosylase . The enzymatic products of the AP endo nicking reaction were a 20-mer with a hydroxyl group at the 3'-terminus and a 28-mer with a phosphodeoxyribose at the 5'-terminus . To obtain maximal enzymatic activity, it was necessary to stabilize the abasic site during treatment with uracil-DNA glycosylase with a reducing agent . Otherwise, a 20-mer with phosphoribose at the 3'-terminus resulted from beta-elimination . In agreement with others, Km and kcat were 100 nM and 10 s(-1), respectively . Heat treatment of the abasic site-containing 49-mer without enzyme also resulted in conversion to the beta-elimination product . The resultant heat degradation product was an efficient inhibitor of AP endo with a Ki of 30 nM . The enzyme required divalent cation (Mg2+) for activity, but bound substrate DNA in the absence of Mg2+ . Electrophoretic mobility shift assays indicated that AP endo bound tightly to DNA containing an abasic site and formed a 1:1 complex at low enzyme concentrations . The association and dissociation rate constants for substrate binding to AP endo were determined by using a challenge assay to follow AP endo-substrate complex formation . Heat degradation product together with heparin served as an effective trap for free enzyme . The results are consistent with a Briggs-Haldane mechanism where k(on) and k(off) are 5 x 10(7) M(-1) s(-1) and 0.04 s(-1), respectively (Kd = 0.8 nM), kcat is 10 s(-1), and product release is very rapid (i.e . k(off,product) >> 10 s(-1)) . This scheme is in excellent agreement with the measured steady-state kinetic parameters.

J Biol Chem, 1997 Jan 10, 272(2), 903 - 10
Identification, cloning, and characterization of cystatin M, a novel cysteine proteinase inhibitor, down-regulated in breast cancer; Sotiropoulou G et al.; A novel human cystatin gene was identified in a differential display comparison aimed at the isolation of transcriptionally regulated genes involved in invasion and metastasis of breast cancer . Messenger RNAs from primary and metastatic tumor cells isolated from the same patient were compared . A partial cDNA was isolated that was expressed in the primary tumor cell line but not in the metastatic line . The full-length cDNA was cloned and sequenced, and the inferred amino acid sequence was found to encode a novel protein, which we named cystatin M, with 40% homology to human family 2 cystatins and similar overall structure . Cystatin M is expressed by normal mammary cells and a variety of human tissues . The mature cystatin M protein was produced in Escherichia coli as a glutathione S-transferase fusion protein using the pGEX-2T expression system and purified by affinity chromatography . The cystatin M fusion protein displayed inhibitory activity against papain . Native cystatin M protein of approximately 14.5 kDa is secreted and was immunoprecipitated from supernatants of mammary cell cultures using affinity-purified antisera raised against recombinant cystatin M . An N-glycosylated form of cystatin M of 20-22 kDa was co-immunoprecipitated and accounted for about 30-40% of total cystatin M protein . Both forms of native cystatin M also occurred intracellularly . Consistent with the mRNA differential expression, no cystatin M protein was detected in metastatic mammary epithelial tumor cells . Loss of expression of cystatin M is likely associated with the progression of a primary tumor to a metastatic phenotype.

J Biol Chem, 1997 Jan 10, 272(2), 804 - 13
Heterologous expression, isolation, and characterization of versicolorin B synthase from Aspergillus parasiticus . A key enzyme in the aflatoxin B1 biosynthetic pathway; Silva JC et al.; Aflatoxin B1 is a potent environmental carcinogen produced by certain strains of Aspergillus . Central to the biosynthesis of this mycotoxin is the reaction catalyzed by versicolorin B synthase (VBS) in which a racemic substrate, versiconal hemiacetal, is cyclized to an optically active product whose absolute configuration is crucial to the interaction of aflatoxin B1 with DNA . Attempted over-production of VBS in Escherichia coli led principally to protein aggregated into inclusion bodies but also small amounts of soluble but catalytically inactive enzyme . Comparisons to wild-type VBS by SDS-polyacrylamide gel electrophoresis and after N-glycosidase F treatment revealed that extensive glycosylation accounted for the mass discrepancy (7,000+/-1,500 Da) between the native and bacterially expressed proteins . Several over-expression systems in Saccharomyces cerevisiae were surveyed in which one that incorporated a secretion signal was found most successful . VBS of indistinguishable mass on SDS-polyacrylamide gel electrophoresis and kinetic properties from the wild-type enzyme could be obtained in 50-100-fold greater amounts and whose catalytic behavior has been examined . The translated protein sequence of VBS showed three potential N-glycosylation sites (Asn-Xaa-Ser/Thr) consistent with the modifications observed above and unexpectedly revealed extensive homology to the ADP-binding region prominently conserved in the glucose-methanol-choline (GMC) family of flavoenzymes . Over-production of VBS in yeast marks the first aflatoxin biosynthetic enzyme to be so obtained and opens the way to direct study of the enzymology of this complex biosynthetic pathway.

J Biol Chem, 1997 Jan 10, 272(2), 798 - 803
Polymerase chain reaction-based cloning of alkyl-dihydroxyacetonephosphate synthase complementary DNA from guinea pig liver; de Vet EC et al.; Peroxisomes are indispensable organelles for ether lipid biosynthesis in mammalian tissues, and the deficiency of these organelles in a number of peroxisomal disorders leads to deficiencies in ether phospholipids . We have previously purified the committed enzyme for ether lipid biosynthesis, i.e . alkyl-dihydroxyacetone-phosphate synthase, to homogeneity . We have now determined the N-terminal amino acid sequence, as well as additional internal sequences obtained after cyanogen bromide cleavage of the enzyme . With primers directed against the N-terminal sequence and against a cyanogen bromide fragment sequence, a 1100-bp cDNA fragment was obtained by conventional polymerase chain reaction using first-strand cDNA from guinea pig liver as a template . The 5' and 3' ends of the cDNA were obtained by rapid amplification of cDNA ends . The open reading frame encodes a protein of 658 amino acids, containing the N-terminal amino acid sequence as well as the cyanogen bromide cleavage fragment sequences . The derived amino acid sequence includes a mature protein 600 amino acids long and a presequence 58 amino acids long . The latter contains a stretch of amino acids known as peroxisomal targeting signal 2 . The size of the mRNA was estimated to be around 4200 nucleotides . Recombinant His-tagged alkyl-dihydroxyacetonephosphate synthase expressed in Escherichia coli was enzymatically active.

J Biol Chem, 1997 Jan 10, 272(2), 754 - 8
Interruption of Escherichia coli heat-stable enterotoxin-induced guanylyl cyclase signaling and associated chloride current in human intestinal cells by 2-chloroadenosine; Parkinson SJ et al.; Diarrhea induced by Escherichia coli heat-stable enterotoxin (STa) is mediated by a receptor guanylyl cyclase cascade . The present study establishes that an intracellular nucleotide-dependent pathway disrupts toxin-induced cyclic GMP (cGMP) production and the associated chloride (Cl-) flux that underlie intestinal secretion . Incubation of Caco 2 human intestinal epithelial cells with the nucleoside analog 2-chloroadenosine (2ClAdo) resulted in a concentration- and time-dependent inhibition of toxin-induced cGMP production . Inhibition of cGMP production correlated with the metabolic conversion of 2ClAdo to 2-chloroadenosine triphosphate . The effect of 2ClAdo did not reflect activation of adenosine receptors, inhibition of adenosine deaminase, or modification of the binding or distribution of STa receptors . Guanylyl cyclase activity in membranes prepared from 2ClAdo-treated cells was inhibited, in contrast to membranes from cells not exposed to 2ClAdo, demonstrating that inhibition of guanylyl cyclase C (GCC) was mediated by a noncompetitive mechanism . Treatment of Caco 2 cells with 2ClAdo also prevented STa-induced Cl- current . Application of 8-bromo-cGMP, the cell-permeant analog of cGMP, to 2ClAdo-treated cells reconstituted the Cl- current, demonstrating that inhibition of Cl- flux reflected selective disruption of ligand stimulation of GCC rather than the chloride channel itself . Thus, the components required for adenine nucleotide inhibition of GCC signaling are present in intact mammalian cells, establishing the utility of this pathway to elucidate the mechanisms regulating ST-dependent guanylyl cyclase signaling and intestinal fluid homeostasis . In addition, these data suggest that the adenine nucleotide inhibitory pathway may be a novel target to develop antisecretory therapy for enterotoxigenic diarrhea.

Nature, 1997 Jan 9, 385(6612), 172 - 6
Unusual Rel-like architecture in the DNA-binding domain of the transcription factor NFATc; Wolfe SA et al.; Transcription factors of the NFAT family regulate the production of effector proteins that coordinate the immune response . The immunosuppressive drugs FK506 and cyclosporin A (CsA) act by blocking a Ca2+-mediated signalling pathway leading to NFAT . Although FK506 and CsA have enabled human organs to be transplanted routinely, the toxic side-effects of these drugs limit their usage . This toxicity might be absent in antagonists that target NFAT directly . As a first step in the structure-based search for NFAT antagonists, we now report the identification and solution structure of a 20K domain of NFATc (NFATc-DBD) that is both necessary and sufficient to bind DNA and activate transcription cooperatively . Although the overall fold of the NFATc DNA-binding domain is related to that of NF-kappaB p50 (refs 2, 3), the two proteins use significantly different strategies for DNA recognition . On the basis of these results, we present a model for the cooperative complex formed between NFAT and the mitogenic transcription factor AP-1 on the interleukin-2 enhancer.

Biochemistry, 1997 Jan 7, 36(1), 274 - 80
The last two cytoplasmic loops in the lactose permease of Escherichia coli comprise a discontinuous epitope for a monoclonal antibody; Sun J et al.; Monoclonal antibody (mAb) 4B11 binds to a conformational epitope in the lactose permease that is exposed on the cytoplasmic face of the membrane with a KD of 2.8 x 10(-7) M . By studying binding of 4B11 to permease mutants containing six contiguous His residues in each cytoplasmic loop, inserted factor Xa protease sites, or a C-terminal deletion, the cytoplasmic loops between helices VIII and IX (loop VIII/IX) and between helices X and XI (loop X/XI) are shown to comprise the epitope . Subsequently, Cys-scanning mutagenesis in conjunction with thiol modification was carried out in order to identify specific residues involved in 4B11 recognition . Glu342 and Arg344 in loop X/XI are primary determinants for 4B11 binding, while Ile283 in loop VIII/IX and Phe334 and Lys335 in loop X/XI are secondary determinants . Consistently, binding of avidin to biotinylated single-Cys replacements in loop VIII/IX or loop X/XI blocks 4B11 binding, but avidin binding to biotinylated Cys residues in other cytoplasmic loops or insertion of cytochrome b562 into cytoplasmic loop VI/VII has no significant effect . The studies demonstrate that the last two cytoplasmic loops in lactose permease comprise a discontinuous epitope for monoclonal antibody 4B11 and thereby provide independent evidence for the conclusion that helices VIII-XI are in close proximity.

Biochemistry, 1997 Jan 7, 36(1), 269 - 73
Cysteine-scanning mutagenesis of helix II and flanking hydrophilic domains in the lactose permease of Escherichia coli; Frillingos S et al.; Using a functional lactose permease mutant devoid of Cys residues (C-less permease), each amino acid residue in putative transmembrane helix II and flanking hydrophilic loops (from Leu34 to Lys74) was replaced individually with Cys . Of the 41 single-Cys mutants, 28 accumulate lactose to > 70% of the steady state observed with C-less permease, and an additional 10 mutants exhibit lower but significant levels of accumulation (25-60% of C-less) . His35-->Cys permease exhibits very low activity (ca . 20% of C-less), while Gly64-->Cys or Asp68-->Cys permease is unable to accumulate lactose . However, His35 can be replaced with Arg without effect on transport activity {Padan, E., Sarkar, H.K., et al . (1985) Proc . Natl . Acad . Sci . U.S.A . 82, 6765-6768} . In addition, even though mutant Gly64-->Cys or Glu68-->Cys is inactive both in the C-less background and in the wild-type, neither Gly64 {Jung, K., Jung, H., et al . (1995) Biochemistry 34, 1030-1039} nor Glu68 {Jessen-Marshall, A.E., & Brooker, R.J . (1996) J . Biol . Chem . 271, 1400-1404} is essential for active lactose transport . Immunoloblot analysis reveals that all of the mutants except His35-->Cys permease are inserted into the membrane at concentrations comparable to that of C-less permease . The transport activity of the single-Cys mutants is altered by N-ethylmaleimide (NEM) treatment in a highly specific manner . Most of the mutants are insensitive, but Cys replacements render the permease sensitive to NEM inactivation at positions that cluster in a manner indicating that they are on one face of an alpha-helix (Thr45-->Cys, Gly46-->Cys, Phe49-->Cys, Ser53-->Cys, Ser56-->Cys, Gln60-->Cys, and Ser67-->Cys) . Interestingly, the same face contains positions where Cys substitution itself leads to low transport activity (Ile52-->Cys, Leu57-->Cys, Gln60-->Cys, and Gly64-->Cys) . The results demonstrate that although no residue per se in this region of the permease is irreplaceable, the surface of one face of helix II is important for active lactose transport.

Biochemistry, 1997 Jan 7, 36(1), 261 - 8
Importance of the gamma-carboxyl group of glutamate-462 of the large alpha-subunit for the catalytic function and the stability of the multienzyme complex of fatty acid oxidation from Escherichia coli; He XY et al.; His450 of the large alpha-subunit of the multienzyme complex of fatty acid oxidation from Escherichia coli was recently identified as an essential catalytic residue of L-3-hydroxyacyl-CoA dehydrogenase {He, X-Y., & Yang, S.-Y . (1996) Biochemistry 35, 9625-9630} . To explore the roles of acidic residues in the dehydrogenase catalysis, every conserved acidic residue in the dehydrogenase functional domain except for those in the NAD-binding motif was replaced with alanine . The resulting mutant complexes were overproduced and characterized . Their component enzymes other than the dehydrogenase were affected very slightly . Removal of the beta-carboxyl group of Asp524 and Asp542 caused only a 3- and 4-fold, respectively, decrease in the catalytic efficiency of the dehydrogenase, thereby showing that their involvement in the dehydrogenase catalysis was limited . In contrast, the alpha/Glu462-->Ala mutant complex showed a greater than 160-fold reduction in the kcat of the dehydrogenase in the forward direction without a significant change of the k(m) for the substrate . The catalytic properties of the alpha/Glu462-->Gln mutant complex were found to be similar to those of the alpha/Glu462-->Ala mutant complex except that the kcat of the dehydrogenase in the backward direction was about 4-fold lower and the Km for the substrate of the thiolase was 6-fold higher . It is concluded that the negative charge of the gamma-carboxyl group of Glu462, but not its ability to form a hydrogen bond, is critical for its interaction with His450, thereby assisting in the catalysis of the dehydrogenase . The pKa of His450 in the E.NADH binary complex was virtually unchanged by the replacement of Glu462 with Ala or Gln . It seems that the binding of substrate is necessary for forming a strong interaction between His450 and Glu462 with the result that the electroneutrality in the active site is maintained and the activation energy of the reaction is lowered . Additionally, the negative charge of Glu462 increases the thermostability of the multienzyme complex.

Biochemistry, 1997 Jan 7, 36(1), 249 - 54
Backbone and side chain dynamics of lac repressor headpiece (1-56) and its complex with DNA; Slijper M et al.; The dynamics of the backbone and (some of) the side chains of lac headpiece (1-56; lac HP56) have been studied for the free protein and for its complex with lac half-operator DNA by 15N T1 and T1p relaxation measurements combined with {1H-15N} NOE experiments . For the structurally well-defined part of the free lac HP56 (i.e., residues 3-49) a rigid backbone was found for residues in the three alpha-helices and for the turn of the helix-turn-helix motif . The loop between helices II and III of lac headpiece, which was characterized by slight disorder in the structure calculations, shows increased mobility . The detected side chains are very mobile . These data are in full agreement with the rms deviations in the structural data of free lac HP56 . When lac HP56 is complexed with DNA, several changes in mobility take place . The most remarkable change was found for the loop region between helices II and III: residue His-29 within this loop interacts with Thy-3 of the operator DNA . As a result this mobile loop adapts itself to the DNA and becomes more rigid . Moreover, most DNA-contacting side chains show a significant decrease in flexibility, although these side chains do not become as rigid as the backbone . These results suggest that the mobility of the regions within lac HP56 important for complexation, i.e., the loop and the DNA-contacting side chains, is essential for a good fit onto the counterparts of the target DNA.

Biochemistry, 1997 Jan 7, 36(1), 173 - 80
Zn2+ promotes the self-association of human immunodeficiency virus type-1 integrase in vitro; Lee SP et al.; It has been recently demonstrated that the Mg(2+)-dependent 3'-processing activity of purified human immunodeficiency virus type-1 (HIV-1) integrase is stimulated by the addition of exogenous Zn2+ {Lee, S . P., & Han, M . K . (1996) Biochemistry 35, 3837-3844} . This activation was hypothesized to result from integrase self-association . In this report, we examine the Zn2+ content of purified HIV-1 integrase by atomic absorption spectroscopy and by application of a thiol modification reagent, p-(hydroxymercuri)benzenesulfonate, with a metallochromic indicator, 4-(2-pyridylazo)resorcinol . We find that the Zn2+ content of HIV-1 integrase varies from 0.1 to 0.92 equiv of Zn2+ per monomer depending on the conditions of protein purification . In vitro activity assays, time-resolved fluorescence emission anisotropy, and gel filtration chromatographic analyses all indicate that EDTA yields an apoprotein which is predominantly monomeric and less active with Mg2+ . Further, sedimentation equilibrium studies reveal that reconstitution of the apoprotein with Zn2+ results in a monomer-tetramer-octamer transition . These results suggest that Zn2+ promotes a conformation with enhanced oligomerization and thereby stimulates Mg(2+)-dependent 3'-processing . This may also imply that multimers larger than dimers (tetramers and possibly octamers) are required for in vitro activity of integrase in the presence of Zn2+ and Mg2+ . It should be noted, however, that the content of Zn2+ did not significantly affect the 3'-processing and strand transfer reactions with Mn2+ in vitro.

Biochemistry, 1997 Jan 7, 36(1), 164 - 72
Energetic and kinetic contributions of contact residues of antibody D1.3 in the interaction with lysozyme; England P et al.; Fully functional variable fragments (Fv) of D1.3, a mouse antibody directed against the hen egg lysozyme, were readily produced as hybrids (Fv-MalE) with the maltose-binding protein of Escherichia coli and purified independently of their antigen-binding properties . We used site-directed mutations of residues in the complementarity-determining regions (CDRs) of D1.3 as local conformational probes, and compared their effects on the binding of Fv and Fv-MalE to lysozyme . We found that the MalE moiety did not significantly interfere with the interaction between the antigen and the antibody Fv fragment . We then determined the contribution of several potential contact residues of D1.3 in the interaction with lysozyme, by assaying the effect of site-directed mutations on the kinetics of association and dissociation of the complex between Fv-MalE and immobilized lysozyme, using the BIAcore apparatus . While the k(on) values were virtually unaffected by the mutations, the k(off) values varied by more than three orders of magnitude . Both charged (aspartate and arginine) and aromatic (tyrosine and tryptophan) residues in the CDR3 regions of the heavy and light chains of D1.3, which form the center of its antigen-combining site, played a preponderant part in the binding of lysozyme . Our results also showed that indirect hydrogen bonds, bridged by water molecules, contributed significantly to the interaction between D1.3 and lysozyme, and that their energy could be estimated at 1 to 2 kcal.mol-1.

Biochemistry, 1997 Jan 7, 36(1), 127 - 38
Interaction of Escherichia coli cobalamin-dependent methionine synthase and its physiological partner flavodoxin: binding of flavodoxin leads to axial ligand dissociation from the cobalamin cofactor; Hoover DM et al.; Cobalamin-dependent methionine synthase from Escherichia coli catalyzes the last step in de novo methionine biosynthesis . Conversion of the inactive cob(II)alamin form of the enzyme, formed by the occasional oxidation of cob(I)alamin during turnover, to an active methylcobalamin-containing form requires a reductive methylation of the cofactor in which an electron is supplied by reduced flavodoxin and the methyl group is derived from S-adenosyl-L-methionine . E . coli flavodoxin acts specifically in this activation reaction, and neither E . coli ferredoxin nor flavodoxin from the cyanobacterium Synechococcus will substitute, despite their highly similar midpoint potentials for one-electron transfer . As assessed by EPR spectroscopy, the binding of flavodoxin to cob(II)alamin methionine synthase results in a change in the coordination geometry of the cobalt from five-coordinate to four-coordinate . Histidine 759 of methionine synthase, which replaces the normal lower ligand dimethylbenzimidazole on binding of methylcobalamin to methionine synthase, is dissociated from the cobalt of the cobalamin by the binding of flavodoxin . The association of flavodoxin and methionine synthase depends on ionic strength and pH; the pH dependence corresponds to the uptake of one proton on association . The formation of a complex between flavodoxin and methionine synthase perturbs the midpoint potentials of the flavin and cobalamin cofactors only marginally and without any significant thermodynamic advantage for electron transfer to the cobalamin of methionine synthase . No significant binding was seen between oxidized flavodoxin and methylcobalamin methionine synthase . A model for the interaction of methionine synthase with flavodoxin is proposed in which flavodoxin binding leads to changes in the distribution of methionine synthase conformations.

Biochemistry, 1997 Jan 7, 36(1), 49 - 56
Analysis of binding interactions in an idiotope-antiidiotope protein-protein complex by double mutant cycles; Goldman ER et al.; The idiotope-antiidiotope complex between the anti-hen egg white lysozyme antibody D1.3 and the anti-D1.3 antibody E5.2 provides a useful model for studying protein-protein interactions . A high-resolution crystal structure of the complex is available {Fields, B . A., Goldbaum, F . A., Ysern, X., Poljak, R.J., & Mariuzza, R . A . (1995) Nature 374, 739-742}, and both components are easily produced and manipulated in Escherichia coli . We previously analyzed the relative contributions of individual residues of D1.3 to complex stabilization by site-directed mutagenesis {Dall'Acqua, W., Goldman, E . R., Eisenstein, E., & Mariuzza, R . A . (1996) Biochemistry 35, 9667-9676} . In the current work, we introduced single alanine substitutions in 9 out of 21 positions in the combining site of E5.2 involved in contacts with D1.3 and found that 8 of them play a significant role in ligand binding (delta Gmutant-delta Gwild type > 1.5 kcal/mol) . Furthermore, energetically important E5.2 and D1.3 residues tend to be juxtaposed in the crystal structure of the complex . In order to further dissect the energetics of specific interactions in the D1.3-E5.2 interface, double mutant cycles were carried out to measure the coupling of 13 amino acid pairs, 9 of which are in direct contact in the crystal structure . The highest coupling energy (4.3 kcal/mol) was measured for a charged-neutral pair which forms a buried hydrogen bond, while side chains which interact through solvated hydrogen bonds have lower coupling energies (1.3-1.7 kcal/mol), irrespective of whether they involve charged-neutral or neutral-neutral pairs . Interaction energies of similar magnitude (1.3-1.6 kcal/mol) were measured for residues forming only van der Waals contacts . Cycles between distant residues not involved in direct contacts in the crystal structure also showed significant coupling (0.5-1.0 kcal/mol) . These weak long-range interactions could be due to rearrangements in solvent or protein structure or to secondary interactions involving other residues.

Biochemistry, 1997 Jan 7, 36(1), 24 - 33
Reaction mechanism of Escherichia coli dihydrodipicolinate synthase investigated by X-ray crystallography and NMR spectroscopy; Blickling S et al.; Dihydrodipicolinate synthase (DHDPS) catalyzes the condensation of pyruvate with L-aspartate beta-semialdehyde . It is the first enzyme unique to the diaminopimelate pathway of lysine biosynthesis . Here we present the crystal structures of five complexes of Escherichia coli DHDPS with substrates, substrate analogs, and inhibitors . These include the complexes of DHDPS with (1) pyruvate, (2) pyruvate and the L-aspartate beta-semialdehyde analog succinate beta-semialdehyde, (3) the inhibitor alpha-ketopimelic acid, (4) dipicolinic acid, and (5) the natural feedback inhibitor L-lysine . The kinetics of inhibition were determined, and the binding site of the L-lysine was identified . NMR experiments were conducted in order to elucidate the nature of the product of the reaction catalyzed by DHDPS . By this method, (4S)-4-hydroxy-2,3,4,5-tetrahydro-(2S)-dipicolinic acid is identified as the only product . A reaction mechanism for DHDPS is proposed, and important features for inhibition are identified.

Proc Natl Acad Sci U S A, 1997 Jan 7, 94(1), 202 - 7
Conversion of inactive glycosylation inhibiting factor to bioactive derivatives by modification of a SH group; Nakano T et al.; Escherichia coli-derived recombinant human glycosylation inhibiting factor (rhGIF) contains three cysteine residues (Cys-57, -60, and -81) . All SH groups in the cysteine residues are free, and the GIF molecule had no biologic activity . Carboxymethylation of the SH group of Cys-60 in the molecule resulted in the generation of bioactivity, although the activity of the carboxymethylated GIF was 10- to 20-fold less than that of suppressor T cell (Ts)-derived GIF . However, treatment of the inactive rhGIF with ethylmercurithiosalicylate or 5,5'-dithiobis(2-nitrobenzoic acid) (DTNB) resulted in the generation of derivatives whose bioactivity was comparable to that of the Ts-derived bioactive GIF . The activity of these derivatives was lost by treatment with DTT . Isolation and chemical analysis of the DTNB-treated GIF derivative revealed that binding the 5-thio-2-nitrobenzoic acid group with Cys-60 was responsible for the generation of the highly bioactive derivative . Inactive cytosolic GIF from mammalian cells could also be converted to bioactive derivative by treatment with the SH reagent, while Ts-derived bioactive GIF was inactivated by DTT . These results, together with an x-ray crystal structure of GIF molecules, strongly suggest that the generation of bioactivity of GIF in Ts cells is due to posttranslational modifications that result in conformational changes in the molecule.

Virology, 1997 Jan 6, 227(1), 220 - 8
Inducible expression of the 2-5A synthetase/RNase L system results in inhibition of vaccinia virus replication; Diaz-Guerra M et al.; Studies of interferon (IFN)-treated virus-infected animal cells have revealed the 2-5A system (2-5A synthetase/RNase L enzymes) as being responsible for virus inhibition only in the case of picornaviridae . To investigate whether those IFN-induced enzymes could be responsible for inhibition of poxvirus replication, we have generated recombinant vaccinia viruses (VV) containing the corresponding genes (VV-2-5AS and VV-RL, respectively) . RNase L produced in cells infected with VV-RL leads to rRNA degradation and inhibition of virus protein synthesis, which correlates with about 92% reduction in virus yields by 48 hr after infection . Combined expression of this enzyme with 2-5A-synthetase further inhibits virus yields . The pattern of rRNA fragments produced by infection with viruses VV-RL and/or VV-2-5AS is the characteristic for activation of the 2-5A pathway by IFN treatment . Combined infection of VV-RL together with vesicular stomatitis virus (VSV) demonstrates this inhibition to be specific for VV and not due to a general effect . Breakdown of rRNA is largely due to the recombinant vector-derived enzyme, since a C-terminal deletion mutant of RNase L is inactive and the extent of rRNA degradation induced by infection with VV-RL is similar in cells treated or not with IFN . Moreover, the anti-VV effects of RNase L is also observed in a cell line lacking the endogenous ds RNA-dependent protein kinase (PKR) . Thus, our findings provide direct evidence for antiviral activity of the 2-5A system on poxviruses.

Virology, 1997 Jan 6, 227(1), 211 - 4
Strongly reduced phage Qbeta replication, but normal phage MS2 replication in an Escherichia coli K12 mutant with inactivated Qbeta host factor (hfq) gene; Su Q et al.; RNA phage production was observed in Escherichia coli hfq mutant strains transformed with plasmids containing genomic cDNA of phages Qbeta or MS2 . Qbeta production was reduced 5000-fold in an insertional mutant containing an omega cassette in the middle of hfq, but was unaffected in a mutant with the cassette near the end of hfq . The insertions had no effect on MS2 replication.

Virology, 1997 Jan 6, 227(1), 198 - 206
Probing phage PRD1-specific proteins with monoclonal and polyclonal antibodies; Hanninen AL et al.; Four new specificity class MAbs against PRD1 proteins (P6, P7/14, P11, P18) and polyclonal antiserum against the minor capsid protein P5 were produced . The antibodies were used to analyze the phage protein distribution inside the host cell during infection as well as in the virion . The minor component of the capsid, P5, was shown to be located on the surface of the virion . The proteins responsible for particle infectivity were localized to the membrane fraction of the host cells . In addition, by detection with MAbs, genes encoding proteins P14 and P18 were positively localized on the PRD1 genome.

Virology, 1997 Jan 6, 227(1), 77 - 87
Complementation of deletion of the vaccinia virus E3L gene by the Escherichia coli RNase III gene; Shors T et al.; This work investigated whether the Escherichia coli RNase III gene, rnc+, could complement vp1080, a mutant vaccinia virus that is deleted of its E3L gene . Like E3L, rnc+ codes for a dsRNA binding protein that contains an additional nucleolytic activity . Rnc genes were cloned into the eukaryotic expression vector pMTVa-, expressed in COS-1 cells, and shown to be functional . Transient rescue experiments in HeLa cells demonstrated that the cleavage function of the rnc+ gene was necessary for full rescue of vp1080 . The rnc 70 gene, which encodes a product deficient in catalytic activity but still capable of binding to dsRNA, rescued vp1080 weakly . The rnc 105 gene, which encodes a product that cannot bind or cleave dsRNA, was unable to rescue vp1080 . The rnc genes were also inserted into the E3L locus of vp1080 . While recombinants containing the rnc+ gene or the rnc 70 gene regained the IFN resistance phenotype in RK13 cells, full host range of vaccinia virus was only restored in the recombinant containing the rnc+ gene . Thus, the ability of RNase III to process dsRNA appears to be necessary to restore the host range phenotype . The vp-rnc 105 recombinant behaved similarly to vp1080.

J Exp Med, 1997 Jan 6, 185(1), 171 - 5
Multiple cytokines and acute inflammation raise mouse leptin levels: potential role in inflammatory anorexia; Sarraf P et al.; Several inflammatory cytokines, most notably tumor necrosis factor (TNF) and IL-1, induce anorexia and loss of lean body mass, common manifestations of acute and chronic inflammatory conditions . In C57BL/6 female mice, the administration of TNF, IL-1, and, to a lesser extent, leukemia inhibitory factor (LIF), produced a prompt and dose-dependent increase in serum leptin levels and leptin mRNA expression in fat . IL-10, IL-4, ciliary neurotrophic factor, and IL-2, cytokines not known to induce anorexia or decrease food intake, had no effect on leptin gene expression or serum leptin levels . After administration of Escherichia coli lipopolysaccharide (LPS), leptin gene expression and leptin levels were increased . These findings suggest that leptin levels may be one mechanism by which anorexia is induced during acute inflammatory conditions.

Clin Chim Acta, 1997 Jan 3, 257(1), 3 - 23
Gene transfer technologies for the production of enzyme and protein reference materials; Oster T et al.; To maintain the success of recommended methods and to allow comparison among various methods of enzyme analysis, enzyme reference materials are required, having catalytic properties as close as possible to those of the corresponding human enzymes . Though human sources are preferable, ethical reasons require the extraction and purification from animal tissues . By providing theoretically unlimited amounts of material, gene transfer technologies and mass culture can overcome the need of human or mammalian tissues . We have used these technologies to produce human gamma-glutamyltransferase (GGT) and pancreatic lipase (PL) in various types of host cells . Different strategies were tested, especially for GGT, depending on the inherent properties and requirements of the human enzyme . Expression and purification protocols were optimized, yielding good amounts of recombinant enzymes which share many physico-chemical and catalytic features with their natural counterparts . Kinetic constants and catalytic behavior were very similar, demonstrating the usefulness of these products as reference materials . We assume recombinant DNA technologies could be successfully applied to most enzymes or proteins assayed in clinical chemistry laboratories.

Biochem Biophys Res Commun, 1997 Jan 3, 230(1), 76 - 80
Myosin subfragment-1 is fully equipped with factors essential for motor function; Iwane AH et al.; The sliding velocity of actin filaments propelled by chicken skeletal myosin subfragment-1 (S1) was measured when the tail end of S1 was specifically bound to the glass surface . To achieve the specific binding, a regulatory light chain was replaced by a recombinant fusion protein of biotin-dependent transcarboxylase (BDTC) and chicken gizzard smooth muscle regulatory light chain (cgmRLC) . The BDTC-cgmRLC of S1 was then attached to the glass surface using a biotin-avidin system . The velocity of actin filaments caused by S1 bound to the surface in this manner was 6.8 +/- 0.6 microm/sec at 29 degrees C, which was 3.5-fold greater than that (1.9 +/- 0.3 microm/sec) when bound directly to the surface as in previous studies, but similar to that caused by native chicken skeletal myosin (6.5 +/- 0.6 microm/sec) . The actin-activated Mg-ATPase activity was similar to that of S1 before the RLC of S1 was exchanged for BDTC-cgmRLC . The results indicate that S1 can produce a normal fast movement of actin filaments as well as hydrolyse ATP and generate force.

Biochem Biophys Res Commun, 1997 Jan 3, 230(1), 211 - 4
Characterization of a fusion protein between human cytochrome P450 1A1 and rat NADPH-P450 oxidoreductase in Escherichia coli; Chun YJ et al.; A cDNA of fusion protein between human cytochrome P450 1A1 and rat NADPH-P450 reductase was genetically engineered and expressed in Escherichia coli DH5alpha cells under the control of an inducible tac promoter (Y . J . Chun, T . Shimada, and F . P . Guengerich, (1996) Arch . Biochem . Biophys . 330, 48-58) . E . coli membranes of transformed cells showed much higher P450 1Al-dependent monooxygenase and NADPH-P450 reductase activities than pCW control vector or P450 1A1 expression vector-transformed cells . Ethoxyresorufin O-deethylase and methoxyresorufin O-demethylase were 22-fold and 11-fold higher than the control activity, respectively . alpha-Naphthoflavone and beta-naphthoflavone strongly inhibited P450 1A1 activity of the fusion protein, with alpha-naphthoflavone being more potent than beta-naphthoflavone . Divalent cations (e.g . Ca2+ and Mg2+) increased P450 1A1 activity as well as NADPH-P450 reductase activity . These results demonstrate that this fusion protein in E . coli membrane may be a useful model for elucidating details of protein-protein interactions between P450 and NADPH-P450 reductase in the endoplasmic reticulum of mammalian cells.

Biochem Biophys Res Commun, 1997 Jan 3, 230(1), 202 - 5
The recombinant catalytic domain of mouse collagenase-3 depolymerizes type I collagen by cleaving its aminotelopeptides; Lemaitre V et al.; The sequence coding for the catalytic domain of mouse collagenase-3 (MMP-13) was amplified by polymerase chain reaction and expressed in Escherichia coli . The recombinant catalytic domain (CCD), mainly recovered as inclusion bodies, was renatured and purified to homogeneity by preparative SDS-PAGE . The purified CCD degraded gelatin, casein and a synthetic peptide . CCD was not able to cleave the triple-helical domain of type I collagen but conserved the specific property of full-length collagenase-3 to cleave the N-telopeptides . These results show that residues involved in the recognition and cleavage of the aminotelopeptides of type I collagen are located in the catalytic domain of mouse collagenase-3 and that the C-terminal domain is not required for this activity.

Biochem Biophys Res Commun, 1997 Jan 3, 230(1), 192 - 5
cDNA sequence analysis and expression of kappa-bungarotoxin from Taiwan banded krait; Chang L et al.; The cDNAs encoding kappa-bungarotoxin was constructed from the cellular RNA isolated from the venom glands of Bungarus multicinctus by reverse transcription-polymerase chain reaction . A high degree of nucleotide sequence homology was observed between kappa-bungarotoxin and other kappa-neurotoxins . The kappa-bungarotoxin was subcloned into the expression vector pET32a(+) and transformed into BL21(DE3) E . coli strain . The recombinant toxin was expressed as a fusion protein . Recombinant kappa-bungarotoxin was separated from the fused protein by cleavage with CNBr and purified by reversed phase high performance liquid chromatography . In addition to kappa-bungarotoxin, the cDNA fragment encoding kappa3-bungarotoxin was also found in the cDNA mixtures prepared from the cellular RNA of the venom glands of the same snake . This result suggests that the venom glands of Taiwanese B . multicinctus should secrete at least two kinds of kappa-neurotoxins.

Biochem Biophys Res Commun, 1997 Jan 3, 230(1), 164 - 6
Sequencing, analysis and expression in Escherichia coli of a gene encoding a 15 kDa Cryptosporidium parvum protein; Khramtsov NV et al.; A previous paper presented data on a cDNA sequence encoding a protein associated with the AIDS related pathogen, Cryptosporidium parvum . However, the position of the start codon was uncertain, and the 5' end was continuous, lending doubt about the size and complete sequence of the final protein product . Herein we present the complete gene sequence and conclude the predicted size of the putative protein to be 16.2 kDa.

Gene, 1997 Jan 3, 184(1), 107 - 14
The locus of enterocyte effacement pathogenicity island of enteropathogenic Escherichia coli encodes secretion functions and remnants of transposons at its extreme right end; Donnenberg MS et al.; The locus of enterocyte effacement (LEE) is necessary for enteropathogenic Escherichia coli to cause characteristic attaching and effacing lesions in host cells . To determine whether sequences at the extreme right end of the LEE downstream of the espB gene are required for attaching and effacing, we constructed a mutant with an omega-interposon insertion immediately downstream of espB . This mutant is incapable of attaching and effacing, of secreting EspA and EspB and of inducing tyrosine phosphorylation of host cell proteins . These phenotypes are restored by a plasmid containing the extreme right end of the LEE . The nucleotide sequence of this region reveals a relatively low G+C content, remnants of transposons, and several open reading frames . The predicted products of these open reading frames include a potential chaperone, a potential component of the secretion apparatus, and a hypothetical peptide with proline rich repeats reminiscent of several eukaryotic proteins . These data indicate that the extreme right end of the LEE is required for attaching and effacing and reveal information relevant to the origin and function of the LEE.

Mutat Res, 1997 Jan 3, 373(1), 61 - 6
Enhanced generation of A:T-->T:A transversions in a recA730 lexA51(Def) mutant of Escherichia coli; Watanabe-Akanuma M et al.; RecA730 belongs to a class of mutant RecA protein that is often referred to as RecA*, since it is constitutively activated for coprotease functions in the absence of exogenous DNA-damage . Escherichia coli strains carrying recA730 (or other recA* alleles) exhibit dramatic increases in SOS-dependent spontaneous mutator activity . We have analyzed the specificity of this mutator phenotype by employing F'-plasmids carrying a set of mutant lacZ genes that can individually detect two types of transitions, four types of transversions, and four kinds of specific frameshift events . Analysis revealed that most of the spontaneous mutagenesis in a recA730 lexA51(Def) strain (which expresses derepressed levels of all LexA-regulated proteins) can be attributed to a specific increase in A:T-->T:A, A:T-->C:G and G:C-->T:A transversions, with the A:T-->T:A transversions occurring most frequently . These transversion events were completely abolished in a delta umuDC strain, indicating that the functionally active UmuD'C proteins are normally required for their generation . The spectrum obtained was similar to that of strains with a defect in the epsilon (3'-->5' proofreading) subunit of DNA polymerase III . Such an observation raises the possibility that the wild-type epsilon protein is in activated in strains expressing the RecA730 and UmuD'C proteins.

Biochim Biophys Acta, 1997 Jan 3, 1350(1), 15 - 20
A novel genetic organization: the leuA-rpoD1 locus in the cyanobacterium Microcystis aeruginosa K-81; Asayama M et al.; We cloned and sequenced the region upstream of rpoD1, which encodes a principal sigma factor in the cyanobacterium Microcystis aeruginosa K-81 . An open reading frame (orf1, 1599 bp) was discovered, the deduced amino-acid sequence of which (533 aa, 58, 016 Da) exhibits homology to another bacterial leuA gene product, 2-isopropylmalate synthase . The leuA (orf1) gene specifically complemented an E . coli leuA mutant . The 5'-upstream region of leuA did not contain possible leader peptide or stem-loop structures for attenuation . These findings indicate that the genetic structure of the leuA-rpoD1 locus in M . aeruginosa K-81 significantly differs from those of known leuA and rpoD loci found in other bacteria.

FEBS Lett, 1997 Jan 3, 400(2), 238 - 42
Demonstration of dimer formation of the cytoplasmic domain of a transmembrane osmosensor protein, EnvZ, of Escherichia coli using Ni-histidine tag affinity chromatography; Hidaka Y et al.; EnvZ is a transmembrane osmosensor which regulates the phosphorylation of OmpR, a transcription factor for ompF and ompC genes which encode the major outer membrane porin proteins, OmpF and OmpC in Escherichia coli . Autophosphorylation of EnvZ occurs through a transphosphorylation reaction between two EnvZ molecules . To elucidate the molecular mechanism of signal transduction by EnvZ, we examined the dimer formation of the EnvZ cytoplasmic domain {EnvZ(C)} . For this purpose, we developed a method to determine the complex formation between the purified EnvZ(C) and the purified His6-EnvZ(C) by means of Ni-6xhistidine tag affinity chromatography . When the mixture of EnvZ(C) and His6-EnvZ(C) was applied to Ni-NTA resin, both His6-EnvZ(C) and EnvZ(C) were bound to the resin, indicating that EnvZ can form an oligomer without the periplasmic and transmembrane domains . Binding experiments using the Ni-NTA resin revealed that EnvZ(C) forms a dimer with the Ka value for dimerization being approximately 10(5) M(-1) in the equilibrium state.

J Biol Chem, 1997 Jan 3, 272(1), 626 - 38
Combinatorial screening and rational optimization for hybridization to folded hepatitis C virus RNA of oligonucleotides with biological antisense activity; Lima WF et al.; We describe our initial application of a biochemical strategy, comprising combinatorial screening and rational optimization, which directly identifies oligonucleotides with maximum affinity (per unit length), specificity, and rates of hybridization to structurally preferred sites on folded RNA, to the problem of design of antisense oligonucleotides active against the hepatitis C virus (HCV) . A fully randomized sequence DNA oligonucleotide (10-mer) library was equilibrated with each of two folded RNA fragments (200 and 370 nucleotides (nt)), together spanning the 5' 440 nt of an HCV transcript (by overlapping 130 nt), which were varied over a range of concentrations . The equilibrations were performed in solution under conditions determined to preserve RNA structure and to limit all RNA-DNA library oligonucleotide interactions to 1:1 stoichiometry . Subsequent Escherichia coli RNase H (endoribonuclease H: EC 3.1.26.4) cleavage analysis identified two preferred sites of highest affinity heteroduplex hybridization . The lengths and sequences of different substitute chemistry oligonucleotides complementary to these sites were rationally optimized using an iterative and quantitative analysis of binding affinity and specificity . Thus, DNA oligonucleotides that hybridized with the same affinity to the preferred sites in the folded RNA fragments found by screening as to short (< or = 25 nt) RNA complements were identified but were found to vary in length (10-18 nt) from site to site . Phosphorothioate (P=S) and 2'-fluoro (2'-F) uniformly substituted oligonucleotides also were found, which hybridized optimally to these sites, supporting the design of short (10-15-nt) and maximally specific oligonucleotides that are more nuclease-resistant (via P=S) and have higher affinity (via 2'-F) than DNA . Finally, the affinities of DNA and uniform 2'-F-, P=S-substituted 10-20-mer oligonucleotide complements for the best hybridization site, from HCV nt 355 to nt 364-374, closely corresponded to antisense mechanism inhibition activities in an in vitro translation assay and in a human cell-based HCV core protein expression assay, respectively . These results validate our strategy for the selection of hybridization-optimized and biologically active antisense oligonucleotides targeting HCV RNA and support the potential for utility in further applications.

J Biol Chem, 1997 Jan 3, 272(1), 617 - 25
Molecular cloning and functional reconstitution of a urate transporter/channel; Leal-Pinto E et al.; Maintenance of urate homeostasis requires urate efflux from urate-producing cells with subsequent renal and gastrointestinal excretion . The molecular basis for urate transport, however, has not been identified . A novel full-length cDNA encoding a 322-amino acid protein, designated UAT (urate transporter), has been cloned from a rat renal cDNA library by antibody screening . UAT mRNA transcripts that approximate 1.55 kilobases are present, but differentially expressed in various rat tissues . Recombinant UAT protein that was expressed from the cloned cDNA in Escherichia coli and purified via immobilized metal affinity chromatography has been functionally reconstituted as a highly selective urate transporter/channel in planar lipid bilayers . The IgG fraction of the polyclonal antibody that was used to select the UAT clone from the cDNA library, but not nonimmune IgG, blocked urate channel activity . Based on the wide tissue distribution of the mRNA for UAT we propose that UAT provides the molecular basis for urate flux across cell membranes, allowing urate that is formed during purine metabolism to efflux from cells and serving as an electrogenic transporter that plays an important role in renal and gastrointestinal urate excretion.

J Biol Chem, 1997 Jan 3, 272(1), 609 - 16
Modulation of the activity of RNase E in vitro by RNA sequences and secondary structures 5' to cleavage sites; Mackie GA et al.; The endoribonuclease RNase E is believed to initiate the degradation of many mRNAs in Escherichia coli, yet the mechanism by which it recognizes cleavage sites is poorly understood . We have prepared derivatives of the mRNA encoding ribosomal protein S20 which contain a single major RNase E cleavage site at residues 300/301 preceded by variable 5' extensions . Three of these RNAs are cleaved in vitro with significantly reduced efficiencies relative to the intact S20 mRNA by both crude RNase E and pure Rne protein (endonuclease component of RNase E) . In all three substrates as well as in the full-length mRNA the major cleavage site itself remains single-stranded . One such substrate (t84D) contains a 5' stem-loop structure characterized by three noncanonical A-G pairs . Removal or denaturation of the stem restores efficient cleavage at the major RNase E site . The other two contain single-stranded 5'-termini but apparently lack cleavage sites near the termini . Our data show that sensitivity to RNase E can be influenced by distant structural motifs in the RNA and also suggest a model in which the initial recognition and cleavage of a substrate near its 5' end facilitates sequential cleavages at more distal sites . The model implies that RNase E contains at least a dimer of the Rne subunit and that the products of the first cleavage are retained by Rne prior to the second cleavage.

J Biol Chem, 1997 Jan 3, 272(1), 422 - 9
Spectroscopic studies of cobalt(II) binding to Escherichia coli bacterioferritin; Keech AM et al.; The iron storage protein bacterioferritin (BFR) consists of 24 identical subunits, each containing a dinuclear metal binding site called the ferroxidase center, which is essential for fast iron core formation . Cobalt(II) binding to wild-type and site-directed variants of Escherichia coli BFR was studied by optical and magnetic techniques . Data from absorption spectroscopy demonstrate the binding of two cobalt(II) ions per subunit of wild-type and heme-free BFR, each with a pseudotetrahedral or pentacoordinate geometry, and EPR studies show that the two cobalt(II) ions are weakly magnetically coupled . Studies of variants of BFR in which a single glutamic acid residue at the ferroxidase center is replaced by alanine confirm that this is the site of cobalt(II) binding, since the altered centers bind only one cobalt(II) ion . This work shows that the electroneutrality of the ferroxidase center is preserved on binding a pair of divalent metal ions . Optical and EPR data show that cobalt(II) binding to BFR exhibits positive cooperativity, with an average Kd of approximately 1 x 10(-5) M . The favored filling of the ferroxidase center with pairs of metal ions may have mechanistic implications for the iron(II) binding process . Discrimination against oxidation of single iron(II) ions avoids odd electron reduction products of oxygen.

J Biol Chem, 1997 Jan 3, 272(1), 326 - 31
Alternate energy coupling of ArsB, the membrane subunit of the Ars anion-translocating ATPase; Kuroda M et al.; The arsenical resistance (ars) operon of the conjugative R-factor R773 confers resistance to arsenical and antimonial compounds in Escherichia coli, where resistance results from active extrusion of arsenite catalyzed by the products of the arsA and arsB genes . Previous in vivo studies on the energetics of arsenite extrusion showed that expression of both genes produced an ATP-coupled arsenite extrusion system that was independent of the electrochemical proton gradient . In contrast, in cells expressing only the arsB gene, arsenite extrusion was coupled to electrochemical energy and independent of ATP, suggesting that the Ars transport system exhibits a dual mode of energy coupling depending on the subunit composition . In vitro the ArsA-ArsB complex has been shown to catalyze ATP-coupled uptake of 73AsO2(-1) in everted membrane vesicles . However, transport catalyzed by ArsB alone has not previously been observed in vitro . In this study we demonstrate everted membrane vesicles prepared from cells expressing only arsB exhibit uptake of 73AsO2(-1) coupled to electrochemical energy.

J Biol Chem, 1997 Jan 3, 272(1), 196 - 202
CspA, the major cold-shock protein of Escherichia coli, is an RNA chaperone; Jiang W et al.; CspA, the major cold-shock protein of Escherichia coli, is dramatically induced during the cold-shock response . The amino acid sequence of CspA shows 43% identity to the "cold-shock domain" of the eukaryotic Y-box protein family, which interacts with RNA and DNA to regulate their functions . Here, we demonstrate that CspA binds to RNA as a chaperone . First, CspA cooperatively binds to heat-denatured single-stranded RNA if it is larger than 74 bases, causing a supershift in gel electrophoresis . A minimal concentration of CspA at 2.7 x 10(-5) M is absolutely required for this cooperative binding, which is sufficiently lower than the estimated cellular concentration of CspA (10(-4) M) in cold-shocked cells . No specific RNA sequences for CspA binding were identified, indicating that it has a broad sequence specificity for its binding . When the 142-base 5'-untranslated region of the cspA mRNA was used as a substrate for ribonucleases A and T1, the addition of CspA significantly stimulated RNA hydrolysis by preventing the formation of RNase-resistant bands due to stable secondary structures in the 5'-untranslated region . These results indicate that binding of CspA to RNA destabilizes RNA secondary structures to make them susceptible to ribonucleases . We propose that CspA functions as an RNA chaperone to prevent the formation of secondary structures in RNA molecules at low temperature . Such a function may be crucial for efficient translation of mRNAs at low temperatures and may also have an effect on transcription.

J Biol Chem, 1997 Jan 3, 272(1), 189 - 95
Influence of acidic residues and the kink in the active-site helix on the properties of the disulfide oxidoreductase DsbA; Hennecke J et al.; The catalytic disulfide bond Cys30-Cys33 of the disulfide oxidoreductase DsbA from Escherichia coli is located at the amino terminus of an alpha-helix, which has a kink caused by insertion of a tripeptide (residues 38-40) . The oxidative force of DsbA (E'O = -125 mV) mainly results from the low pKa of 3.4 of its Cys30 thiol . To investigate the role of the kink and the electrostatic contribution of Glu37 and Glu38 to the redox properties of DsbA, we have characterized a series of DsbA variants (delta38-40, delta38-40/H41P, E37Q, E38Q, and E37Q/E38Q) . In contrast to theoretical predictions, the redox potentials of the variants are almost unchanged, and the pKa values of Cys30 do not differ by more than 0.5 units from that of DsbA wild type . All variants show the same in vivo activity and dependence of redox potential on ionic strength as the wild type . The mutations have no influence on the polypeptide specificity of the protein, which is independent of the isoelectric point of the polypeptide substrate and most pronounced at acidic pH . We conclude that neither the kink in the active-site helix nor Glu37 and Glu38 are critical for the physical properties of DsbA.

J Biol Chem, 1997 Jan 3, 272(1), 83 - 8
Occlusion of RNA polymerase by oligomerization of DnaA protein over the dnaA promoter of Escherichia coli; Lee YS et al.; DnaA protein, the initiator protein for initiation of Escherichia coli chromosomal replication, has been shown to repress its own expression from two dnaA promoters, 1P and 2P . The sequence-specific binding of DnaA protein to the DnaA box, located between the two promoters, results in subsequent oligomerization of DnaA protein . Upon increasing the concentration of DnaA protein, the oligomerization proceeds to both dnaA promoters from the DnaA box and inhibits RNA polymerase binding to both promoters . This results in the repression of transcription, suggesting that the extent of oligomerization of DnaA proteins over two dnaA promoters contributes to the autoregulation of expression of the dnaA gene . When the two dnaA promoters were bound and repressed by DnaA protein, the interaction of RNA polymerase with IciA protein, which is a specific inhibitor of initiation of in vitro E . coli chromosomal replication, appeared to dissociate the oligomerized DnaA proteins from the 1P promoter and allowed RNA polymerase to be loaded for its transcription.

Brain Res Dev Brain Res, 1997 Jan 2, 98(1), 91 - 101
Carbonic anhydrase II and carbonic anhydrase-related protein in the cerebellar cortex of normal and lurcher mice; Nogradi A et al.; The developmental profiles of carbonic anhydrase II (CA-II) and a carbonic anhydrase related protein (CARP) were studied in rat and mouse cerebella . Enzyme histochemistry, immunohistochemistry, in situ hybridisation and Western blotting were used to study the synthesis and expression of these enzymes in cerebellar sections from age matched control, CA-II deficient and lurcher mice, the latter being characterised by Purkinje cell degeneration . Both CA-II and CARP were first found to be expressed in the Purkinje cells in the 9 day old mouse, and the immunoreactivity of both peptides increased with time . Immunohistochemistry showed more intense staining of CARP than of CA-II in Purkinje cells throughout the developmental profile of the mouse, and this was mirrored by the mRNA levels determined by in situ hybridisation . Immunohistochemistry of CA-II and CARP also demonstrated the progressive dendritic growth of the mouse and rat Purkinje cells . CA-II and CARP immunoreactivity ceased by the end of cerebellar maturation . The onset of Purkinje cell degeneration was detected at day 10 in the lurcher mouse, with concomitant marked decrease in CA-II level: however CARP expression was found to be unchanged . By postnatal day 16 neither CA-II mRNA, protein, nor activity was detectable in contrast to CARP which remained at a decreased level unit the Purkinje cells population had completely degenerated . Our findings suggest a role of CA-II in the degenerative processes of the lurcher Purkinje cells, with CARP playing an important role in the development and maturation of the cerebellar cortex.

EMBO J, 1997 Jan 2, 16(1), 203 - 9
ATP-dependent resolution of R-loops at the ColE1 replication origin by Escherichia coli RecG protein, a Holliday junction-specific helicase; Fukuoh A et al.; The RecG protein of Escherichia coli is a DNA helicase that promotes branch migration of the Holliday junctions . We found that overproduction of RecG protein drastically decreased copy numbers of ColE1-type plasmids, which require R-loop formation between the template DNA and a primer RNA transcript (RNA II) for the initiation of replication . RecG efficiently inhibited in vitro ColE1 DNA synthesis in a reconstituted system containing RNA polymerase, RNase HI and DNA polymerase I . RecG promoted dissociation of RNA II from the R-loop in a manner that required ATP hydrolysis . These results suggest that overproduced RecG inhibits the initiation of replication by prematurely resolving the R-loops formed at the replication origin region of these plasmids with its unique helicase activity . The possibility that RecG regulates the initiation of a unique mode of DNA replication, oriC-independent constitutive stable DNA replication, by its activity in resolving R-loops is discussed.

EMBO J, 1997 Jan 2, 16(1), 154 - 62
Molecular anatomy of a transcription activation patch: FIS-RNA polymerase interactions at the Escherichia coli rrnB P1 promoter; Bokal AJ et al.; FIS, a site-specific DNA binding and bending protein, is a global regulator of gene expression in Escherichia coli . The ribosomal RNA promoter rrnB P1 is activated 3- to 7-fold in vivo by a FIS dimer that binds a DNA site immediately upstream of the DNA binding site for the C-terminal domain (CTD) of the alpha subunit of RNA polymerase (RNAP) . In this report, we identify several FIS side chains important specifically for activation of transcription at rrnB P1 . These side chains map to positions 68, 71 and 74, in and flanking a surface-exposed loop adjacent to the helix-turn-helix DNA binding motif of the protein . We also present evidence suggesting that FIS activates transcription at rrnB P1 by interacting with the RNAP alphaCTD . Our results suggest a model for FIS-mediated activation of transcription at rrnB P1 that involves interactions between FIS and the RNAP alphaCTD near the DNA surface . Although FIS and the transcription activator protein CAP have little structural similarity, they both bend DNA, use a similarly disposed activation loop and target the same region of the RNAP alphaCTD, suggesting that this is a common architecture at bacterial promoters.

EMBO J, 1997 Jan 2, 16(1), 54 - 8
Cooperation of enzymatic and chaperone functions of trigger factor in the catalysis of protein folding; Scholz C et al.; The trigger factor of Escherichia coli is a prolyl isomerase and accelerates proline-limited steps in protein folding with a very high efficiency . It associates with nascent polypeptide chains at the ribosome and is thought to catalyse the folding of newly synthesized proteins . In its enzymatic mechanism the trigger factor follows the Michaelis-Menten equation . The unusually high folding activity of the trigger factor originates from its tight binding to the folding protein substrate, as reflected in the low Km value of 0.7 microM . In contrast, the catalytic constant kcat is small and shows a value of 1.3 s(-1) at 15 degrees C . An unfolded protein inhibits the trigger factor in a competitive fashion . The isolated catalytic domain of the trigger factor retains the full prolyl isomerase activity towards short peptides, but in a protein folding reaction its activity is 800-fold reduced and no longer inhibited by an unfolded protein . Unlike the prolyl isomerase site, the polypeptide binding site obviously extends beyond the FKBP domain . Together, this suggests that the good substrate binding, i.e . the chaperone property, of the intact trigger factor is responsible for its high efficiency as a catalyst of proline-limited protein folding.

EMBO J, 1997 Jan 2, 16(1), 1 - 14
The crystal structure of Escherichia coli maltodextrin phosphorylase provides an explanation for the activity without control in this basic archetype of a phosphorylase; Watson KA et al.; In animals, glycogen phosphorylase (GP) exists in an inactive (T state) and an active (R state) equilibrium that can be altered by allosteric effectors or covalent modification . In Escherichia coli, the activity of maltodextrin phosphorylase (MalP) is controlled by induction at the level of gene expression, and the enzyme exhibits no regulatory properties . We report the crystal structure of E . coli maltodextrin phosphorylase refined to 2.4 A resolution . The molecule consists of a dimer with 796 amino acids per monomer, with 46% sequence identity to the mammalian enzyme . The overall structure of MalP shows a similar fold to GP and the catalytic sites are highly conserved . However, the relative orientation of the two subunits in E . coli MalP is different from both the T and R state GP structures, and there are significant changes at the subunit-subunit interfaces . The sequence changes result in loss of each of the control sites present in rabbit muscle GP . As a result of the changes at the subunit interface, the 280s loop, which in T state GP acts as a gate to control access to the catalytic site, is held in an open conformation in MalP . The open access to the conserved catalytic site provides an explanation for the activity without control in this basic archetype of a phosphorylase.

FEBS Lett, 1997 Jan 2, 400(1), 136 - 40
Activation of protein phosphatase 5 by limited proteolysis or the binding of polyunsaturated fatty acids to the TPR domain; Chen MX et al.; Protein phosphatase 5 (PP5) exhibits very low phosphatase activity, which can be stimulated > 25-fold by proteolysis . Since proteolysis cleaves the N-terminal tetratricopeptide repeat (TPR) domain from the catalytic domain, these results indicate that the TPR domain shields the active site . Polyunsaturated fatty acids, such as arachidonic acid, and lipids containing polyunsaturated fatty acids, such as phosphatidylinositol, stimulate both bacterially expressed human and native rabbit PP5 activity > 25-fold towards casein and myelin basic protein . Phosphatidylinositol binds to the TPR domain, and not to the catalytic domain, indicating that activation by polyunsaturated fatty acids is allosteric and that it may occur by movement of the TPR domain to allow substrate access.

FEBS Lett, 1997 Jan 2, 400(1), 108 - 12
Instability of expressed Cu/Zn superoxide dismutase with 2 bp deletion found in familial amyotrophic lateral sclerosis; Watanabe Y et al.; The mutant Cu/Zn superoxide dismutase (SOD1) associated with familial amyotrophic lateral sclerosis (FALS) with a 2 bp deletion was produced in two protein expression systems . The mutant SOD1, expressed as a fusion protein in E . coli, had immunoreactivity to an anti-human SOD1 antibody but no SOD activity . It was more susceptible to proteolysis and its immunoreactivity decreased more rapidly than the wild type . The mutant SOD1, expressed in Cos1 cells, was not detected by either SOD activity staining or Western blot analysis, although expression of its mRNA was confirmed . These results suggest that the mutant SOD1 is seriously unstable in mammalian cells.

FEBS Lett, 1997 Jan 2, 400(1), 45 - 50
Casein kinase II phosphorylates Ser468 in the PEST domain of the Drosophila IkappaB homologue cactus; Packman LC et al.; Cactus protein is a Drosophila homologue of the mammalian IkappaB family of cytoplasmic anchor proteins . In unstimulated cells they function to retain rel/NFkappaB transcription factors in the cytoplasm but are rapidly degraded in response to signalling . The destruction of cactus or IkappaBalpha allows the rel/NFkappaB transcription factor to relocalise to the nucleus . Cactus is a phosphoprotein and has in its C-terminus a PEST protein stability domain . In this paper we show that, like mammalian IkappaBalpha, the PEST domain of cactus is phosphorylated by casein kinase II . We have localised the site of modification to a single residue, Ser468, and find no evidence for additional phosphorylation sites . The conservation of these sites in mammalian and invertebrate cytoplasmic anchor proteins suggests that phosphorylation by casein kinase II may play a critical functional role, plausibly in the regulation of constitutive or inducible proteolysis.

Nature, 1997 Jan 2, 385(6611), 93 - 7
Regulatory intramolecular association in a tyrosine kinase of the Tec family; Andreotti AH et al.; The T-cell-specific tyrosine kinase Itk is a member of the Tec family of non-receptor tyrosine kinases, and is required for signalling through the T-cell antigen receptor (TCR) . The role of Itk in TCR signalling and the manner in which Itk activity is regulated are not well understood . Substrate binding and enzymatic activity of the structurally related Src kinases are regulated by an intramolecular interaction between the Src-homology-2 (SH2) domain and a phosphotyrosine . Although Itk also contains SH3, SH2 and tyrosine kinase domains, it lacks the corresponding regulatory phosphorylation site, and therefore must be regulated by an alternative mechanism . The proline-rich sequence adjacent to the SH3 domain of Tec family kinases contains an SH3 ligand, potentially allowing a different intramolecular interaction . By using multidimensional nuclear magnetic resonance we have determined the structure of a fragment of Itk, confirming that these domains interact intramolecularly . Formation of this intramolecular SH3-ligand complex prevents the Itk SH3 domain and proline-rich region from interacting with their respective protein ligands, Sam68 and Grb-2 . We believe that this structure represents the first example of an intramolecular interaction between an SH3 domain and a proline-rich ligand, and has implications for the regulation of Tec family kinases.

Nature, 1997 Jan 2, 385(6611), 37 - 41
Hydrolysis of GTP by elongation factor G drives tRNA movement on the ribosome; Rodnina MV et al.; Elongation factor G (EF-G) is a GTPase that is involved in the translocation of bacterial ribosomes along messenger RNA during protein biosynthesis . In contrast to current models, EF-G-dependent GTP hydrolysis is shown to precede, and greatly accelerate, the rearrangement of the ribosome that leads to translocation . Domain IV of the EF-G structure is crucial for both rapid translocation and subsequent release of the factor from the ribosome . By coupling the free energy of GTP hydrolysis to translocation, EF-G serves as a motor protein to drive the directional movement of transfer and messenger RNAs on the ribosome.

Chin J Biotechnol, 1997, 13(1), 9 - 15
Protein engineering on subtilisin E; Zhu L et al.; Protein engineering was carried out by site-directed and random mutagenesis on subtilisin E gene . Four mutants were obtained . They are M222A; M222A, N118S; M222A, N118S, Q103R; and M222A, N118S, Q103R, D60N . The mutant genes were recombined in pBE-2, an E . coli-B . subtilis shuttle vector, and transformed into B . subtilis DB104, an alkaline and neutral proteinase deficient strain . The subtilisin E mutations obtained from their gene expressions were purified . The properties of these mutants showed that the M222A mutation made the enzyme resistant to oxidation, N118S mutation increased the thermal stability, while Q103R and D60N mutations enhanced the specific activity of the enzyme but decreased the thermal stability and, in particular, D60N mutation caused the enzyme to be very unstable . The IEF-PAGE showed that the wild type and M222A mutant had the same pI of 8.92, while those of double mutant, triple mutant, and quadruple mutant were 8.88, 9.10, and 9.17, respectively . The optimum pH range was 7.5-9.5 for suc-AAPF-pNA substrate and was 10-12 for casein substrate.

Chin J Biotechnol, 1997, 13(1), 59 - 62
Immobilized cells with endocellular enzymes improved by the permealizing and crosslinking treatment; Shi Y et al.; In order to achieve high activity of enzymatic catalysis, the method of permealizing and crosslinking treatment has been suggested for immobilizing cells with endocellular enzymes . Polyethene-polyamine and glutardehyde were used to enhance the activity and stability of E . coli having aspartase entrapped in k-carrageenan gel and intact P . dacunhae cells having aspartate-beta-decarbonoxylase . The strength and enlargement effects, which favor the performance of catalysis in a packed bed reactor and a membrane reactor, respectively, were also obtained from the experiment results.

Biofactors, 1997, 6(3), 305 - 9
A non-radioactive and two radioactive assays for selenophosphate synthetase activity; Liu SY et al.; Selenophosphate synthetase catalyzes the formation of monoselenophosphate (SePO3(3-)) from ATP and selenide (reaction 1) . {formula: see text} In one assay frequently used, {8-14C}AMP formation from {8-14C}ATP is estimated after separation of the nucleotides by thinlayer chromatography . An alternative non-radioactive assay in which the AMP product is estimated using AMP deaminase is described . The highly oxygen-labile selenophosphate product can be estimated in an assay employing {gamma-32P}ATP . The 32P-labeled selenophosphate is converted to {32P}orthophosphate by treatment with iodine and estimated after removal of residual {32P}ATP on charcoal.

Urol Res, 1997, 25(4), 251 - 5
Beta-galactosidase as a marker of HSP70 promoter induction in Dunning R3327 prostate carcinoma cells; Roigas J et al.; Hyperthermia is known to improve the response of tumors to radiation or chemotherapeutic treatment when combined in multimodal strategies . The cellular response to hyperthermia is associated with the synthesis of heat shock proteins (HSP) . To study the stress response in prostate cancer we have developed a clone of Dunning R3327 rat prostate carcinoma cells stably transfected with a gene construct containing the E . coli beta-galactosidase gene driven by the Drosophila HSP70 promoter . The measurement of beta-galactosidase serves as a rapid and semiquantitative assay of HSP70 gene activation . The Dunning cell clone showed evidence of incorporation of the HSP70/beta-galactosidase construct within the genomic DNA by Southern blot analysis . When compared to mock-transfected control cells, the clone showed minimal baseline beta-galactosidase activity, which significantly increased following a hyperthermic stress . The time course of beta-galactosidase elevation following heat stress paralleled the time course of cellular HSP70 elevation by Western blot analysis . These stably transfected Dunning R3327 cells may provide a useful tool to study the effects of hyperthermia, radiation, and chemotherapeutic agents on the cellular stress response and in the establishment of HSP70 as a marker of cellular resistance in the multimodal treatment of prostate cancer.

Plasmid, 1997, 38(1), 25 - 33
A novel retron that produces RNA-less msDNA in Escherichia coli using reverse transcriptase; Lima TM et al.; Bacterial retroelements, or retrons, use reverse transcriptase (RT) to produce a multicopy single-stranded DNA (msDNA) molecule that is covalently linked to RNA . In these studies we show that a retron from Escherichia coli 110, a clinical isolate, produces a novel RNA-less msDNA with a 5' phosphate residue . The msDNA is a 74-nucleotide single-stranded DNA molecule with a stable stem-loop structure without a mismatched base pair . Only the genes encoding msDNA (msd), msdRNA (msr), and RT (ret) are required to produce the msDNA molecule . The organization of these genes on the retron was similar to that of other elements producing branched msDNA-RNA . The conserved guanine, which is the branched residue in msDNA-RNA complexes and is essential for branch formation, is also present . Site-directed mutagenesis showed that this guanine is essential for the production of RNA-less msDNA . We postulate that the RNA-less msDNA in strain 110 is produced by nucleolytic cleavage of the branched msDNA-RNA compound.

Plasmid, 1997, 38(1), 1 - 11
Requirement of a limited segment of the sog gene for plasmid R64 conjugation; Narahara K et al.; The sog gene of the IncI1 plasmid R64 was sequenced and characterized . The sog gene was shown to express two acidic proteins, SogL and SogS, with 1255 and 844 amino acid residues, respectively . The SogS protein was expressed by translational reinitiation within the SogL reading frame . Analysis of dnaG-suppression activity using the Escherichia coli dnaG strain indicated that the domain for this activity was located within the N-terminal one-third segment of the SogL protein . A Deltasog mutation was constructed by replacing most of the sog coding sequence with a DNA fragment encoding a tetracycline resistance gene . Introduction of the Deltasog mutation into an R64 derivative resulted in approximately a 50-fold reduction in transfer frequency . It was observed that only a limited portion of the SogL or SogS protein corresponding to an internal 0.94-kb EcoRV-SnaBI segment of the sog gene was required for the conjugal transfer of R64.

Indian J Exp Biol, 1997 Jan, 35(1), 99 - 102
Cloning of PCR synthesized 191 bp DNA overlapping lac promoter for plasmid construction; Biswas SR; E coli genomic DNA was amplified by polymerase chain reaction (PCR) using two 5' and 3' oligonucleotide primers (27-mer) . Amplified DNA was 191 bp . The region of amplified DNA on lac operon in E coli was 14 bp upstream from the transcription initiation site and 177 bp downstream . Amplified DNA was cloned into a phagemid vector for construction of plasmid, suitable for use as template for making strand-specific probe to detect initiated lac transcript by RNase protection assay, labelling for Southern and Northern hybridization . Another use of this probe made from this construct is to detect strand-specific DNA repair . The construct was verified by DNA sequencing.

Rapid Commun Mass Spectrom, 1997, 11(12), 1279 - 85
Rapid separation and characterization of protein and peptide mixtures using 1.5 microns diameter non-porous silica in packed capillary liquid chromatography/mass spectrometry; MacNair JE et al.; Octadecyl-modified 1.5 microns diameter non-porous silica particles were packed in 150 microns i.d . (360 microns o.d.) capillaries with lengths of 20 cm which were used to separate proteins and peptides generated from enzymatic digests of proteins . Gradients were produced using an exponential dilution method at pressures of 520 Bar (7500 psi) and electrospray ionization mass spectrometry was used for detection . This system was similar to packed capillary perfusion chromatography with respect to chromatographic resolution and analysis time and had a limit of detection comparable to traditional packed capillaries which use 5 microns diameter porous particles . The analyses required as little as 250 femtomol of protein or 500 femtomol of peptide on-column in approximately 30 min . This technique was then applied to verify the existence of an overexpressed protein in an E . coli cell lysate and to confirm the presence of four glycoforms of a peptide generated in the proteolytic digest of an antibody.

Life Sci, 1997, 61(8), 813 - 8
Tolerance to lipopolysaccharide is not related to the ability of the hypothalamus to produce prostaglandin E2; Chemo A et al.; The effects of repeated administration of Escherichia coli lipopolysaccharide (LPS) on body temperature and hypothalamic prostaglandin E2 (PGE2) production was examined in male Sprague-Dawley rats to elucidate whether the development of endotoxin tolerance is related to the ability of the hypothalamus to produce PGE2 . Initial injection of LPS resulted in hyperthermia, preceded by short-termed hypothermia, while no changes in body temperature were observed after the second injection (administered 48 h later) . In contrast, LPS induced elevation in hypothalamic PGE2 production after both the first and second injections of the pyrogen . This led us to conclude that endotoxin tolerance is independent of the hypothalamic production of PGE2 in response to repeated administration of LPS.

Microbiol Immunol, 1997, 41(7), 519 - 25
Production of monoclonal antibody discriminating serological difference in Escherichia coli O9 and O9a polysaccharides; Kido N et al.; A monoclonal antibody (mAb) with a unique antigenic specificity against Escherichia coli O9 was produced . The O9a mAb was reactive with a part of the strains in E . coli O9 . The O9a mAb did not react with LPS from the E . coli O9 test strain Bi316-42 . The distribution of the antigen defined by the O9a mAb in E . coli O9 was consistent with that of E . coli O9a present in E . coli O9 strains . The chemical structure of the repeating unit of the O-specific polysaccharide detected by the mAb was demonstrated to be a mannotetraose by two-dimensional nuclear magnetic resonance spectroscopy . It was confirmed that the mAb recognized E . coli O9a serotype in E . coli O9 serotype strains, suggesting that E . coli O9a serotype might be a dominant strain in E . coli O9.

Acta Microbiol Pol, 1997, 46(1), 7 - 17
Escherichia coli defects caused by null mutations in dnaK and dnaJ genes; Paciorek J et al.; Escherichia coli dnaJ and dnaKdnaJ mutants are defective in cell growth and survival at high temperature . Lack of DnaK and DnaJ proteins results in cell filamentation and leads to the defect in motility . Synthesis of DNA and protein is inhibited at 42 degrees C, especially in double-deletion mutant . Degradation of protein was observed in both mutants at high temperature . Complementation of these defects can be achieved by the expression of the wild-type alleles from low-copy number plasmid.

Dev Biol Stand, 1997, 90, 211 - 20
Immunization with viral antigens: infectious haematopoietic necrosis; Winton JR; Infectious haematopoietic necrosis (IHN) is one of the most important viral diseases of salmonids, especially among juvenile fish where losses can be high . For over 20 years, researchers have tested a variety of preparations for control of IHN . Early vaccines consisted of killed virus and were effective when delivered by injection, but too costly to be practical on a large scale . Attenuated vaccines were developed by serial passage in cell culture and by monoclonal antibody selection . These offered excellent protection and were cost-effective, but residual virulence and uncertainty about their effects on other aquatic species made them poor candidates for licensing . Subunit vaccines using part of the IHNV glycoprotein gene cloned into E . coli or into an attenuated strain of A . salmonicida have been tested, appeared safe and were inexpensive . These vaccines were reported to provide some protection when delivered by immersion . Information on the location of antigenic sites on the glycoprotein led to trials using synthetic peptides, but these did not seem to be economically viable . Recently, plasmid vectors encoding the glycoprotein gene under control of a cytomegalovirus promoter were developed for genetic immunization . The constructs were highly protective when delivered by injection, but a more practical delivery system is needed . Thus, while several vaccine strategies have been tried in order to stimulate specific immunity against IHN, more research is needed to develop a commercially viable product for control of this important disease.

Arch Virol, 1997, 142(7), 1351 - 63
Grapevine berry inner necrosis, a new trichovirus: comparative studies with several known trichoviruses; Yoshikawa N et al.; Biological, morphological and serological properties of grapevine berry inner necrosis virus (GINV), the casual virus of grapevine berry inner necrosis disease occurring in Japan, were compared with those of several known trichoviruses . Host range and particle length of GINV were quite similar to those of apple chlorotic leaf spot trichovirus (ACLSV) . In ultrathin sections of the infected tissues, GINV particles existed in aggregated masses in the cytoplasm of vascular parenchyma and mesophyll cells . No virus specific inclusion bodies, such as pinwheels, viroplasmas or vesicles were observed . Serological relationships were not found between GINV and ACLSV, grapevine virus A or grapevine virus B . The cDNAs of the 3'-terminal region of the GINV genome were synthesized from poly (A)+RNAs extracted from infected tissues by PCR-selected cDNA subtraction and 3'-RACE PCR . The sequence of the 3'-terminal 2469 nucleotides contained three open reading frames (ORF) encoding a protein with the conserved motifs of RNA polymerase (ORF1), a 39 kDa putative movement protein (ORF2) and a 22 kDa protein (ORF3) . The 22 kDa protein expressed in Escherichia coli reacted with antiserum against GINV, indicating that it is the coat protein of GINV . Polymerase and coat protein amino acid sequence comparisons and phylogenetic analyses with nine species of the genera Trichovirus and Capillovirus indicated that GINV is a new trichovirus relatively close to ACLSV.

Blood Purif, 1997, 15(3), 188 - 94
Chlorine dioxide: a new agent for dialysis monitor disinfection in a pediatric center; Palo TD et al.; In order to evaluate the bacterial and endotoxin contamination in the dialysis fluids of our pediatric center and the effectiveness of chlorine dioxide (CD) compared with a conventional method, (1) deionized water, (2) dialysate fluid, (3) basic concentrate, and (4) acid concentrate were tested in 4 dialysis machines . Monitor sterilization was made using CD in protocol A and sodium hypochlorite/acetic acid in protocol B . Once every 2 weeks the deionized water set of distribution was routinely disinfected with peracetic acid . Each protocol lasted 1 months and the samples were taken, under aseptic conditions, on the 15th, 22nd and 27th day . All samples, at all stages of the study, showed an endotoxin concentration below the limits recommended by the Canadian Standard Association . Fifty-nine out of 72 samples in A and 62 out of 72 samples in B showed a bacterial count within the range recommended by the Association for the Advancement of Medical Instrumentation . The data show that both protocols produced the same results . However, protocol A is to be preferred for its simultaneous disinfecting-cleaning and descaling activity which proves time-saving.

Environ Mol Mutagen, 1997, 30(1), 65 - 71
Mutational specificity of 1-(2-chloroethyl)-3-cyclohexyl-1-nitrosourea in Escherichia coli: comparison of in vivo with in vitro exposure of the supF gene; Luque-Romero FL et al.; Forward mutations induced by 1-(2-chloroethyl)-3-cyclohexyl-1-nitrosourea (CCNU) in the supF gene of Escherichia coli were recovered from bacteria deficient in nucleotide excision repair and in DNA-alkyltransferase activity . Bacteria were exposed to 0.4 mM CCNU (in vivo supF mutagenesis), increasing the overall mutation frequency 15.7-fold above the spontaneous value . A total of 73 independent supF- mutants were sequenced . The resulting mutation spectrum was compared with those obtained in bacteria and mammalian cells following the classical shuttle-vector approach (in vitro supF mutagenesis) . In vivo CCNU mutagenesis in E . coli yielded a large number of deletions (20/73), in agreement with mammalian data but distinct from in vitro bacterial spectra, which are almost exclusively composed of G:C-->A:T transitions . A substantial proportion (6/18) of CCNU-induced deletions (> 3 bp) involved repeated DNA sequences, suggesting a contribution of a slippage-misalignment process in the generation of this mutation class . Substitutions occurred primarily at G:C base pairs (44/53) and were predominantly G:C-->A:T transitions (39/53) . This mutational change was attributed to the mispair potential of the O6-chloroethylguanine lesion with thymine . Most G:C-->A:T transitions (34/39) were located at three 5'-GG-3' hotspot sites (positions 123, 160, and 168) . The distribution of hotspot sites for G:C-->A:T substitutions differed as a function of the in vivo or in vitro chemical modification of the supF-bearing plasmids and revealed significant differences in the DNA strand distribution of this mutational event . Our data suggest that the transcriptional status of the target gene has strong influence on the probability of O6-chloroethylguanine formation, reducing its incidence in the transcribed DNA strand.

Membr Cell Biol, 1997, 11(1), 121 - 9
Electrofusion of Escherichia coli cells; Tyurin MV et al.; Bacterial cell fusion was observed under conditions optimal for highly efficient electrotransformation of untreated Escherichia coli cells . E . coli clones possessing the joint phenotype were found after electric treatment of genetically marked parental strains . The phenotypes of the clones segregated due to a subsequent cultivation of the clones, whereas some of them turned out to be recombinants . Electron microscopy carried out 1 h after electric treatment of the cells revealed physical contacts between the cells being in parallel arrangement . The electrofusion processes detected are important for the estimation of electrotransformation efficiency . A possibility to use electrofusion of untreated bacterial cells for in vivo transfer of plasmid and chromosomal DNAs is discussed.

Semin Thromb Hemost, 1997, 23(3), 281 - 93
Hemolytic uremic syndromes in childhood; Gordjani N et al.; The hemolytic uremic syndrome (HUS) comprises hemolytic anemia, acute renal failure, and thrombocytopenia . It is the most frequent cause of acute renal failure in childhood . Ninety percent of the patients have a diarrheal prodrome, and are referred to as having typical HUS . Approximately 10% exhibit the so-called atypical HUS . Typical HUS is caused by shigatoxin-producing Escherichia coli . The toxin, bound to the globotriosyl ceramide cell receptor and internalized, interferes with protein synthesis, predominantly of endothelial cells . The main target is the kidney, but nearly every organ system can be involved . The most common extrarenal involvement is damage to the central nervous system . The central event is probably an insult to the endothelial cell with consecutive loss of antithrombogenic properties . The von Willebrand factor, activation of platelets via platelet-activating factor, other growth factors (e.g., interleukins 1, 6, 8), nitric oxide, lipopolysaccharides, activated polymorphonucleated neutrophils, and the metabolites of the arachidonic acid cascade (e.g., prostaglandin I2) are believed to be involved in the pathogenic cascade . Controlled therapeutic trials with heparin, dipyridamole, aspirin, and urokinase have not been associated with improved outcome . Antibiotics have not yielded any benefit . Plasma infusions and plasma exchange appear to be efficacious, and are justified in cases of atypical HUS and thrombotic thrombocytopenic purpura . Binding of the toxin to the intestinal lumen, and thereby inhibition of enteral reabsorption, is under investigation.

Dev Genet, 1997, 20(4), 338 - 47
Green fluorescent protein/beta-galactosidase double reporters for visualizing Drosophila gene expression patterns; Timmons L et al.; We characterized 120 novel yeast Ga14-targeted enhancer trap lines in Drosophila using upstream activating sequence (UAS) reporter plasmids incorporating newly constructed fusions of Aequorea victoria green fluorescent protein (GFP) and Escherichia coli beta-galactosidase genes . Direct comparisons of GFP epifluorescence and beta-galactosidase staining revealed that both proteins function comparably to their unconjugated counterparts within a wide variety of Drosophila tissues . Generally, both reporters accumulated in similar patterns within individual lines, but in some tissues, e.g., brain, GFP staining was more reliable than that of beta-galactosidase, whereas in other tissues, most notably tests and ovaries, the converse was true . In cases of weak enhancers, we occasionally could detect beta-galactosidase staining in the absence of discernible GFP fluorescence . This shortcoming of GFP can, in most cases, be alleviated by using the more efficient S65T GFP derivative . The GFP/beta-gal reporter fusion protein facilitated monitoring several aspects of protein accumulation . In particular, the ability to visualize GFP fluorescence enhances recognition of global static and dynamic patterns in live animals, whereas beta-galactosidase histochemistry affords sensitive high resolution protein localization . We present a catalog of Ga 14-expressing strains that will be useful for investigating several aspects of Drosophila melanogaster cell and developmental biology.

DNA Seq, 1997, 7(3-4), 193 - 7
An ABC-transporter system homolog in an organism of the phylogenetic domain Archaea; Jovell RJ et al.; A cluster of genes was identified in an archaeal organism, the methanogen Methanosarcina mazei S-6, that was a homolog of the ABC-transporter system loci of several organisms belonging to the phylogenetic domain Bacteria . The gene number, size, and organization were also similar . The proteins encoded by these genes were similar in structure, hydrophilicity-hydrophobicity profiles, and motifs to the equivalent components of homolog systems in bacteria and eucarya.

Methods Enzymol, 1997, 281, 336 - 40
Expression and purification of mammalian 5-aminolevulinate synthase; Dailey HA et al.; We have described a procedure for production and purification of recombinant, mature-length mouse ALAS-2 . The fact that E . coli utilizes the C5 path for ALA production means that there is no problem with contamination of the recombinant ALAS-2 by host cell enzyme, such as one may have with a yeast expression system . While the detailed procedure produces enzyme in good yield with relatively common protein purification techniques, future expression systems may be developed to take advantage of the rapid purification achieved by the use of a 6-histidine (His6) aminoterminal tag and metal chelate chromatography . Such approaches in this laboratory with protoporphyrinogen oxidase, coproporphyrinogen oxidase, and uroporphyrinogen decarboxylase have resulted in the production and purification of enzymes whose kinetic and physical parameters are essentially identical to those of proteins lacking the His6 tag.

Biochem Cell Biol, 1997, 75(2), 143 - 51
A consensus sequence for a functional human endogenous retrovirus K (HERV-K) dUTPase; Harris JM et al.; Amino acid sequence comparisons have revealed that a potential dUTPase gene is encoded by the retrovirus HERV-K, a defective multicopy virus that is transmitted vertically in humans . This gene is distinct from the human cellular dUTPase gene and thus two potential sources of the enzyme exist in human cells . dUTPases characterized from various sources each contain five conserved amino acid sequence motifs that form the active site of the enzyme . The protein sequence of the putative HERV-K dUTPase deduced from previous DNA sequence data from one proviral clone (HERV-K10) shows marked deviations at highly conserved residues in four of five of these motifs . Therefore, the reported DNA sequence may represent a mutated form of the viral dUTPase gene . To address this possibility, we cloned and sequenced 22 copies of the HERV-K dUTPase gene from human DNA . The results of this analysis indicate that variations evident in the HERV-K10 dUTPase amino acid sequence represent mutations of the wild-type viral DNA sequence . A version of the HERV-K dUTPase gene that corresponds to the ancestral, wild-type DNA sequence was constructed and adapted for expression in Escherichia coli . The resulting enzyme was found to exhibit properties similar to those of dUTPases isolated from other systems . A possible role of the HERV-K dUTPase in human disease is discussed.

Folia Microbiol (Praha), 1997, 42(3), 161 - 4
The Escherichia coli cell cycle, cell division and ppGpp: regulation and mechanisms; D'Ari R; The literature demonstrating tight regulation of the Escherichia coli cell cycle is reviewed . Recent evidence is presented indicating that the normal rod cell shape can be abandoned, allowing growth as a coccus, either by increasing the amount of the division proteins FtsZ, FtsA and FtsQ, or by increasing the pool of the nucleotide ppGpp . It is argued that ppGpp may be a cell cycle signal in E . coli.

Morfologiia, 1997, 111(2), 69 - 72
{The mechanisms of the stimulating effect of tissue basophils on the reparative processes in inflammation}; Klimenko NA et al.; Mast cells stimulating effect on reparative processes in inflammation involves histamine, serotonin and heparin . This was shown on the model of E . coli-induced acute infectious peritonitis in rats using antagonists of major biologically active mast cells products (histamine, serotonin and heparin)-dimedrole, cimetidine, ciproheptadine and protamine sulfate . Fibroblast accumulation is associated with histamine, influencing predominantly H1- and H2-receptors as well as with serotonin . Fibroblast proliferative synthetic ability increases through histamine action on H1-receptors and serotonin and heparin influence . Accumulation of monocytes-macrophages and their proliferative synthetic ability stimulation is affected by histamine (through H1-receptors) and serotonin.

Annu Rev Biochem, 1997, 66, 863 - 917
Protein import into mitochondria; Neupert W; Mitochondria import many hundreds of different proteins that are encoded by nuclear genes . These proteins are targeted to the mitochondria, translocated through the mitochondrial membranes, and sorted to the different mitochondrial subcompartments . Separate translocases in the mitochondrial outer membrane (TOM complex) and in the inner membrane (TIM complex) facilitate recognition of preproteins and transport across the two membranes . Factors in the cytosol assist in targeting of preproteins . Protein components in the matrix partake in energetically driving translocation in a reaction that depends on the membrane potential and matrix-ATP . Molecular chaperones in the matrix exert multiple functions in translocation, sorting, folding, and assembly of newly imported proteins.

Annu Rev Biochem, 1997, 66, 199 - 232
Molecular basis for membrane phospholipid diversity: why are there so many lipids?
Dowhan W.
Phospholipids play multiple roles in cells by establishing the permeability barrier for cells and cell organelles, by providing the matrix for the assembly and function of a wide variety of catalytic processes, by acting as donors in the synthesis of macromolecules, and by actively influencing the functional properties of membrane-associated processes . The function, at the molecular level, of phosphatidylethanolamine, phosphatidylglycerol, and cardiolipin in specific cellular processes is reviewed, with a focus on the results of combined molecular genetic and biochemical studies in Escherichia coli . These results are compared with primarily biochemical data supporting similar functions for these phospholipids in eukaryotic organisms . The wide range of processes in which specific involvement of phospholipids has been documented explains the need for diversity in phospholipid structure and why there are so many membrane lipids.

Annu Rev Biophys Biomol Struct, 1997, 26, 223 - 58
Molecular mechanism of photosignaling by archaeal sensory rhodopsins; Hoff WD et al.; Two sensory rhodopsins (SRI and SRII) mediate color-sensitive phototaxis responses in halobacteria . These seven-helix receptor proteins, structurally and functionally similar to animal visual pigments, couple retinal photoisomerization to receptor activation and are complexed with membrane-embedded transducer proteins (HtrI and HtrII) that modulate a cytoplasmic phosphorylation cascade controlling the flagellar motor . The Htr proteins resemble the chemotaxis transducers from Escherichia coli . The SR-Htr signaling complexes allow studies of the biophysical chemistry of signal generation and relay, from the photobiophysics of initial excitation of the receptors to the final output at the level of the flagellar motor switch, revealing fundamental principles of sensory transduction and more broadly the nature of dynamic interactions between membrane proteins . We review here recent advances that have led to new insights into the molecular mechanism of signaling by these membrane complexes.

Acta Biochim Pol, 1997, 44(1), 153 - 7
Characterization of the Escherichia coli gene encoding a new member of the short-chain dehydrogenase/reductase (SDR) family; Sirko A et al.; The nucleotide sequence of a chromosomal DNA fragment located upstream from the cysPTWAM operon of Escherichia coli was established . Sequence analysis indicates the presence of an open reading frame which has been designated ucpA (upstream cys P) . The potential protein products exhibits strong sequence homology to the members of a large protein family, short-chain dehydrogenases/reductases . Involvement of Crp, FruR and IHF in the regulation of ucpA transcription in vivo was demonstrated.

Virus Genes, 1997, 14(2), 131 - 6
Complete sequence and transposon mutagenesis of the BamHI J fragment of Cydia pomonella granulosis virus; Kang W et al.; The BamHI J fragment of Cydia pomonella granulosis virus was subcloned and subjected to transposon mutagenesis in Escherichia coli using a Tn3 derivative . After screening by restriction endonuclease digestion and polymerase chain reaction (PCR), 44 clones were selected representing insertions every 100 to 300 bp . The complete sequence was compiled and the transposon insertion sites mapped precisely by sequencing . Analysis of the sequence revealed the presence of 7 potential open reading frames (ORFs) . The BamHI J fragment was already known to encode IAP and OPDV-E6 . Three other ORFs encode products similar to known proteins, viz . an Autographa californica nuclear polyhedrosis virus 8.6 kDa protein, a Lymantria dispar nuclear polyhedrosis virus 34 kDa protein, and vertebrate reovirus omega-1 proteins . The ORF with similarity to omega-1 is also similar to baculovirus p10 proteins . In both cases, the similarity occurs in regions likely to form a coiled-coil structure.

Planta, 1997, 202(3), 349 - 56
Cloning of the cDNA for glutaredoxin, an abundant sieve-tube exudate protein from Ricinus communis L . and characterisation of the glutathione-dependent thiol-reduction system in sieve tubes; Szederkenyi J et al.; Sieve-tube exudate protein (STEP) from Ricinus communis L . seedlings consists of a characteristic set of more than 100 different polypeptides, against which a complex antiserum was raised . This antiserum cross-reacted with dominant protein species (molecular weights 10-30 kDa) present in the sieve-tube exudate and, to a lesser extent, with proteins in tissue extracts of Ricinus and a wide range of other plant species . For further elucidation of the nature of individuals STEPs in the sieve tubes the anti-STEP serum was used to screen a cDNA expression library constructed from Ricinus cotyledon mRNA . Two clones that differed in the 3' untranslated region encoded a protein of 11 kDa which showed striking homology to bacterial and eucaryotic glutaredoxin sequences . Glutaredoxin activity was confirmed for the recombinant protein after overexpression in Escherichia coli and characterised in detail in sieve-tube exudate . Michaelis Menten constants (Km) for reduced glutathione and cysteine were 2 mM and 50 microns, respectively . Besides L-cysteine, dehydroascorbate and protein disulphides were also reduced by the activity present in the sieve-tube exudate . Glutathione, which is the obligate donor of reduced thiols for glutaredoxin, was present in sieve-tube sap in millimolar concentrations (up to 3 mM) with a ratio of total to oxidised glutathione of 3:1 . It is suggested that glutaredoxin and glutathione in sieve tubes prevent oxidative damage and may be involved in redox regulation of sieve-tube proteins.

Lupus, 1997, 6(5), 436 - 40
The effect of OM-89 (Subreum) on the murine model of systemic lupus erythematosus MRL-lpr/lpr; Dhaher YY et al.; OM-89 (Subreum), an E . coli extract, is used clinically in the treatment of rheumatoid arthritis . In this study, the authors examined the effect of OM-89 on some aspects of SLE in the murine model MRL-lpr/lpr . Animals were given OM-89 orally at a dose of 400 mg/kg weight (40 mg active substance) 5 d a week from six weeks old . It was found that mice receiving the drug reached the point of 55% (6/11) survival at the age of 33 weeks compared with 27 weeks for the control group (54%; 7/13) . There was a significant increase in the delay before developing alopecia in the treated group (P < 0.01) . The increase in proteinuria in the control group was significantly higher than in the treated group (P < 0.03) . In a second set of experiments sacrificing the animals at week 22, a significant decrease in anti-dsDNA auto-antibodies was also found in the treated group (P < 0.05), histopathologically a less severe tubular destruction in the kidney was observed in the treated group . It can be concluded that the oral treatment of OM-89 can significantly reduce the severity of SLE in this strain of mice . It can be postulated that the administration of the bacterial extract could modulate the immune response, modifying the Th1 and Th2 balance and inducing oral tolerance.

Lasers Surg Med, 1997, 21(1), 88 - 93
In vitro measurements of cytotoxic effects of 193 nm and 213 nm laser pulses at subablative fluences; Ediger MN et al.; BACKGROUND AND OBJECTIVE: The frequency-quintupled q-switched Nd:YAG laser is being studied as an alternative to the ArF excimer laser for photorefractive procedures . The present report describes two experiments comparing biologic effects of these laser devices . STUDY DESIGN/MATERIALS AND METHODS: Bovine corneas were irradiated with subablative laser pulses in liquid nitrogen and analyzed by electron paramagnetic resonance spectroscopy to assess free radical production . Aqueous bacterial suspensions were irradiated with low-intensity laser pulses and assayed for cell survival . RESULTS: Electron paramagnetic resonance spectra were very similar in both amplitude and shape for exposure at the two wavelengths . Bacterial survival was markedly less for 213 nm irradiation than 193 nm exposure and displayed a different dependence on cumulative exposure . CONCLUSION: Free radical production by 213 nm laser exposure is quite comparable to that seen previously for 193 nm irradiation . However, cell lethality appears to be significantly greater at the longer ultraviolet wavelength . This difference may contribute to complications observed after corneal photoablation with the 213 nm device.

Membr Cell Biol, 1997, 10(5), 543 - 51
Use of hydrated reversed micelles of surfactant in organic solvent for stabilization of individual oligomeric forms of uridine phosphorylase from Escherichia coli K-12; Burlakova AA et al.; The catalytic activity of uridine phosphorylase from Escherichia coli K-12 entrapped in hydrated reversed micelles of aerosol OT (AOT) in octane has been studied as a function of the degree of hydration of the micelles . It was shown that the catalytic activity of uridine phosphorylase reached maximum values at {H2O/{AOT} ratios equal to 8.4, 12.9, 16.1 and 18.6 . Based on the sedimentation data the conclusion has been made that the maxima of the catalytic activity correspond to the monomeric, dimeric, trimeric and tetrameric forms of the enzyme . The measurements of the rate of the enzymatic reaction catalyzed by uridine phosphorylase entrapped in hydrated reversed micelles at various concentrations of AOT indicate that the monomeric enzyme form, in contrast to the trimeric and tetrameric forms, exhibits the membranotropic properties.

J Cancer Res Clin Oncol, 1997, 123(6), 325 - 30
Reconstruction and expression of chimeric anti-HBx antibody in Escherichia coli; Zhou G et al.; The variable regions of murine monoclonal anti-HBx immunoglobulin and the constant region of human antibody were cloned by reverse transcript-polymerase chain reaction (RT-PCR) . The heavy-chain and light-chain variable regions were connected and coexpressed with human constant region C-r3 and C-k3 in the reconstructed vector of E . coli . The products showed high specificity and binding ability with HBx . Which is closely associated with hepatocarcinogenesis . This makes it possible to humanize the mouse monoclonal antibodies and express the fusion protein in E.coli for potential radioimmunotherapy in patients with hepatocellular carcinoma.

Fiziol Zh, 1997, 43(1-2), 83 - 8
{Mast cells in a focus of carrageenan-induced acute aseptic inflammation}; Klymenko MO et al.; On the model of carrageenen-induced acute aseptic peritonitis in rats the regularities of the functional state of mast cells (MC), of the content of free and cellular histamine in exudate and mesenterium and histamine in blood from the 5th minute up to 10th day after injection of phlogogene have been studied . Also as in infectious inflammation, the phasic reaction of MC which is observed at least during 10 days after action of phlogogene has been shown . The quick short first phase during half an hour after induction of inflammation has been observed and the gradual long second phase from the 1st hour up to 10th day has been noted . At least during 1st day the regularities of reaction of MC were extremely similar to the ones in infection peritonitis . Also as in infectious inflammation the second phase of reaction of MC correlates with the accumulation of leukocytes in inflammatory focus . At the same time the dependence of dynamics of functional changes of MC in aseptic inflammation on the properties of phlogogene (irritating or relatively indifferent phlogenes) has been established.

Free Radic Biol Med, 1997, 23(4), 627 - 36
Role of rpoS regulon in resistance to oxidative stress and near-UV radiation in delta oxyR suppressor mutants of Escherichia coli; Ivanova AB et al.; Escherichia coli delta oxyR mutants are hyper-sensitive to oxidative agents but this sensitivity is reversed to hyper-resistance in delta oxyR suppressor strains (delta oxyRsup; Greenberg, J.T . and Demple, B . 1988 . EMBO J . 7:2611-2618) . Also, delta oxyR mutants have increased mutation rates that are also reversed in delta oxyRsup . We now report that the rpoS regulon may have a role in determining hyper-resistance and loss of hyper-mutability of delta oxyRsup . Delta oxyRsup cells were also resistant to near-ultraviolet radiation (near-UV) and survived longer in stationary phase than delta oxyR cells . In delta oxyRsup cells elevated beta-galactosidase expression from a rpoS::lacZ promoter fusion and significant overproduction of RpoS protein was observed . These increases were accompanied by substantial elevation in transcription of rpoS-dependent genes as determined by beta-galactosidase expression from katE::lacZ, dps::lacZ, and xthA::lacZ promoters . Catalase HPI and HPII activities were also increased . When rpoS::Tn10 was transduced into delta oxyRsup, phenotypes switched back to hyper-sensitive, hyper-mutable and reduced catalases I and II . Individual delta oxyR colonies exhibited significant clonal variability in beta-galactosidase expression from rpoS::lacZ promoter . These results provide further evidence of the functional and regulatory overlap between two major anti-oxidant defense systems of bacteria.

Virus Genes, 1997, 14(1), 31 - 40
A major antigenic domain of hantaviruses is located on the aminoproximal site of the viral nucleocapsid protein; Gott P et al.; Hantavirus nucleocapsid protein has recently been shown to be an immunodominant antigen in hemorrhagic with renal syndrome (HFRS) inducing an early and long-lasting immune response . Recombinant proteins representing various regions of the nucleocapsid proteins as well as segments of the G1 and the G2 glycoproteins of hantavirus strains CG18-20 (Puumala serotype) and Hantaan 76-118 have been expressed in E . coli . The antigenicity of these proteins was tested in enzyme immunoassays and immunoblots . These studies revealed that human IgG immune response is primarily directed against epitopes located within the amino acid residues 1 to 119 of the amino terminus of viral nucleocapsid proteins . This fragment was recognized by all HFRS patient sera tested (n = 128) . The corresponding enzyme immunoassays proved to be more sensitive than the indirect immunofluorescence assays . Furthermore, the majority of bank vole monoclonal antibodies raised against Puumala virus reacted specifically with this site . A recombinant G1 protein (aa 59 to 401) derived from the CG 18-20 strain was recognized by 19 out of 20 sera from HFRS patients.

Lung, 1997, 175(4), 253 - 63
Involvement of tachykinins in endotoxin-induced airway hyperresponsiveness; Loeffler BS et al.; Inhaled endotoxin, lipopolysaccharide (LPS), has been shown to result in bronchial hyperresponsiveness (BHR) to endogenous bronchoconstrictive mediators such as histamine . To determine the role of sensory neuropeptides released from bronchopulmonary C-fibers in LPS-induced BHR, 24 guinea pigs were allocated randomly to the following four groups . Animals in Groups I and IV were challenged with intratracheal instillation of 100 microliters of saline vehicle, and those in Groups II and III with 1 mg of LPS (Escherichia coli, 0111:B4) in 100 microliters of saline . Groups III and IV also received a high dose capsaicin (HDC) treatment to deplete tachykinins from C-fibers 1-2 weeks prior to the experiment . Animals were anesthetized and paralyzed, and total lung resistance (RL) and compliance (Cdyn) were measured continuously during the experiment . Dose responses of RL and Cdyn to histamine (0-8 micrograms/kg, intravenously) and capsaicin (0-1.6 micrograms/kg, intravenously), a specific C-fiber stimulant, were obtained prior to and at 1, 2, and 3 h following LPS/saline vehicle challenge . At 2 h after LPS, delta RL caused by histamine (8 micrograms/kg) was significantly higher in Group II (1.145%) than that in Group I (280%; p < 0.05); similarly, delta RL caused by capsaicin (1.6 micrograms/kg) was also increased after LPS (Group I, 107%; Group II, 267%; p < 0.05) . Although HDC treatment completely abolished the bronchomotor response to capsaicin in both Groups III and IV, it enhanced the LPS-induced BHR to histamine (8 micrograms/kg; Group III, 1.834%; p < 0.05) . In conclusion, these results suggest that the role of tachykinins in LPS-induced BHR may be dependent upon the type and the route of administration of the bronchoactive substance studied.

Biochimie, 1997, 79(1), 13 - 22
Site directed DNA joining; Guilliams TG et al.; We have previously identified a cDNA encoding part of the amino terminal portion of the large subunit of replication factor c (RF-C, AP-1), which we have named VDJP . Analysis of VDJP demonstrated that it has amino acid homology to bacterial DNA ligases and specific binding to the nonamer portion of the V(D)J recombination signal sequence motif . In this report, we demonstrate that VDJP is capable of forming a covalent bond between DNA fragments in a sequence dependent fashion . The VDJP mediated DNA ligation reaction is neither dependent on the presence of compatible DNA ends nor on sequence homology between the DNA fragments that are joined . Furthermore, we show that the covalent junction between the DNA fragments is resistant to proteases and phenol, and therefore not protein linked.

Adv Exp Med Biol, 1997, 419, 181 - 3
Molecular characterization of rat T lymphocyte alloantigen RT6.1 as an ADP-ribosyltransferase; Maehama T et al.; A family of glycosylphosphatidylinositol-linked ADP-ribosyltransferases, of which cDNAs were cloned from various mammalian cells, possess a common Glu-rich motif (EEEVLIP) near their carboxyl termini . Although the first Glu in the common motif is replaced by Gln (Q207EEVLIP) in rat T lymphocyte alloantigens RT6.1 and RT6.2, the two RT6s appear to have ADP-ribosyltransferase activity . To investigate the significance of the Glu-rich motif in the enzyme activity, we produced a mutant RT6.1, in which Gln207 was replaced by Glu (Q207E), together with wild-type RT6s, in Escherichia coli . The recombinant RT6.1 and RT6.2 displayed extremely low auto-ADP-ribosylation, though the latter modification was somewhat higher than the former one . In contrast, much higher the auto-modification was observed for Q207E mutant . Moreover, the mutant could effectively ADP-ribosylate agmatine as a substrate . Thus, the single amino acid mutation of RT6.1 caused remarkable increase in its ADP-ribosyltransferase activity, indicating that the Glu-rich motif near the carboxy terminus plays an important role in the enzyme activity.

Adv Exp Med Biol, 1997, 419, 145 - 54
Purification, characterisation, and molecular cloning of a chicken erythroblast mono(ADP-ribosyl)transferase; Davis T et al.; We have purified an arginine-specific mono(ADP-ribosyl)transferase from chicken erythrocytes . The purified transferase was free from poly (ADP-ribose) polymerase activity . The molecular weight of the purified enzyme was estimated to be 27.5 kDa by gel filtration through Sephadex G-75 in a non-denaturing solvent . Activity gel experiments indicate that the active enzyme has an apparent molecular weight in SDS gels of about 28 kDa . The optimum pH of the reaction is about 8.0 . The K(m) value for NAD+ of the purified enzyme is about 130 microM . Small molecular weight inhibitors of poly (ADP-ribose) polymerase have no significant effect on the mono ADP-ribosyl transferase enzyme activity . A number of inhibitors of the arginine-specific mono(ADP-ribosyl)transferase activity have been identified . Among the more effective inhibitors are 1,4 naphthoquinone, 5,8-dihydroxy-1,4-naphthoquinone, 4-amino-1-naphthol and 1,2-naphthoquinone . We have also cloned a mono(ADP-ribosyl)transferase from chicken erythroblasts . This gene has been expressed in E . coli and ADP-ribosylation activity has been demonstrated using histones as substrate . The activity is shown to be arginine-specific by the use of poly-L-arginine as substrate . Use of a specific inhibitor has shown that this enzyme is indeed a mono(ADP-ribosyl)transferase and not a NAD glycohydrolase activity . The sequence of this gene is very similar to several other mono(ADP-ribosyl)transferase genes . There are thus at least three different chicken mono(ADP-ribosyl)transferase genes in the blood system alone; this suggests that there is a quite large family of mono(ADP-ribosyl)transferase genes in animals . We have also isolated the promoter region of this chicken gene and are able to identify several standard motifs in this promoter.

Adv Exp Med Biol, 1997, 412, 405 - 11
Maternal immunization of pregnant cattle with recombinant VP8* protein of bovine rotavirus elicits neutralizing antibodies to multiple serotypes . Colostral neutralizing antibody by rotavirus VP8*; Yoo D et al.; Neonatal calf diarrhoea caused by bovine rotavirus is one of the most common diseases in cattle . VP8 portion of the rotavirus VP4 protein is known to contain neutralizing epitopes and hemagglutination activity . We expressed the VP8* portion of bovine rotavirus strain C486 (G6P1 serotype) in E . coli, and examined potential of the recombinant VP8* protein for induction of neutralizing antibody responses in host animals . One hundred twenty pregnant beef cows were selected and immunized eight weeks prior to parturition with the recombinant VP8* protein . Colostral and milk samples were collected 12 hours and 10 days post-calving, respectively, and the virus neutralizing titers elicited by the recombinant subunit antigen were determined by plaque reduction assay . Colostral antibody titres of the vaccinated group were significantly higher than those of the unvaccinated control group, and these titers were equivalent to the titers elicited by a commercial vaccine . While titers of the control group rapidly decreased to 220 after 10 days of calving, neutralizing titers in the milk of the vaccinees remained 510 . Rabbit and bovine antibodies induced by the recombinant VP8* protein were also able to neutralize bovine rotavirus P5 serotype (B641) at significant level and P11 serotype (B223) moderately . Our results suggest that the E . coli-produced recombinant VP8* protein can be an useful subunit vaccine candidate to prevent rotavirus infection in new-born calves.

Adv Exp Med Biol, 1997, 412, 311 - 6
Pathotypes of bovine verotoxigenic Escherichia coli isolates producing attaching/effacing (AE) lesions in the ligated intestinal loop assay in rabbits; China B et al.; Effacement of the microvilli and intimate attachment to the enterocytes (AE lesions) are two common properties of enteropathogenic (EPEC) and many verotoxigenic (VTEC) E . coli isolates from humans and animals . However not all of the several chromosomal and plasmidic genes and loci involved in the pathogenesis of the human EPEC strain E2348/69 are present in EPEC and VTEC isolates from animal species . We here report that in addition to verotoxin-encoding genes, bovine VTEC isolates harbour a variant of the original eaeA gene, confirming previous results, but neither the eaf nor the hfp loci which are involved in early attachment stage, and that not all of them possess an eaeB gene, as determined by the colony hybridization assay . Have these bovine VTEC isolates lost some of the loci or are they not necessary for the production of AE lesions in vivo? We also report the results of the ligated intestinal loop assay in rabbits with several bovine VTEC isolates . The production of AE lesions was correlated with the presence of an eaeA gene, but not with the presence of an eaeB gene, and was of course independent of the presence of the eaf and bfp loci . The eaeA-negative VTEC isolates produced no AE lesions . Either the eaeB gene is unnecessary for the production of AE lesions in the rabbit ligated intestinal loop assay or bovine VTEC possess other loci coding for similar functions . As to the adhesins involved in the early attachment step of bovine VTEC, they are most probably specific to cattle.

Adv Exp Med Biol, 1997, 412, 303 - 10
Regulators of Escherichia coli K99 region 1 genes; Lo-Tseng T et al.; Expression of K99 is highly regulated being dependent on 8 K99-specific genes and several host-specific genes including cyclic AMP receptor protein (Crp) and leucine responsive protein (Lrp) . The 8 K99-specific genes are organized into 3 separately regulated clusters (regions I-III) with region I and II genes being dependent on Crp . Using TnphoA tagged K99 genes, Lrp was shown to be required for expression of region I genes . Differential methylation of GATC boxes is a common method by which Lrp functions as a regulator . Two GATC boxes are present adjacent to the 5' end of fanA, the first gene . Using the restriction enzymes Dpn I and Mbo I which recognize methylated and non-methylated GATC boxes, respectively, it was shown that differential methylation was not a mechanism regulating K99 expression . Using a gel mobility shift assay and protein extracts from various strains, it was shown that a 625 bp DNA fragment adjacent to the 5' end of fanA bound protein prepared from Lrp+ strains but not from Lrp- strains . While region I genes also require CRP for expression, the same degree of gel shift was observed when the extract was prepared from a Crp- strain . The products of fanA and fanB are believed to be positive regulators . However, protein extracts from strains with or without fanA and fanB caused the same degree of gel shift . Thus, while there are a variety of regulators necessary for region I gene expression, only Lrp or Lrp regulated proteins bind to the promoter region 5' to fanA.

Adv Exp Med Biol, 1997, 412, 295 - 302
Hemolysin phenotypes and genotypes of eaeA-positive and eaeA-negative bovine verotoxigenic Escherichia coli; Sandhu KS et al.; Intimin or EaeA protein has been implicated in the attaching/effacing lesion caused by entero-hemorrhagic Escherichia coli (EHEC) in the intestine but it is not produced by all EHEC and is therefore not adequate as a marker for EHEC . Hemolysins are produced by a high percentage of verotoxigenic E . coli (VTEC) and could be a marker for EHEC, but their distribution and relation to virulence are not known . We used PCR amplification to determine the presence or absence of eaeA sequences in 281 VTEC isolates from the feces of healthy cattle . There were 101 eaeA-positive isolates, which belonged to O groups 5, 26, 69, 80, 84, 98, 103, 111, 119, 145, 157 and 108 eaeA-negative isolates, which belonged to O groups 8, 22, 38, 113, 119, 116, 132, 153, 156 or untypable . All isolates were tested for hemolysis on horse blood agar and on washed sheep blood agar . PCR amplification was used to test for EHEC hemolysin, Ehly1, Ehly2 and alpha-hemolysin D sequences . Among eaeA-positive isolates 98% were positive for EHEC hemolysin sequences and were hemolytic on washed sheep red blood cell agar; the corresponding percentage for eaeA-negative isolates was 36% . Ehly1 and Ehly2 sequences were present in only 11 isolates (O groups 26, 84, 119 and 132) . None of the eaeA-positive and 13 of the eaeA-negative isolates (including all 11 isolates of O group 132) were positive for alpha-hemolysin D gene sequences by PCR and alpha-hemolysin production on horse blood agar . We conclude that since Ehly1, Ehly2 and alpha-hemolysin occur at low frequency among bovine VTEC and serotypes implicated in human disease they are unlikely to be significant virulence factors . In contrast, EHEC hemolysin was present in almost all eaeA-positive VTEC isolates and in approximately one-third the eaeA-negative ones; it may be both a virulence marker and virulence factor but further testing is required . The study identified isolates with all combinations of eaeA and EHEC hly, which may be useful for further testing.

Adv Exp Med Biol, 1997, 412, 241 - 5
Cloning of the RDEC-1 locus of enterocyte effacement (LEE) and functional analysis of the phenotype on HEp-2 cells; Karaolis DK et al.; EPEC Escherichia coli are an important cause of epidemic diarrhea in infants . The disease is characterized by attaching and effacing (A/E) lesions where the bacteria attach intimately to the enterocyte surface resulting in localized destruction of microvilli . A 35-kb chromosomal locus termed LEE (locus of enterocyte effacement) in an EPEC strain (E2348/69) has recently been found and is thought to contain all the necessary genes for A/E lesions . RDEC-1 is a strain of E . coli that causes diarrhea in rabbits by a similar mechanism and serves as a model for human EPEC disease . We report 1) the cloning of the RDEC-1 LEE, 2) show that the RDEC-1 LEE is similar in size to the LEE in E2348/69, 3) the RDEC-1 LEE possesses all four regions of the E2348/69 LEE, 4) there are restriction site polymorphisms between the RDEC-1 LEE and that of E2348/69, and 5) the RDEC-1 LEE clone is functionally similar to E2348/69 in its fluorescent actin staining test and suggests that the LEE may be sufficient for the production of A/E lesions by these strains.

Adv Exp Med Biol, 1997, 412, 175 - 83
Fimbrial colonisation factors F18ab and F18ac of Escherichia coli isolated from pigs with postweaning diarrhea and edema disease; Imberechts H et al.; During the last 5 years at least four new types of colonisation factors have been described in association with porcine postweaning diarrhea and edema disease strains of E . coli . Recently, evidence was presented that these fimbrial factors are closely related to each other, and therefore the common denomination F18 was proposed . Until now, two variants F18ab and F18ac were identified that can be distinguished by serology . Alternatively, to circumvent elaborate growth conditions for the optimal expression of F18 fimbriae in vitro, PCR and subsequent restriction enzyme digestion of the amplification product can be used to differentiate F18ab from F18ac positive isolates . Reports that studied the prevalence of F18 positive E . coli show that this factor is present in about 30% to more than 50% of the PWD or ED strains negative for F4, F5, F6 or F41 . Susceptibility of pigs to colonisation depends on the availability of intestinal receptors, and is under the control of a chromosomal locus . In young pigs susceptibility increases with age . Intestinal infection with F18 positive E . coli induces protection against repeated colonisation with E . coli bearing the homologous or the heterologous fimbrial variant of F18 . Finally, preliminary passive protection studies suggest that F18 antibodies inhibit the colonisation of the pig's intestine by F18ab and F18ac positive strains.

Adv Exp Med Biol, 1997, 412, 167 - 73
A three-receptor model for the interaction of the K88 fimbrial adhesin variants of Escherichia coli with porcine intestinal epithelial cells; Erickson AK et al.; Four phenotypes of pigs distinguished by the variant(s) of K88 fimbrial adhesin (K88ab, K88ac, K88ad) that bind to their intestinal epithelial cells (I-none of the variants, II-K88ad, III-K88ab and K88ac, and IV-all three variants) have been identified . We hypothesize that the differences between the phenotypes are defined by the presence or absence of K88 adhesin receptors . We propose a three-receptor model to account for the observed phenotypes: 1) Receptor bed which binds all three variants and is found in phenotype IV, 2) Receptor be which binds K88ab and K88ac and is found in phenotype III and IV, and 3) Receptor d which binds K88ad and is found in phenotype II . We have identified the be receptor activity as a pair of mucin-type sialoglycoproteins (210 and 240 kDa) . Although neither the bcd nor d receptor has been identified biochemically, their presence has been established using both blocking and receptor localization studies . Blocking studies using phenotype IV brush borders demonstrated that K88ab and K88ac fimbriae block the binding of E . coli expressing any of the K88 variants, but K88ad fimbriae block only K88ad E . coli binding . These results indicate that two receptors (bcd and bc) exist in the phenotype IV animals . Receptor localization studies on intestinal sections from phenotype IV animals showed that K88ab and K88ac adhesin binding is continuous from the crypt to the tip of the villus . The binding of the K88ad adhesin binding is multifocal in phenotype IV pigs, but continuous from crypt to tip of the villus in sections of phenotype II pigs . These studies verify the presence of two receptors (bcd and bc) in phenotype IV animals, and indicate that the K88ad receptor in phenotype IV animals (bcd) is different than in phenotype II animals (d).

Adv Exp Med Biol, 1997, 412, 99 - 102
A chick model for the study of "attaching and effacing Escherichia coli" infection; Sueyoshi M et al.; Young chicks were experimentally infected with 6 strains of AEEC isolated from calves, pigs, chicks, and humans . AEEC colonized the cecum of chicks and induced the AE lesions on the mucosal surface . In the early stages of the AE lesions, AEEC attached to the enterocyte were enfolded with the microvilli . In the advanced stages, microvilli and cytoskeletons of the enterocytes were disrupted, and cytoplasmic cups and pedestal-like protrusions were formed on the cell surface . The AE lesions interconnected with the adjacent lesions, and it formed the network on the mucosal surface . Leukocytes infiltrated in the mucosa associated with AE lesions, and lymphatic nodules also developed . The results of these studies support the conclusion that chicks can be used as a model for the study of the lesions caused by AEEC.

Folia Parasitol (Praha), 1997, 44(1), 71 - 6
Carbohydrate-binding specificities and physico-chemical properties of lectins in various tissue of phlebotominae sandflies; Palanova L et al.; Physico-chemical properties and carbohydrate-binding specificity of hemagglutination activity (HA) were compared in tissue lysates and haemolymph of unfed and bloodfed females of five sandfly species . Sandfly gut lectins were found to be heat-labile, sensitive to dithiotreitol treatment, freezing/thawing procedures and were affected by divalent cations . The pH optimum of HA ranged between 7.0-7.5 . Specificity of gut HA of all species studied was directed towards aminosugars and some glycoconjugates, mainly lipopolysaccharide from Escherichia coli K-235, heparin and fetuin . Gut HA of Phlebotomus papatasi (Scopoli, 1786) was strongly inhibited by lipophosphoglycan (LPG) from Leishmania major promastigotes . In females, that took blood, the HA was higher but the carbohydrate-binding specificity remained the same; this suggests that the same lectin molecule was present, at different levels, both in unfed and fed flies . High HA was found in ovaries of fed females of Lutzomyia longipalpis (Lutz et Nieva, 1912), P . papatasi and P . duboscqi Neveu-Lemaire, 1906 . In P . papatasi and P . duboscqi the HA was present also in the haemolymph and head lysates of both fed and unfed females . Carbohydrate-binding specificity of HA present in these tissues was similar with the gut lectin.

Eur Biophys J, 1997, 25(5-6), 431 - 5
Further analysis of the role of spectrin repeat motifs in alpha-actinin dimer formation; Flood G et al.; Protein constructs consisting of repeats 1-4, repeats 1-3 and repeats 2-4 of the rod domain of chicken alpha-actinin were expressed as fusion proteins in Escherichia coli . Based on the evidence of circular dichroism spectra and cooperative thermal unfolding profiles both truncated rod fragments were judged to have assumed the native structural fold . The thermal stabilities were in both cases significantly lower than that of the intact rod (repeats 1-4) . Analyses by sedimentation equilibrium and velocity provided further evidence to show that fragment 1-4 is entirely dimeric in the concentration range of these experiments, resembling therefore the rod domain isolated by proteolytic digestion of native alpha-actinin . Fragment 2-4, and probably also 1-3, show concentration-dependent association, with dissociation constants, estimated by sedimentation equilibrium, in the 1-10 microM range . Thus, in confirmation of earlier work, all four repeats are required to generate a maximally stable anti-parallel dimer (Kd approximately 10 pM), suggesting the presence of binding sites in all of them to allow for aligned pairing.

Mol Immunol, 1997 Jan, 34(1), 9 - 20
Isolation of anti-mesothelin antibodies from a phage display library; Chowdhury PS et al.; Phage display has been used to select single-chain Fvs (scFvs) against mesothelin, a surface antigen present on mesothelial cells as well as mesotheliomas and non-mucinous ovarian cancers . Several attempts to produce anti-mesothelin hybridomas from spleen cells of mice immunized with recombinant mesothelin were unsuccessful . This report describes the isolation of anti-mesothelin scFvs from a phage display library made from the mRNA of the same spleens . Panning on recombinant antigen produced in E . coli or on antigen positive cells was employed . Several scFvs which bind specifically to mesothelin were isolated . Panning on recombinant antigen yielded five different scFvs . Panning on cells selected two different scFvs which also differ from the scFvs selected by recombinant antigen . The heavy chains of the scFvs selected on recombinant antigen are derived from four different heavy chain gene families and the scFvs selected on cells are derived from two of these families . In contrast, the light chains of all of these scFvs are derived from family XI . Moreover, the light chains of the two scFvs selected on cells are very similar to the light chains of two of the scFvs selected by panning on recombinant mesothelin except for a few point mutations . One of these scFvs which have been studied in detail has been shown to bind specifically to mesothelin positive transfected cells.

Arch Virol, 1997, 142(4), 795 - 805
Foot-and-mouth disease virus-infected but not vaccinated cattle develop antibodies against recombinant 3AB1 nonstructural protein; Silberstein E et al.; Foot-and-mouth disease (FMD) vaccines induce antibodies against structural and some nonstructural proteins present in vaccine preparations . To differentiate between FMDV-infected and vaccinated animals, we developed immunochemical assays capable of detecting antibodies against a FMDV nonstructural protein . Recombinant nonstructural 3AB1 protein was expressed in E.coli and in insect cells and used to detect anti-3AB1 antibodies . ELISA and Western blot analysis showed that sera from cattle infected with FMDV reacted with recombinant 3AB1 protein whereas sera from cattle which had been vaccinated against FMDV, mock-infected, or infected with different bovine viruses did not recognize the 3AB1 protein . In contrast, anti-virus infection associated antigen (VIAA) antibodies were present in both FMDV-infected and vaccinated animals . Detection of anti-3AB1 antibodies in sera of experimentally infected cattle obtained between 7 and 560 days postinfection indicated that immunological tests based on the detection of recombinant 3AB1 protein could be used for the diagnosis of FMDV infection.

Arch Virol, 1997, 142(4), 781 - 93
Serological comparison of tospoviruses with polyclonal antibodies produced against the main structural proteins of tomato spotted wilt virus; Feldhoff A et al.; A new purification procedure for the tospoviruses of serogroups II and III was developed . SDS-polyacrylamide gel electrophoresis of purified tomato spotted wilt virus (TSWV; serogroup I), groundnut ringspot virus (GRSV: serogroup II), tomato chlorotic spot virus (TCSV; serogroup II) and impatiens necrotic spot virus (INSV; serogroup III) showed that the glycoprotein G2 of serogroup II members differs significantly in size from that of the serogroup I virus . Western immunoblot analysis using polyclonal antisera produced against purified glycoproteins TSWV-G1 and G2 as well as peptides of hydrophilic sequences of TSWV-G1 and G2 expressed in Escherichia coli demonstrated a higher homology amongst G1 of different serogroups than for G2 . These results are supported by comparing the glycoprotein gene sequences of different serogroups.

Plasmid, 1997, 37(2), 155 - 8
An expression vector encoding a lacZ reporter gene facilitates identification of stable, high-producing CHO cell clones; Grossman TH et al.; We describe a vector and a streamlined procedure for isolating high-producing stable mammalian cell transfectants . The vector encodes the Escherichia coli lacZ gene as a reporter . We show that levels of beta-galactosidase activity, assayed in situ in clonal isolates, can be used to identify clones producing high levels of the protein of interest (in this case, soluble human CD4 protein).

Int Rev Cytol, 1997, 174, 127 - 93
The role of molecular chaperones in mitochondrial protein import and folding; Ryan MT et al.; Molecular chaperones play a critical role in many cellular processes . This review concentrates on their role in targeting of proteins to the mitochondria and the subsequent folding of the imported protein . It also reviews the role of molecular chaperons in protein degradation, a process that not only regulates the turnover of proteins but also eliminates proteins that have folded incorrectly or have aggregated as a result of cell stress . Finally, the role of molecular chaperones, in particular to mitochondrial chaperonins, in disease is reviewed . In support of the endosymbiont theory on the origin of mitochondria, the chaperones of the mitochondrial compartment show a high degree of similarity to bacterial molecular chaperones . Thus, studies of protein folding in bacteria such as Escherichia coli have proved to be instructive in understanding the process in the eukaryotic cell . As in bacteria, the molecular chaperone genes of eukaryotes are activated by a variety of stresses . The regulation of stress genes involved in mitochondrial chaperone function is reviewed and major unsolved questions regarding the regulation, function, and involvement in disease of the molecular chaperones are identified.

Microbiol Immunol, 1997, 41(4), 371 - 6
Antibodies are generated during infection to Coxiella burnetii macrophage infectivity potentiator protein (Cb-Mip); Seshu J et al.; Antisera from rabbits immunized with formalin inactivated Coxiella burnetii isolates associated with either acute (Nine Mile, phase I or phase II) or chronic (Priscilla) Q fever showed reactivity to a C . burnetii macrophage infectivity potentiator protein (Cb-Mip) cloned in Escherichia coli . Further, antisera generated in BALB/c mice after infection with live Nine Mile phase I or Priscilla isolates also showed reactivity to Cb-Mip by immunoblot analysis . In addition, human serum from an individual with previous serological and clinical evidence of Q fever showed reactivity to Cb-Mip . This study indicates that Cb-Mip is immunogenic in both experimental and natural infections, and is the first report on the presence of antibodies to Mip/Mip-like proteins of intracellular bacteria in human sera . Cb-Mip may serve as a potential target antigen for developing recombinant vaccines or diagnostic assays for Q fever.

Eur J Hum Genet, 1997 Jan-Feb, 5(1), 15 - 21
Characterisation of five missense mutations in the cystathionine beta-synthase gene from three patients with B6-nonresponsive homocystinuria; Dawson PA et al.; Homocystinuria, due to a deficiency of the enzyme cystathionine beta-synthase (CBS), is an inborn error of sulphur-amino acid metabolism . This is an autosomal recessive disease which results in hyperhomocysteinaemia and a wide range of clinical features, including optic lens dislocation, mental retardation, skeletal abnormalities and premature thrombotic events . We report the identification of 5 missense mutations in the protein-coding region of the CBS gene from 3 patients with pyridoxine-nonresponsive homocystinuria . Reverse-transcription PCR was used to amplify CBS cDNA from each patient and the coding region was analysed by direct sequencing . The mutations detected included 3 novel (1058C-->T, 992C-->A and 1316G-->A) and 2 previously identified (430G-->A and 833C-->T) base alterations in the CBS cDNA . Each of these mutations predicts a single amino acid substitution in the CBS polypeptide . Appropriate cassettes of patient CBS cDNA, containing each of the above defined mutations, were used to replace the corresponding cassettes of normal CBS cDNA sequence within the bacterial expression vector pT7-7 . These recombinant mutant and normal CBS constructs were expressed in Escherichia coli cells and the catalytic activities of the mutant proteins were compared with normal . All of the mutant proteins exhibited decreased catalytic activity in vitro, which confirmed the association between the individual mutation and CBS dysfunction in each patient.

J Basic Microbiol, 1997, 37(2), 105 - 14
The metabolism of gluconate in Escherichia coli . The subsidiary system and the nature of the gntS gene; Isturiz T et al.; The transport and phosphorylation of gluconate in E . coli occurs through two systems (GntI and GntII) which duplicate activities . bioH-asd deletion mutants do not grow on media with gluconate as sole carbon source because they lack the GntI system and do not express GntII . Although E . coli c177 is a delta (bioH-asd) mutant, it carries the pyrB linked mutation gnt177 that enables it to metabolize this substrate through inducible expression of the GntII system . Several gntS derivatives which are unable to grow on gluconate were isolated from E . coli C177 by spontaneous curing of the transposon Tn10 previously inserted at the gntS locus (zjf::Tn10, min 95.3) . A representative gntS mutant, E . coli TI141A retained the ability to take up gluconate but had lost the thermosensitive gluconokinase activity (gene gntV, min 96.9) . Furthermore, it could be demonstrated that gntV is repressed in E . coli TI141A . The results indicate that gntS might specify a trans-acting positive regulator involved in the control of at least the expression of the thermosensitive gluconokinase (GntII), instead of a gluconate uptake system as it was previously postulated . Likewise, these results can be used to reconsider whether the locus altered by the gnt177 lesion is allelic with that of the GntII permease instead of a regulator, as it was originally postulated.

Endothelium, 1997, 5(1), 21 - 35
Optimization of transfection of human endothelial cells; Teifel M et al.; Recently developed transfection methods for mammalian cells provide a powerful means for the study of gene function . Unfortunately, human endothelial cells were relative refractory to the classic transfection techniques . In this study we compared the usability of calcium phosphate, DEAE-dextran transfection, transferrinfection, lipofection, and electroporation for the transfection of early passage HUVECs and for the human endothelial cell lines ECV 304 and EA.hy 926 . The classic transfection methods resulted in no or only marginal expression of the reporter gene E . coli beta-galactosidase . For lipofection experiments we compared the commercially available liposome formulations DOTAP and Transfectam with liposomes prepared of dimethyldioctadecylammoniumbromide (DDAB) or 1,2-dimyristyloxypropyl-3-dimethylhydroxyethyl ammonium bromide (DMRIE) as the cationic lipid compound and dioleylphosphatidylethanolamine (DOPE) or Azolectin (a crude fraction of soybean lipids, commercially available as phosphatidylcholine II) as neutral co-lipid . Because the protocol for the chemical synthesis of DMRIE has not been published yet, we developed a protocol for the chemical synthesis of this cationic lipid . With transfection protocols optimized for each cell line, we could achieve transfection efficiencies up to 2% . Compared to the other methods used, the lipofection proved to be a reliable technique for the efficient transfection of the human endothelial cell lines ECV 304 and EA.hy 926 . Although we achieved a maximum transfection efficiency of 0.45% for the lipofection of HUVEC, the electroporation seemed to be the better choice for these cells.

Environ Mol Mutagen, 1997, 29(3), 221 - 9
Sources of variability in mutant frequency determinations in different organs of lacZ plasmid-based transgenic mice: experimental features and statistical analysis; Boerrigter ME et al.; Sources of variability that may affect the measurement of mutant frequencies in lacZ plasmid-based transgenic mice, including rescue-to-rescue, animal-to-animal (within organ), and organ-to-organ, were studied in the context of some of the experimental features that distinguish this model from other currently used systems . Statistical analysis of repeated determinations of DNA from kidney, spleen, liver, lung, and brain indicated that 350,000 colony-forming units from each of three animals should be analyzed per treatment group in order to detect a 50% difference in mutant frequencies . Consideration is given to some of the experimental features of the transgenic assay system, including its positive selection system, its rescue efficiency, the ability to detect large deletions in the lacZ target genes, the contribution of Escherichia coli-derived mutations to the spontaneous mutant frequencies observed in vivo, and cost effectiveness of mutant frequency determinations.

Bioorg Khim, 1997 Jan, 23(1), 18 - 20
{Integration of IS186 element into the -10-region of the promoter of heat shock lon-gene in Escherichia coli}; Ignatov KB et al.; The insertion element IS186 was found to be incorporated into the -10 region of the promoter of the E . coli lon gene . The integration represses lon gene expression . Homology between the integration sites of IS186, the -10 region of E . coli heat shock promoters, and the terminal inverted repeat of IS186 was revealed.

Fold Des, 1997, 2(2), 89 - 92
Detecting native-like properties in combinatorial libraries of de novo proteins; Roy S et al.; BACKGROUND: Combinatorial methods based on binary patterning of polar and nonpolar residues have been used to generate large libraries of de novo alpha-helical proteins . Within such libraries, the ability to find structures that resemble natural proteins requires a rapid method to sort through large collections of proteins and detect those possessing 'native-like' features . The current paper presents such a method and applies it to an initial collection of de novo proteins . RESULTS: We present a method to identify proteins with native-like properties from libraries of de novo sequences expressed in vivo . A novel 'rapid prep' freeze/thaw procedure was used to prepare samples; chromatographic purification was not required . The semi-crude samples were analyzed for native-like features by one-dimensional 1H NMR spectroscopy . Using this method, we demonstrate that native-like features can readily be observed for several proteins among a collection of sequences designed by binary patterning of polar and nonpolar amino acids . CONCLUSIONS: Native-like properties can be detected using a method that requires neither isotopic enrichment nor chromatographic purification . The method is inexpensive, rapid, and suitable for parallel processing . It can therefore be employed to screen for native-like properties among large collections of de novo sequences . Using this method, we demonstrate that although the binary code strategy does not explicitly design tertiary packing, it can nonetheless generate proteins that possess native-like properties . The use of combinatorial methods to produce large collections of proteins coupled with the availability of a rapid assay for detecting native-like properties will facilitate the design and isolation of novel proteins with desirable properties.

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