Escherichia coli

Biochim Biophys Acta, 1998 Apr 23, 1384(1), 121 - 9
Functional implications of the 21-24 loop in recombinant prochymosin; Li H et al.; To investigate the role of the 21-24 (pepsin numbering) loop in prochymosin, the amino acid residues GTPP at positions 21 through 24 were replaced with GG, the equivalent loop residues from its homologous protein, penicillopepsin, or SG, GS by site-directed mutagenesis . The mutants except GTPP(21-24)GS could be expressed in Escherichia coli . Activation studies indicated that the refolded prochymosin mutants were capable of undergoing autocatalytic activation to produce pseudochymosin by cleaving its N-terminal 27 amino acid residues at pH 2 . The resulting pseudochymosin mutants were able to convert into chymosin at pH 5.5 by further autocatalytic cleavage to remove additional 15 amino acid residues . These results demonstrate that the prochymosin analogs can fold into an active state from an unfolded state and that the pseudochymosin analogs can proceed in the transformation from one active form into another active form . Spectroscopic analyses revealed that after mutation the far UV CD spectrum of prochymosin was considerably modified, showing less negative ellipticity values, and the fluorescence emission intensities of prochymosin and pseudochymosin were remarkably reduced . The stabilities of prochymosin and pseudochymosin, especially, were dramatically decreased . The stabilization energy of prochymosin was reduced by 7-8 kJ/mol . The inactivation temperature of pseudochymosin was decreased by 15-20 degrees C . The wild-type pseudochymosin was stable at pH 1.5 and 6.5, whereas the mutants were completely inactivated at the same pH values . Taken together, it is reasonable to conclude that the 21-24 loop (GTPP) plays an important role in determining the stability of prochymosin and pseudochymosin, although the mutants with mutated loop (GG or SG) still can refold into an active conformation.

Infect Immun, 1998 Jun, 66(6), 2905 - 13
Signal transduction during Legionella pneumophila entry into human monocytes; Coxon PY et al.; Legionella pneumophila causes Legionnaires' disease by replication in alveolar macrophages and monocytes . The bacteria are internalized most efficiently by opsonin-dependent, CR3-mediated phagocytosis . This investigation focused on determining the role of actin polymerization and phosphorylation signals in this uptake mechanism . Uptake inhibition assays and confocal microscopic analysis indicated that entry of L . pneumophila activated tyrosine kinase (TK) and protein kinase C (PKC) and induced actin polymerization at the site of bacterial entry . Upon L . pneumophila entry, six major cellular proteins (75, 71, 59, 56, 53, and 52 kDa) were TK phosphorylated in soluble fractions of monocytes, and three of these proteins (52, 53, and 56 kDa) were consistently found in insoluble (i.e., cytoskeletal) fractions of monocytes as well . Tyrosine phosphorylation was suppressed when cells were pretreated with the kinase inhibitor genistein, tyrphostin, or staurosporine . A similar tyrosine-phosphorylated protein pattern was observed with CR3-mediated entry of avirulent L . pneumophila, Escherichia coli, or zymosan into monocytes . This study has shown that PKC and TK signals which activate actin polymerization during the process of phagocytosis are induced upon L . pneumophila entry . In addition, CR3 receptor-mediated phagocytosis into monocytes may involve tyrosine phosphorylation of similar proteins, regardless of the particle being phagocytosed . Therefore, the tyrosine-induced phosphorylation observed during opsonized L . pneumophila entry is not a virulence-associated event.

Infect Immun, 1998 Jun, 66(6), 2879 - 86
Rectal and intranasal immunizations with recombinant urease induce distinct local and serum immune responses in mice and protect against Helicobacter pylori infection; Kleanthous H et al.; To determine the optimal inductive sites for immunization against Helicobacter pylori infection, the protective efficacy of recombinant urease (rUre) was assessed for mice given the vaccine by either the oral (p.o.), intranasal (i.n.), or rectal route . When mice were immunized with rUre (25 microg p.o . or rectally or 10 microg i.n.) plus heat-labile toxin from Escherichia coli as the mucosal adjuvant, all routes afforded protection against challenge with H . pylori, as indicated by a significant reduction in gastric urease activity (P < 0.0005) compared to that of sham-immunized controls . Quantitative H . pylori culture of stomach tissue demonstrated a >97% reduction in bacterial burden in mice immunized by all routes (P < 0.05) . Induction of antiurease immunoglobulin A (IgA) levels in gastric luminal secretions after p.o . immunization was greater than after i.n . administration (means, 6.0 and 1.02 ng/ml, respectively) and was dependent upon challenge with H . pylori . However, immunization by the rectal route resulted in the generation of the highest levels of gastric antiurease IgA (mean, 40 . 89 ng/ml), which was detectable prior to challenge with H . pylori . Immunohistochemical staining of stomach tissue for cells secreting urease-specific antibody and CD4(+) T cells showed levels of recruitment to be dependent upon challenge with H . pylori and equivalent for all routes . These results identify both the rectum and nasal passages as suitable inductive sites for urease immunization.

Infect Immun, 1998 Jun, 66(6), 2782 - 90
Expanded CD14+ CD16+ monocyte subpopulation in patients with acute and chronic infections undergoing hemodialysis; Nockher WA et al.; Infections are frequent complications in end-stage renal failure patients undergoing hemodialysis (HD), and peripheral blood monocytes are important cells in host defense against infections . The majority of circulating monocytes express high levels of lipopolysaccharide receptor antigen CD14 and are negative for the immunoglobulin Fcgamma receptor type III (CD16) . We studied the occurrence of a minor subpopulation coexpressing low levels of CD14 together with CD16 in HD patients . In healthy controls CD14+ CD16+ monocytes account for 8% +/- 4% of CD14+ monocytes, with an absolute number of 29 +/- 14 cells/microl . In stable HD patients the CD14+ CD16+ subpopulation was significantly elevated (14% +/- 3%, or 66 +/- 28 cells/microl), while the number of CD14(++) monocytes (monocytes strongly positive for CD14) remained constant . In HD patients suffering from chronic infections a further rise in CD14+ CD16+ monocytes was observed (128 +/- 71 cells/microl; P < 0.01) such that this subpopulation constituted 24% of all blood monocytes . In contrast, numbers of CD14++ cells did not change compared to those for stable HD patients, indicating that the CD14+ CD16+ monocyte subpopulation was selectively expanded . During acute infections the CD14+ CD16+ cell subpopulation always expanded . A whole-blood assay revealed that CD14+ CD16+ monocytes exhibited a higher phagocytosis rate for Escherichia coli bacteria than CD14++ monocytes, underlining their role during host defense . In addition, CD14+ CD16+ monocytes expressed higher levels of major histocompatibility complex (MHC) class II antigens (HLA-DR, -DP, and -DQ) and equal amounts of MHC class I antigens (HLA-ABC) . Thus, CD14+ CD16+ cells constitute a potent phagocytosing and antigen-presenting monocyte subpopulation, which is expanded during acute and chronic infections commonly observed in chronic HD patients.

Infect Immun, 1998 Jun, 66(6), 2655 - 9
Differential effects of myeloperoxidase-derived oxidants on Escherichia coli DNA replication; Rosen H et al.; The microbicidal myeloperoxidase (MPO)-H2O2-chloride system strongly inhibits Escherichia coli DNA synthesis . Also, cell envelopes from MPO-treated E . coli cells lose their ability to interact with hemimethylated DNA sequences of oriC, the chromosomal origin of replication, raising the prospect that suppression of DNA synthesis involves impairment of oriC-related functions (H . Rosen, et al . Proc . Natl . Acad . Sci . USA, 87:10048-10052, 1990) . To evaluate whether origin-specific DNA sequences play a role in the MPO effect on E . coli DNA synthesis, plasmid DNA replication was compared to total (chromosomal) DNA replication for six plasmids with three distinct origins of replication . Plasmid pCM700 replication, replicating from oriC, was as sensitive to MPO-mediated inhibition as was total (chromosomal) DNA replication . A regression line describing this relationship had a slope of 0.90, and the r2 was 0.89 . In contrast, the replication activities of three of four non-oriC plasmids, pUC19, pACYC184, and pSC101, demonstrated significant early resistance to inhibition by MPO-derived oxidants . The exception to this resistance pattern was plasmid pSP102, which has an origin derived from P1 phage . pSP102 replication declined similarly to that of total DNA synthesis . The regression line for pSP102 replication versus total DNA synthesis had a slope of 0.95, and the r2 was 0.92 . The biochemical requirements for P1-mediated replication are strikingly similar to those for oriC-mediated replication . It is proposed that one of these requirements, common to oriC and the P1 origin but not critical to the replication of the other non-oriC plasmids, is an important target for MPO-mediated oxidations that mediate the initial decline in E . coli chromosomal DNA synthesis.

Infect Immun, 1998 Jun, 66(6), 2576 - 86
Molecular characterization of Mycoplasma arthritidis variable surface protein MAA2; Washburn LR et al.; Earlier studies implied a role for Mycoplasma arthritidis surface protein MAA2 in cytadherence and virulence and showed that it exhibited both size and phase variability . Here we report the further analysis of MAA2 and the cloning and sequencing of the maa2 gene from two M . arthritidis strains, 158p10p9 and H606, expressing two size variants of MAA2 . Triton X-114 partitioning and metabolic labeling with {3H}palmitic acid suggested lipid modification of MAA2 . Surface exposure of the C terminus was indicated by cleavage of monoclonal antibody-specific epitopes from intact cells by carboxypeptidase Y . The maa2 genes from both strains were highly conserved, consisting largely of six (for 158p10p9) or five (for H606) nearly identical, 264-bp tandem direct repeats . The deduced amino acid sequence predicted a largely hydrophilic, highly basic protein with a 29-amino-acid lipoprotein signal peptide . The maa2 gene was expressed in Escherichia coli from the lacZ promoter of vector pGEM-T . The recombinant product was approximately 3 kDa larger than the native protein, suggesting that the signal peptide was not processed in E . coli . The maa2 gene and upstream DNA sequences were cloned from M . arthritidis clonal variants differing in MAA2 expression state . Expression state correlated with the length of a poly(T) tract just upstream of a putative -10 box . Full-sized recombinant MAA2 was expressed in E . coli from genes derived from both ON and OFF expression variants, indicating that control of expression did not include alterations within the coding region.

Infect Immun, 1998 Jun, 66(6), 2494 - 500
Escherichia coli cytotoxic necrotizing factor 1 effaces microvilli and decreases transmigration of polymorphonuclear leukocytes in intestinal T84 epithelial cell monolayers; Hofman P et al.; Cytotoxic necrotizing factor type 1 (CNF1), a 110-kDa toxin-like protein from pathogenic Escherichia coli strains, induces an actin cytoskeleton reorganization consisting of the formation of prominent stress fibers by permanent activation of the small GTP-binding protein Rho . Since p21Rho regulates tight-junction permeability and perijunctional actin reorganization in epithelial intestinal cells (A . Nusrat, M . Giry, J . R . Turner, S . P . Colgan, C . A . Parkos, E . Lemichez, P . Boquet, and J . L . Madara, Proc . Natl . Acad . Sci . USA 92:10629-10633, 1995), we used polarized T84 epithelial intestinal cell monolayers to examine whether CNF1 could affect microvillus structure, transepithelial resistance, and polymorphonuclear leukocyte (PMN) transmigration . Incubation of T84 cells with CNF1 did not influence transepithelial resistance, suggesting that barrier function and surface polarity were not affected by the toxin . However, CNF1 effaced intestinal cell microvilli and induced a strong decrease of PMN transepithelial migration in either the luminal-to-basolateral or the basolateral-to-luminal direction . CNF1 could thus be a virulence factor exhibiting a new type of combined activity consisting of effacing of microvilli and occlusion of the epithelial barrier to PMNs . Attenuated transepithelial migration of PMNs could result in the enhanced growth and protection of luminal bacteria.

Genes Dev, 1998 May 15, 12(10), 1425 - 37
clr-1 encodes a receptor tyrosine phosphatase that negatively regulates an FGF receptor signaling pathway in Caenorhabditis elegans; Kokel M et al.; Receptor tyrosine phosphatases have been implicated in playing important roles in cell signaling events by their ability to regulate the level of protein tyrosine phosphorylation . Although the catalytic activity of their phosphatase domains has been well established, the biological roles of these molecules are, for the most part, not well understood . Here we show that the Caenorhabditis elegans protein CLR-1 (CLeaR) is a receptor tyrosine phosphatase (RTP) with a complex extracellular region and two intracellular phosphatase domains . Mutations in clr-1 result in a dramatic Clr phenotype that we have used to study the physiological requirements for the CLR-1 RTP . We show that the phosphatase activity of the membrane-proximal domain is essential for the in vivo function of CLR-1 . By contrast, we present evidence that the membrane-distal domain is not required to prevent the Clr phenotype in vivo . The Clr phenotype of clr-1 mutants is mimicked by activation of the EGL-15 fibroblast growth factor receptor (FGFR) and is suppressed by mutations that reduce or eliminate the activity of egl-15 . Our data strongly indicate that CLR-1 attenuates the action of an FGFR-mediated signaling pathway by dephosphorylation.

Gene, 1998 Apr 28, 211(1), 57 - 62
Cloning of three Caenorhabditis elegans genes potentially encoding novel matrix metalloproteinases; Wada K et al.; Three genes potentially encoding novel matrix metalloproteinases (MMPs) were identified by sequence similarity searching of Caenorhabditis elegans genome database, and cDNAs for these MMPs were cloned . The predicted gene products (MMP-C31,-H19 and -Y19) display a similar domain organization to human MMPs . MMP-H19 and -Y19 are unique in that they have an RXKR motif between the propeptide and catalytic domains that is a furin-like cleavage site, and conserved only in stromelysin-3 and membrane-type MMPs . The amino acid sequence homology with MMP-1/human interstitial collagenase at the catalytic domain is 45%, 34% and 23% for MMP-C31, -H19 and -Y19, respectively . Recombinant proteins of C . elegans MMPs cleaved an MMP peptide substrate with efficiency proportional to their amino acid homology with human MMPs . Digestion of gelatin was observed only with MMP-C31 . Enzyme activity of MMP-C31 and -H19 was inhibited by human tissue inhibitor of MMPs (TIMP)-1, TIMP-2 and synthetic MMP inhibitors, BB94 and CT543, indicating that the catalytic sites of these C . elegans MMPs are structurally closely related with those of mammalian MMPs.

Genetics, 1998 May, 149(1), 7 - 16
Single-strand DNA-specific exonucleases in Escherichia coli . Roles in repair and mutation avoidance; Viswanathan M et al.; Mutations in the genes encoding single-strand DNA-specific exonucleases (ssExos) of Escherichia coli were examined for effects on mutation avoidance, UV repair, and conjugational recombination . Our results indicate complex and partially redundant roles for ssExos in these processes . Although biochemical experiments have implicated RecJ exonuclease, Exonuclease I (ExoI), and Exonuclease VII (ExoVII) in the methyl-directed mismatch repair pathway, the RecJ- ExoI- ExoVII- mutant did not exhibit a mutator phenotype in several assays for base substitution mutations . If these exonucleases do participate in mismatch excision, other exonucleases in E . coli can compensate for their loss . Frameshift mutations, however, were stimulated in the RecJ- ExoI- ExoVII- mutant . For acridine-induced frameshifts, this mutator effect was due to a synergistic effect of ExoI- and ExoVII- mutations, implicating both ExoI and ExoVII in avoidance of frameshift mutations . Although no single exonuclease mutant was especially sensitive to UV irradiation, the RecJ- ExoVII- double mutant was extremely sensitive . The addition of an ExoI- mutation augmented this sensitivity, suggesting that all three exonucleases play partially redundant roles in DNA repair . The ability to inherit genetic markers by conjugation was reduced modestly in the ExoI- RecJ- mutant, implying that the function of either ExoI or RecJ exonucleases enhances RecBCD-dependent homologous recombination.

J Biol Chem, 1998 May 22, 273(21), 13307 - 12
Ada protein-RNA polymerase sigma subunit interaction and alpha subunit-promoter DNA interaction are necessary at different steps in transcription initiation at the Escherichia coli Ada and aidB promoters; Landini P et al.; The methylated form of the Ada protein (meAda) binds the ada and aidB promoters between 60 and 40 base pairs upstream from the transcription start and activates transcription of the Escherichia coli ada and aidB genes . This region is also a binding site for the alpha subunit of RNA polymerase and resembles the rrnB P1 UP element in A/T content and location relative to the core promoter . In this report, we show that deletion of the C-terminal domain of the alpha subunit severely decreases meAda-independent binding of RNA polymerase to ada and aidB, affecting transcription initiation at these promoters . We provide evidence that meAda activates transcription by direct interaction with the C-terminal domain of RNA polymerase sigma70 subunit (amino acids 574-613) . Several negatively charged residues in the sigma70 C-terminal domain are important for transcription activation by meAda; in particular, a glutamic acid to valine substitution at position 575 has a dramatic effect on meAda-dependent transcription . Based on these observations, we propose that the role of the alpha subunit at ada and aidB is to allow initial binding of RNA polymerase to the promoters . However, transcription initiation is dependent on meAda-sigma70 interaction.

J Biol Chem, 1998 May 22, 273(21), 13264 - 72
Assembly of iron-sulfur clusters . Identification of an iscSUA-hscBA-fdx gene cluster from Azotobacter vinelandii; Zheng L et al.; An enzyme having the same L-cysteine desulfurization activity previously described for the NifS protein was purified from a strain of Azotobacter vinelandii deleted for the nifS gene . This protein was designated IscS to indicate its proposed role in iron-sulfur cluster assembly . Like NifS, IscS is a pyridoxal-phosphate containing homodimer . Information gained from microsequencing of oligopeptides obtained by tryptic digestion of purified IscS was used to design a strategy for isolation and DNA sequence analysis of a 7,886-base pair A . vinelandii genomic segment that includes the iscS gene . The iscS gene is contained within a gene cluster that includes homologs to nifU and another gene contained within the major nif cluster of A . vinelandii previously designated orf6 . These genes have been designated iscU and iscA, respectively . Information available from complete genome sequences of Escherichia coli and Hemophilus influenzae reveals that they also encode iscSUA gene clusters . A wide conservation of iscSUA genes in nature and evidence that NifU and NifS participate in the mobilization of iron and sulfur for nitrogenase-specific iron-sulfur cluster formation suggest that the products of the iscSUA genes could play a general role in the formation or repair of iron-sulfur clusters . The proposal that IscS is involved in mobilization of sulfur for iron-sulfur cluster formation in A . vinelandii is supported by the presence of a cysE-like homolog in another gene cluster located immediately upstream from the one containing the iscSUA genes . O-Acetylserine synthase is the product of the cysE gene, and it catalyzes the rate-limiting step in cysteine biosynthesis . A similar cysE-like gene is also located within the nif gene cluster of A . vinelandii . The likely role of such cysE-like gene products is to increase the cysteine pool needed for iron-sulfur cluster formation . Another feature of the iscSUA gene cluster region from A . vinelandii is that E . coli genes previously designated as hscB, hscA, and fdx are located immediately downstream from, and are probably co-transcribed with, the iscSUA genes . The hscB, hscA, and fdx genes are also located adjacent to the iscSUA genes in both E . coli and H . influenzae . The E . coli hscA and hscB gene products have previously been shown to bear primary sequence identity when respectively compared with the dnaK and dnaJ gene products and have been proposed to be members of a heat-shock-cognate molecular chaperone system of unknown function . The close proximity and apparent co-expression of iscSUA and hscBA in A . vinelandii indicate that the proposed chaperone function of the hscBA gene products could be related to the maturation of iron-sulfur cluster-containing proteins . Attempts to place non-polar insertion mutations within either A . vinelandii iscS or hscA revealed that such mutations could not be stably maintained in the absence of the corresponding wild-type allele . These results reveal a very strong selective pressure against the maintenance of A . vinelandii iscS or hscA knock-out mutations and suggest that such mutations are either lethal or highly deleterious . In contrast to iscS or hscA, a strain having a polar insertion mutation within the cysE-like gene was readily isolated and could be stably maintained . These results show that the cysE-like gene located upstream from iscS is not essential for cell growth and that the cysE-like gene and the iscSUA-hscBA-fdx genes are contained within separate transcription units.

J Biol Chem, 1998 May 22, 273(21), 13255 - 63
Rrp6p, the yeast homologue of the human PM-Scl 100-kDa autoantigen, is essential for efficient 5.8 S rRNA 3' end formation; Briggs MW et al.; The eukaryotic 25 S, 18 S, and 5.8 S rRNAs are synthesized as a single transcript with two internal transcribed spacers (ITS1 and ITS2), which are removed by endo- and exoribonucleolytic steps to produce mature rRNA . Genetic selection for suppressors of a polyadenylation defect yielded two cold-sensitive alleles of a gene that we named RRP6 (ribosomal RNA processing) . Molecular cloning of RRP6 revealed its homology to a 100-kDa human, nucleolar PM-Scl autoantigen and to Escherichia coli RNase D, a 3'-5' exoribonuclease . Recessive mutations in rrp6 result in the accumulation of a novel 5 . 8 S rRNA processing intermediate, called 5.8 S*, which has normal 5' ends, but retains approximately 30 nucleotides of ITS2 . Pulse-chase analysis of 5.8 S rRNA processing in an rrp6- strain revealed a precursor-product relationship between 5.8 S* and 5.8 S rRNAs, suggesting that Rrp6p plays a role in the removal of the last 30 nucleotides of ITS2 from 5.8 S precursors . A portion of 5.8 S* rRNA assembles into 60 S ribosomes which form polyribosomes, suggesting that they function in protein synthesis . These findings indicate that Rrp6p plays a role in 5.8 S rRNA 3' end formation, and they identify a functional intermediate in the rRNA processing pathway.

J Biol Chem, 1998 May 22, 273(21), 13230 - 5
Characterization of recombinant human fibroblast growth factor (FGF)-10 reveals functional similarities with keratinocyte growth factor (FGF-7); Igarashi M et al.; A newly identified member of the fibroblast growth factor (FGF) family, designated FGF-10, is expressed during development and preferentially in adult lung . The predicted FGF-10 protein is most related to keratinocyte growth factor (KGF, or FGF-7) . The latter is unique among FGFs in that it binds and signals only through the FGF receptor (FGFR2b) isoform KGF receptor (KGFR) expressed specifically by epithelial cells . In order to examine the biological and biochemical properties of human FGF-10, we isolated the cDNA and expressed its encoded protein in bacteria . The recombinant protein (rFGF-10) was a potent mitogen for Balb/MK mouse epidermal keratinocytes with activity detectable at 0.1 nM and maximal at around 5 nM . Within this concentration range, FGF-10 did not stimulate DNA synthesis in NIH/3T3 mouse fibroblasts . rFGF-10 bound the KGFR with high affinity comparable to that of KGF, and did not bind detectably to either the FGFR1c (Flg) or FGFR2c (Bek) receptor isoforms . The mitogenic activity of FGF-10 could be distinguished from that of KGF by its different sensitivity to heparin and lack of neutralization by a KGF monoclonal antibody . These results indicate that FGF-10 and KGF have similar receptor binding properties and target cell specificities, but are differentially regulated by components of the extracellular matrix.

J Biol Chem, 1998 May 22, 273(21), 13037 - 46
Identification of a nickel(II) binding site on hemoglobin which confers susceptibility to oxidative deamination and intramolecular cross-linking; Levine J et al.; Complexation of Ni(II) with native state recombinant hemoglobin is shown to produce NH2-terminal deamination and globin cross-linking in the presence of the oxidant potassium peroxymonosulfate (OxoneTM) . Both the oxidative deamination and cross-linking are exclusive to the beta chains . Recombinant hemoglobin mutants have been created to identify protein sequence requirements for these reactions . It was found that His-2 of the beta globin is required for redox active Ni(II) complexation, oxidative deamination, and cross-linking . The oxidative deamination results in the formation of a free carbonyl in place of the NH2-terminal amine of the beta chain . Most cross-linking of the beta globin occurs intramolecularly, forming beta globin dimers . Structural characterization of the beta globin dimers indicates the presence of heterogeneous cross-links within the central hemoglobin cavity between the NH2 terminus of one beta chain and the COOH-terminal region of the other.

J Biol Chem, 1998 May 22, 273(21), 12887 - 92
Involvement of molecular chaperonins in nucleotide excision repair . Dnak leads to increased thermal stability of UvrA, catalytic UvrB loading, enhanced repair, and increased UV resistance; Zou Y et al.; UvrA is one of the key Escherichia coli proteins involved in removing DNA damage during the process of nucleotide excision repair . The relatively low concentrations (nanomolar) of the protein in the normal cells raise the potential questions about its stability in vivo under both normal and stress conditions . In vitro, UvrA at low concentrations is shown to be stabilized to heat inactivation by E . coli molecular chaperones DnaK or the combination of DnaK, DnaJ, and GrpE . These chaperone proteins allow sub-nanomolar concentrations of UvrA to load UvrB through >10 cycles of incision . Guanidine hydrochloride-denatured UvrA was reactivated by DnaK, DnaJ, and GrpE to as much as 50% of the native protein activity . Co-immunoprecipitation assays showed that DnaK bound denatured UvrA in the absence of ATP . UV survival studies of a DnaK-deficient strain indicated an 80-fold increased sensitivity to 100 J/m2 of ultraviolet light (254 nm) as compared with an isogenic wild-type strain . Global repair analysis indicated a reduction in the extent of pyrimidine dimer and 6-4 photoproduct removal in the DnaK-deficient cells . These results suggest that molecular chaperonins participate in nucleotide excision repair by maintaining repair proteins in their properly folded state.

Biochem Cell Biol, 1997, 75(6), 771 - 5
Molecular cloning and expression of avian smooth muscle S100A11 (calgizzarin, S100C); Schonekess BO et al.; S100A11 (calgizzarin or S100C), a member of the S100 family of Ca(2+)-binding proteins, was first identified in chicken gizzard smooth muscle and subsequently detected in several mammalian species and tissues . We now report the full-length coding sequence of avian smooth muscle S100A11 . The cloned nucleotide sequence is 515 bases in length, which includes in-frame start and stop codons and encodes a protein of 101 amino acids . The chicken S100A11 sequence differs from human S100A11 at 25 positions (9 conserved) and is four residues shorter (overall identity 72.4%, similarity 81%) . The protein contains two EF hand and conserved hydrophobic residues involved in dimer formation . Cloned avian S100A11 expressed in Escherichia coli and purified by Ca(2+)-dependent hydrophobic interaction chromatography and ion-exchange chromatography was recognized by polyclonal antibodies raised against tissue-purified protein and, like tissue-purified S100A11, bound 45Ca2+ in a gel overlay assay.

Anal Chem, 1998 May 1, 70(9), 1838 - 46
Electrospray mass spectrometry studies of non-heme iron-containing proteins; Lei QP et al.; The oligomeric state and the metal atom stoichiometry of a series of non-heme iron-containing, multimeric proteins have been measured using electrospray ionization (ESI) in a time-of-flight (TOF) mass spectrometer . The proteins were obtained both from natural sources and by overexpression of recombinant DNA in Escherichia coli . ESI-TOF mass spectra of the metalloproteins present in nondenaturing solutions exhibit peaks corresponding to the multimeric forms of the holoproteins containing the expected number of metal atoms . Capillary-skimmer dissociation of the holoproteins produces a series of ions, which allows an exact count of the number of metal atoms present in each subunit, and also provides an indication of the oxidation state of the metal atoms . Two recombinant proteins, Phascolopsis gouldii hemerythrin (Pg-Hr) and Desulfovibrio vulgaris rubrerythrin (Dv-Rr), have been examined as well as hemerythrin isolated from Lingula reevii (Lr-Hr) . ESI-TOF measurements of the aqueous solution of Pg-Hr at pH 6 yields ions of mass 108,783 Da, in close agreement with the calculated average molecular mass of an intact octameric holoprotein . Capillary-skimmer dissociation of the ions of the holoprotein produces a mass spectrum that contains peaks corresponding to a low m/z monomer and a high m/z heptamer . The masses of the monomer ions produced in this manner are assigned to the aposubunit, {subunit + Fe - 3H}+, and {subunit + 2Fe - 6 H}+ . Naturally occurring Lr-Hr is composed of two subunits with average molecular masses measured under denaturing conditions by ESI-TOF to be 13,877.0 Da for the alpha-subunit and 13,517.5 Da for the beta-subunit . Under nondenaturing conditions, a multimeric species with a molecular weight of 110,663 Da is measured by ESI-TOF, corresponding to an alpha 4 beta 4 octamer . Capillary-skimmer dissociation of the alpha 4 beta 4 oligomer produces ions corresponding to both types of monomers (alpha and beta) and the corresponding heptamers (alpha 3 beta 4 and alpha 4 beta 3) . In ESI-TOF measurements of recombinant rubrerythrin Dv-Rr using nondenaturing conditions, the principal ion observed corresponds to a homotetramer with an average molecular mass of 86,844 Da . Capillary-skimmer dissociation of the rubrerythrin tetramer leads to formation of a series of peaks corresponding to the subunit of the apoprotein and to subunits containing from one to three specifically bound iron atoms.

Int J Food Microbiol, 1998 Mar 3, 40(1-2), 57 - 64
An interlaboratory study to find an alternative to the MPN technique for enumerating Escherichia coli in shellfish; Ogden ID et al.; Nine laboratories in eight countries tested 16 batches of common mussels (Mytilus edulis) over a 32 week period in order to find an alternative to the Most Probable Number (MPN) technique to enumerate E . coli . The alternatives investigated included the 3M Petrifilm system, the Merck Chromocult agar method and a Malthus conductance technique . The Petrifilm was found to be unsuitable and was subsequently dropped from the trial . After 669 analyses, a correlation of 0.83 was observed for log E . coli counts between the MPN and Chromocult methods and there was no significant evidence that either method tended to give higher readings than the other . The MPN was slightly better than the Chromocult method for repeatability but the Chromocult was slightly better for reproducibility . However, the observed differences are probably too small to be of practical importance . On the basis of these data therefore, the two methods appear equally suitable for E . coli enumeration in shellfish . There were poor correlations between these methods and the Malthus technique . A small but significant number of samples tested positive on the Malthus instrument but were recorded negative on the MPN and Chromocult tests . Subsequent analysis positively identified E . coli from these Malthus assays . After statistical analysis, errors were noted in both the MPN and Chromocult methods but it was found that there would be no statistical differences if the Chromocult agar were used as an alternative to the MPN technique.

FEBS Lett, 1998 Apr 24, 426(3), 309 - 13
Site-directed mutagenesis and chemical modification of the two cysteine residues of the UDP-N-acetylmuramoyl:L-alanine ligase of Escherichia coli; Nosal F et al.; Site-directed mutagenesis and chemical modification of the two cysteine residues of the MurC L-alanine-adding enzyme from Escherichia coli were undertaken to study their possible role in activity and stability . Their replacement by alanine was not critical for activity . However, C230 played a role in enzyme stability and substrate binding . N-Ethylmaleimide alkylation led to monoalkylated and dialkylated proteins . The monoalkylated protein had mostly unmodified C230 residues . The extent of alkylation of C230 paralleled the loss of activity, whereas that of C426 did not . Protection against inactivation by beta,gamma-imidoadenosine 5'-triphosphate implied the involvement of C230 in the ATP binding site.

FEBS Lett, 1998 Apr 24, 426(3), 297 - 300
The isolated H4-H5 cytoplasmic loop of Na,K-ATPase overexpressed in Escherichia coli retains its ability to bind ATP; Obsil T et al.; The H4-H5 loop of the alpha-subunit of mouse brain Na,K-ATPase was expressed and isolated from Escherichia coli cells . Using fluorescence analogues of ATP, this loop was shown to retain its capability to bind ATP . Isolation of a soluble H4-H5 loop with the native ATP binding site is a crucial step for detailed studies of the molecular mechanism of ATP binding and utilisation.

Hum Genet, 1998 Apr, 102(4), 430 - 4
A missense mutation (His42Arg) in the T-protein gene from a large Israeli-Arab kindred with nonketotic hyperglycinemia; Kure S et al.; Nonketotic hyperglycinemia (NKH) is caused by a mutation in the genes encoding the components of the glycine cleavage multi-enzyme system . More than 80% of the patients have defects in the gene encoding P-protein, whereas the rest of the patients have defects in the gene encoding T-protein . We have found a large Israeli-Arab kindred with NKH . At least 14 children were affected, and all the patients had seizures and respiratory failure within 2 days after birth . Enzymatic analysis revealed that T-protein activity was deficient in the liver specimen from one propositus . We screened this family for a mutation in the protein-coding region and exon/intron boundaries of T-protein gene by direct sequencing analysis . A missense mutation was found in exon 2; this resulted in an amino acid substitution from histidine to arginine at position 42 (H42R) . Histidine 42 is conserved in human, bovine, chicken, pea, and Escherichia coli, suggesting that it has an important role in catalytic functions . Genotype analyses of 26 family members confirmed that the homozygous H42R mutation was completely associated with the onset of NKH . The availability of DNA testing facilitates the prenatal diagnosis of NKH and the identification of carriers, which is necessary for genetic counseling in the affected families.

Biochem Biophys Res Commun, 1998 May 8, 246(1), 238 - 42
Multiple phosphorylation of chicken protein tyrosine phosphatase 1 and human protein tyrosine phosphatase 1B by casein kinase II and p60c-src in vitro; Jung EJ et al.; We have cloned a soluble chicken protein tyrosine phosphatase, named CPTP1, from the cDNA library of chicken intestine . The CPTP1 showed 92% sequence identity to the corresponding 321 amino acid residues of human PTP1B (HPTP1B) . CPTP1 lacked 13 amino acids of the N-terminal region compared with HPTP1B, while the C-terminal 48 amino acid sequence of this protein was distinct from those of other PTPs . In vitro phosphorylation and phosphoamino acid analysis showed that both CPTP1 and HPTP1B were phosphorylated on serine and threonine residues near their N-terminus by casein kinase II (CKII) . Furthermore, phosphorylation of CPTP1 by CKII resulted in an inhibition of its phosphatase activity in vitro . Interestingly, both CPTP1 and HPTP1B were also tyrosine-phosphorylated near their N-terminus by p60c-src . When we examined the vanadate effect, in the absence of vanadate, the tyrosine-phosphorylated CPTP1 by p60c-src was autodephosphorylated by its own phosphatase activity . These results suggest that both CPTP1 and HPTP1B might play an important role in CKII- and p60c-src-induced signal transduction cascades.

Planta, 1998 May, 205(1), 121 - 31
A maize FK506-sensitive immunophilin, mzFKBP-66, is a peptidylproline cis-trans-isomerase that interacts with calmodulin and a 36-kDa cytoplasmic protein; Hueros G et al.; A member of a eukaryotic gene superfamily, encoding a peptidylproline cis-trans-isomerase (rotamase) has been isolated from a maize (Zea mays L . A69Y+) endosperm cDNA library . The maize sequence (mzFKBP-66) encodes a 66-kDa polypeptide most closely related to the subclass of rotamases which bind an immunosuppressive drug, FK506, (termed FK506-binding proteins FKBPs), and possesses four tandem copies of the FKBP-like binding domain . The sequence mzFKBP-66 is expressed ubiquitously in the maize plant, and the protein encoded is present in both cytosolic and nuclear compartments within the cell . Both the native mzFKBP-66 and a recombinant protein overexpressed in Escherichia coli showed peptidylproline cis-trans-isomerase (PPIase) activity at rates comparable to those reported for mammalian immunophilins . This activity was also sensitive to inhibition by FK506 . Immunoaffinity chromatography using anti-mzFKBP66 demonstrated an association of the protein with an unknown 36-kDa polypeptide, and affinity chromatography of mzFKBP-66 on calmodulin-agarose beads indicated the presence of a calmodulin-binding site . The existence of mzFKBP-66-associated proteins suggests that plant immunophilins may act as part of multicomponent complexes, as has been shown for other representatives of this class of enzyme.

Planta, 1998 May, 205(1), 12 - 22
Rice phloem thioredoxin h has the capacity to mediate its own cell-to-cell transport through plasmodesmata; Ishiwatari Y et al.; Rice (Oryza sativa L.) phloem sieve tubes contain RPP13-1, a thioredoxin h protein that moves around the plant via the translocation stream . Such phloem-mobile proteins are thought to be synthesized in the companion cells prior to being transferred, through plasmodesmata, to the enucleate sieve-tube members . In this study, in-situ hybridization experiments confirmed that expression of RPP13-1 is restricted to companion cells within the mature phloem . To test the hypothesis that RPP13-1 enters the sieve tube, via plasmodesmata, recombinant RPP13-1 was expressed in Escherichia coli, extracted, purified and fluorescently labeled with fluorescein isothiocyanate (FITC) for use in microinjection experiments into tobacco (Nicotiana tabacum L.) mesophyll cells . The FITC-RPP13-1 moved from the injected cell into surrounding cells, whereas the E . coli thioredoxin, an evolutionary homolog of RPP13-1, when similarly labeled and injected, failed to move in this same experimental system . In addition, co-injection of RPP13-1 and FITC-dextrans established that RPP13-1 can induce an increase in plasmodesmal size exclusion limit to a value greater than 9.4 but less than 20 kDa . Nine mutant forms of RPP13-1 were constructed and tested for their capacity to move from cell to cell; two such mutants were found to be incapable of movement . Crystal-structure prediction studies were performed on wild-type and mutant RPP13-1 to identify the location of structural motifs required for protein trafficking through plasmodesmata . These studies are discussed with respect to plasmodesmal-mediated transport of macromolecules within the companion cell-sieve tube complex.

J Mol Biol, 1998 Apr 24, 278(1), 267 - 78
Asymmetry, commitment and inhibition in the GroE ATPase cycle impose alternating functions on the two GroEL rings; Kad NM et al.; The ATPase cycle of GroE chaperonins has been examined by transient kinetics to dissect partial reactions in complexes where GroEL is asymmetrically loaded with nucleotides . The occupation of one heptameric ring by ADP does not inhibit the loading of the other with ATP nor does it prevent the consequent structural rearrangement to the "open" state . However, ADP binding completely inhibits ATP hydrolysis in the asymmetric complex, i.e . ATP cannot by hydrolysed when ADP is bound to the other ring . This non-competitive inhibition of the ATPase by ADP is consistent with a ring-switching, or "two-stroke", mechanism of the type: ATP:GroEL --> ADP:GroEL --> ADP:GroEL:ATP --> GroEL:ATP --> GroEL:ADP, i.e . with respect to the GroEL rings, ATP turns over in an alternating fashion . When the ATP-stabilized, "open" state is challenged with hexokinase and glucose, to quench the free ATP, the open state relaxes slowly (0.44 s-1) back to the apo (or closed) conformation . This rate, however, is three times faster than the hydrolytic step, showing that bound ATP is not committed to hydrolysis . When GroES is bound to the GroEL:ATP complex and the system is quenched in the same way, approximately half of the bound ATP undergoes hydrolysis on the chaperonin complex showing that the co-protein increases the degree of commitment . Thus, non-competitive inhibition of ATP hydrolysis, combined with the ability of the co-protein to block ligand exchange between rings has the effect of imposing a reciprocating cycle of reactions with ATP hydrolysing, and GroES binding, on each of the GroEL rings in turn . Taken together, these data imply that the dominant, productive steady state reaction in vivo is: GroEL:ATP:GroES --> GroEL:ADP:GroES --> ATP:GroEL:ADP:GroES --> ATP:GroEL:ADP --> GroES:ATP:GroEL:ADP --> GroES:ATP:GroEL for a hemi-cycle, and that significant inhibi tion of hydrolysis may arise through the formation of a dead-end ADP:GroEL:ATP:GroES complex .

J Mol Biol, 1998 Apr 24, 278(1), 239 - 52
The three-dimensional structure of an avian class-mu glutathione S-transferase, cGSTM1-1 at 1.94 A resolution; Sun YJ et al.; Glutathione S-transferase cGSTM1-1, an avian class-mu enzyme with high sequence identity with rGSTM3-3, was expressed heterologously in Escherichia coli . The three-dimensional structure of this protein that co-crystallized with an inhibitor, S-hexylglutathione, was determined by the molecular replacement method and refined to 1.94 A resolution . The three-dimensional structure and the folding topology of the dimeric cGSTM1-1 closely resembles those of other class-mu GSTs . The bound inhibitor, S-hexylglutathione, orients in disparate directions in the two subunits . The combined space occupied by the hexyl moiety of the inhibitors overlaps with that reported for rGSTM1-1 co-crystallized with (9 S,10 S)-9-(S-glutathionyl)-10-hydroxy-9,10-dihydrophenanthrene . Conformational differences at a flexible loop (residue 35 to 40) were also observed between the crystal structures of cGSTM1-1 and rGSTM1-1.cGSTM1-1 has the highest epoxidase activity among all the class-mu enzymes reported . Tyr115, has been identified as a residue that participates in the epoxidase activity of class-mu glutathione S-transferase and is conserved in cGSTM1-1 . The epoxidase and trans-4-phenyl-3-buten-2-one conjugating activity of cGSTM1-1 are decreased drastically but not abolished by replacing Tyr115 with phenylalanine . The specificity constant of the cGSTM1-1(Y115F) mutant, with 1-chloro-2,4-dinitrobenzene as substrate, is 15-fold higher than that of the wild-type enzyme .

J Mol Biol, 1998 Apr 24, 278(1), 117 - 33
Structural recognition and distortion by the DNA junction-resolving enzyme RusA; Giraud-Panis MJ et al.; RusA is a relatively small DNA junction-resolving enzyme of lambdoid phage-origin . Many of the physical characteristics of this enzyme are similar to those of junction-resolving enzymes of different origins . RusA binds to DNA junctions as a dimer, with a dissociation constant of 2 to 7 nM . RusA also exists in dimeric form in free solution, with a half time for subunit exchange of 4.2 minutes . We find that RusA can cleave both fixed junctions and those that can undergo a number of steps of branch migration, and confirm that the enzyme exhibits a strong preference for cleavage 5' to a CpC sequence . We have isolated a mutant protein, RusA D70N, that is completely inactive in cleavage while binding normally to DNA junctions, suggesting a role for aspartate 70 in the cleavage reaction . Constraining the conformation of the junction by means of tethering the helical ends leads to a marked reduction in cleavage rate by RusA, suggesting that the structure must be altered for cleavage . Using comparative gel electrophoresis we find that the global structure of the DNA junction is altered on RusA binding, into a structure that is different from any that is formed by the free junction . Moreover, the structure of the complex is the same irrespective of the presence or absence of magnesium ions . Thus, like all the junction-resolving enzymes, RusA both recognises and distorts the structure of DNA junctions .

J Mol Biol, 1998 Apr 24, 278(1), 105 - 16
Structural similarities between Escherichia coli RuvA protein and other DNA-binding proteins and a mutational analysis of its binding to the holliday junction; Rafferty JB et al.; Comparison of the structure of Escherichia coli RuvA with other proteins in the Protein Data Bank gives insights into the probable modes of association of RuvA with the Holliday junction during homologous recombination . All three domains of the RuvA protein possess striking structural similarities to other DNA-binding proteins . Additionally, the second domain of RuvA contains two copies of the helix-hairpin-helix (HhH) structural motif, which has been implicated in non-sequence-specific DNA binding . The two copies of the motif are related by approximate 2-fold symmetry and may form a bidentate DNA-binding module . The results described provide support for the organization of the arms of the DNA in our RuvA/Holliday junction complex model and support the involvement of the HhH motifs in DNA binding .

J Mol Biol, 1998 Apr 24, 278(1), 89 - 104
Functions of the ATP hydrolysis subunits (RecB and RecD) in the nuclease reactions catalyzed by the RecBCD enzyme from Escherichia coli; Chen HW et al.; The RecBCD enzyme from Escherichia coli is an ATP-dependent nuclease and helicase . Two of its subunits, the RecB and RecD proteins, are DNA-dependent ATPases . We have purified RecB and RecD proteins with mutations in their consensus ATP binding sites to study the functions of these subunits in the ATP-dependent nuclease activities of RecBCD . Reconstituted heterotrimeric enzymes were prepared by mixing wild-type RecB or RecB-K29Q mutant protein (RecB*) with purified RecC protein, and with a histidine-tagged wild-type RecD (hD) or mutant hRecD-K177Q (hD*) protein . RecBCD and all four reconstituted enzymes (wild-type, two single mutants, and the double mutant) cleave a single-stranded DNA oligomer substrate (25-mer) in the absence of ATP at rates of 0.03 to 0.06 min-1 . The nuclease reaction catalyzed by RecB*ChD* is not stimulated significantly by ATP, while the reactions catalyzed by RecBCD, RecBChD, RecBChD*, and RecB*ChD are 300 to 3000 fold faster in the presence of 0.5 mM ATP . RecB*ChD* also has very low ATP hydrolysis activity (approximately 10(3)-fold less than RecBCD), as do the individual mutant RecB* and hRecD* proteins (approximately 100-fold less than RecB or hRecD) . The products from the ATP-stimulated nuclease reaction with the oligomer substrate suggest a mechanism where two DNA molecules bind to the enzyme in opposite orientations and are cleaved by the nuclease active site . Cleavage towards the 3'-end of one oligomer (observed with RecBChD*) depends on the wild-type RecB subunit, while RecD-dependent cleavage (observed with RecB*ChD) occurs towards the 5'-end of the second bound oligomer .

EMBO J, 1998 Apr 15, 17(8), 2436 - 49
Devoted to the lagging strand-the subunit of DNA polymerase III holoenzyme contacts SSB to promote processive elongation and sliding clamp assembly; Kelman Z et al.; Escherichia coli DNA polymerase III holoenzyme contains 10 different subunits which assort into three functional components: a core catalytic unit containing DNA polymerase activity, the beta sliding clamp that encircles DNA for processive replication, and a multisubunit clamp loader apparatus called gamma complex that uses ATP to assemble the beta clamp onto DNA . We examine here the function of the psi subunit of the gamma complex clamp loader . Omission of psi from the holoenzyme prevents contact with single-stranded DNA-binding protein (SSB) and lowers the efficiency of clamp loading and chain elongation under conditions of elevated salt . We also show that the product of a classic point mutant of SSB, SSB-113, lacks strong affinity for psi and is defective in promoting clamp loading and processive replication at elevated ionic strength . SSB-113 carries a single amino acid replacement at the penultimate residue of the C-terminus, indicating the C-terminus as a site of interaction with psi . Indeed, a peptide of the 15 C-terminal residues of SSB is sufficient to bind to psi . These results establish a role for the psi subunit in contacting SSB, thus enhancing the clamp loading and processivity of synthesis of the holoenzyme, presumably by helping to localize the holoenzyme to sites of SSB-coated ssDNA.

Pharmacology, 1998 May, 56(5), 230 - 6
Pharmacological modulation of LPS-induced MIP-1 alpha production by peripheral blood mononuclear cells; Kimata M et al.; In the present study, we investigated the effects of some anti-asthmatic drugs on the production of the CC chemokine, macrophage inflammatory protein-1 alpha (MIP-1 alpha), in response to lipopolysaccharide (LPS) by peripheral blood mononuclear cells (PBMC) . MIP-1 alpha production was induced by LPS in a concentration-dependent fashion and reached the maximum at 10 micrograms/ml LPS (27.5 +/- 2.3 ng MIP-1 alpha/10(6) PBMC) . At a submaximal concentration of LPS (1 microgram/ml), the release of MIP-1 alpha increased with time and reached the maximum 24 h after LPS stimulation . Actinomycin D and cycloheximide inhibited MIP-1 alpha production completely, but glucocorticoids did not completely inhibit MIP-1 alpha production, with a maximum inhibition of 70% . We examined the effect of beta-stimulants and phosphodiesterase inhibitors, which upregulate intracellular cyclic AMP levels, on MIP-1 alpha production . When PBMC were treated with beta-stimulants alone, beta-stimulants showed a slightly inhibitory effect on MIP-1 alpha production . However, the coadministration of roliplam significantly potentiated the inhibitory effect of beta-stimulants on MIP-1 alpha production . Moreover, db-cAMP suppressed MIP-1 alpha production dose-dependently . The above data indicate that the production of MIP-1 alpha is regulated by cyclic AMP and that cyclic AMP could provide a useful target for therapeutic treatment in asthmatic diseases and other diseases where MIP-1 alpha is involved in their etiology.

Am J Hum Genet, 1998 May, 62(5), 1023 - 33
Human meiotic recombination products revealed by sequencing a hotspot for homologous strand exchange in multiple HNPP deletion patients; Reiter LT et al.; The HNPP (hereditary neuropathy with liability to pressure palsies) deletion and CMT1A (Charcot-Marie-Tooth disease type 1A) duplication are the reciprocal products of homologous recombination events between misaligned flanking CMT1A-REP repeats on chromosome 17p11 . 2-p12 . A 1.7-kb hotspot for homologous recombination was previously identified wherein the relative risk of an exchange event is 50 times higher than in the surrounding 98.7% identical sequence shared by the CMT1A-REPs . To refine the region of exchange further, we designed a PCR strategy to amplify the recombinant CMT1A-REP from HNPP patients as well as the proximal and distal CMT1A-REPs from control individuals . By comparing the sequences across recombinant CMT1A-REPs to that of the proximal and distal CMT1A-REPs, the exchange was mapped to a 557-bp region within the previously identified 1.7-kb hotspot in 21 of 23 unrelated HNPP deletion patients . Two patients had recombined sequences suggesting an exchange event closer to the mariner-like element previously identified near the hotspot . Five individuals also had interspersed patches of proximal or distal repeat specific DNA sequence indicating potential gene conversion during the exchange of genetic material . Our studies provide a direct observation of human meiotic recombination products . These results are consistent with the hypothesis that minimum efficient processing segments, which have been characterized in Escherichia coli, yeast, and cultured mammalian cells, may be required for efficient homologous meiotic recombination in humans.

FEBS Lett, 1998 Apr 17, 426(2), 217 - 20
Stability and functionality of cysteine-less F(0)F1 ATP synthase from Escherichia coli; Kuo PH et al.; All 21 native cysteines in the Escherichia coli F(0)F1 ATP synthase were replaced by alanines . In isolated E . coli membranes, ATP-dependent proton pumping, turnover of ATP hydrolysis and steady-state transition state thermodynamic parameters of the cysteine-less enzyme were similar to wild-type . The cysteine-less enzyme was solubilized in n-octyl beta-D-glucopyranoside, purified by affinity chromatography, and reconstituted into pre-formed liposomes made from E . coli lipids . The properties of the reconstituted, purified enzyme were not significantly different from the membranous enzyme . These data demonstrate that cysteine-less F(0)F1 is biochemically stable and has functionality similar to wild-type.

FEBS Lett, 1998 Apr 17, 426(2), 191 - 5
Regulation of capsular polysialic acid biosynthesis by N-acetyl-D-mannosamine, an intermediate of sialic acid metabolism; Revilla-Nuin B et al.; N-Acetyl-D-mannosamine (ManNAc) is a specific substrate for the synthesis of N-acetylneuraminic acid, the essential precursor of bacterial capsular polysialic acid (PA) . When Escherichia coli K92 used ManNAc as a carbon source, we observed a dramatic reduction (up to 90%) in in vivo PA production . Experiments in which the carbon source was changed revealed that the maximal inhibitory effect occurred when this sugar was present in the medium before the logarithmic phase of bacterial growth had started . Enzymatic analysis revealed that high concentrations of ManNAc-6-phosphate inhibit NeuAc lyase, the enzyme that synthesizes NeuAc for PA biosynthesis in E . coli . These results indicate that ManNAc-6-phosphate is able to regulate NeuAc lyase activity and modulate the PA synthesis.

FEBS Lett, 1998 Apr 10, 426(1), 135 - 9
Aminoacylation of tRNA gene transcripts is strongly affected by 3'-extended and dimeric substrate RNAs; Kholod N et al.; Kinetic parameters of aminoacylation by E . coli phenylalanyl-tRNA synthetase vary for phage T5 tRNA(Phe) gene transcript from 0.950 to 2.545 microM for Km and from 550 to 400 min(-1) for kcat . To reveal the source of this variability for various RNA preparations, homogeneity of the transcripts has been examined . Presence of 3' extensions and dimer formation in transcript preparations reduced the catalytic efficiency kcat/Km several-fold . We have shown that the proportion of dimers and 3'-extended transcripts in tRNA preparations is sensitive to single-base substitutions in tRNA . While wild-type phage T5 tRNA(Phe) gene transcript contains about half of dimeric molecules, for some mutants this value increases up to 90% or drops to 0% . Phage T5 tRNA(Phe) gene with anticodon stem nucleotide substitutions used as a template in run-off transcription produces 5 times less 3'-extended molecules than the wild-type gene . In view of all these results kinetic parameters of aminoacylation reaction for many wild-type and mutant tRNA gene transcripts should be reevaluated.

FEBS Lett, 1998 Apr 10, 426(1), 52 - 6
Expression, purification and preliminary crystal analysis of the human low Mr phosphotyrosine protein phosphatase isoform 1; Marzocchini R et al.; The genes of the human low Mr phosphotyrosine protein phosphatase (PTPase) isoforms 1 (IF1) and 2 (IF2) were isolated by screening a human placenta cDNA library, cloned in pGEX and expressed in E . coli as fusion proteins with glutathione S-transferase . The recombinant proteins were purified by a rapid one-step procedure allowing each enzyme to purify with high final yield and specific activity . This result is important for IF1, whose purification from natural sources is difficult, due to precipitation propensity, thus hindering structural studies . The enzymes obtained showed kinetic parameters very similar to those previously determined for the enzymes purified by classical procedures from both human erythrocytes and rat liver . These recombinant enzymes can therefore be used in place of those purified from natural sources for every purpose . IF1 and IF2 crystals were also grown . IF1 crystals were X-ray-grade, diffracted to better than 2.4 A and were suitable for high resolution X-ray structure determination.

FEBS Lett, 1998 Apr 10, 426(1), 37 - 40
Trapping of conformations of the Escherichia coli F1 ATPase by disulfide bond formation . A state of the enzyme with all three catalytic sites of equal and low affinity for nucleotides; Aggeler R et al.; A mutant of Escherichia coli F1F0-ATPase, alphaS411C/betaY331W/betaE381C/gammaC87S, has been generated . CuCl2 treatment of this mutant led to cross-linking between alpha and beta subunits in yields of up to 90% . This cross-linking across non-catalytic site interfaces inhibited ATP hydrolysis activity . In the absence of cross-linking, MgATP bound in catalytic sites of the mutant with three different affinities of 0.1 microM, 6 microM and 60 microM, respectively, values that are comparable to wild-type . For MgADP, there was one tight site (0.34 microM) and two sites of lower affinity (each 27 microM), again comparable to wild-type enzyme . After cross-linking all three catalytic sites bound MgATP or MgADP with the same relatively low affinity (approximately 60 microM) . Thus cross-linking fixed all three catalytic sites in the same conformation . Trypsin cleavage experiments showed that cross-linking fixed the epsilon subunit in the ATP+EDTA conformation.

FEBS Lett, 1998 Apr 10, 426(1), 21 - 3
IncI1 plasmid R64 encodes the ArsR protein that alleviates type I restriction; Rastorguev SM et al.; The host-controlled EcoK restriction of unmodified phage lambda was five-fold alleviated in the wild-type Escherichia coli strain K12 carrying the R64 plasmid of the incompatibility group I1 . The relevant gene was mapped between the origin of vegetative replication (rep, oriV) and the tet(r) gene about 60 kbp downstream from the origin of transfer, oriT . We cloned this gene inside the 613 bp long EcoRI-PstI fragment and sequenced it . Only one 351 bp long open reading frame (ORF) starting at 124 bp from the beginning of the insert was found in the sequence . Computer search in the current databases revealed that the putative protein is identical to the ArsR protein specified by the IncFI plasmid R773 . ArsR is a repressor of the arsenical resistance (ars) operon, arsRDABC . There are no arsABC genes in the R64 plasmid since plasmid R64- (or pSR8)-mediated resistance of E . coli K12 cells to the arsenicals arsenate and arsenite was not detected . The gene arsR and the antirestriction genes ard (ardA and ardB) are non-homologous . However, comparison of the deduced amino acid sequence of ArsR with the ArdA and ArdB sequences revealed only one small region of similarity, a 9 amino acid motif found in different antirestriction proteins that is hypothesized to be an interaction site for antirestriction proteins with restriction endonucleases.

Am J Trop Med Hyg, 1998 May, 58(5), 655 - 62
Evaluation of the protective efficacy of a recombinant dengue envelope B domain fusion protein against dengue 2 virus infection in mice; Simmons M et al.; A recombinant protein containing part of the dengue (DEN) 2 envelope protein was evaluated as a subunit immunogen for vaccination against DEN virus infection . A gene fragment encoding amino acids 298-400 (B domain) of the DEN-2 virus envelope was expressed as a fusion protein with the maltose binding protein (MBP) of Escherichia coli . This recombinant, DEN-2(B)/MBP, was purified and analyzed for its antigenicity, immunogenicity, and ability to protect mice against lethal challenge . The recombinant antigen reacted with a DEN-2 type-specific neutralizing monoclonal antibody (3H5), DEN-2 hyperimmune mouse ascitic fluid, and DEN-2 immune human sera . When administered to mice, DEN-2(B)/MBP elicited a DEN-2 virus neutralizing antibody response that conferred partial protection against challenge infection with a lethal dose of DEN-2 virus administered by intracranial inoculation . In addition, no replication of DEN-2 virus was detectable in the brains of the immunized mice as compared with control mice that were killed six days after challenge . Sera from immunized mice revealed no cross-neutralizing antibody to any of the other DEN serotypes in the plaque-reduction neutralization test . These findings warrant further studies with the DEN-2(B)/MBP antigen as a potential human vaccine candidate . An effective vaccine could prevent thousands of cases of illness and many deaths each year resulting from DEN virus infections.

Crit Rev Biochem Mol Biol, 1998, 33(2), 95 - 149
Ribosomal tRNA binding sites: three-site models of translation; Burkhardt N et al.; The first models of translation described protein synthesis in terms of two operationally defined tRNA binding sites, the P-site for the donor substrate, the peptidyl-tRNA, and the A-site for the acceptor substrates, the aminoacyl-tRNAs . The discovery and analysis of the third tRNA binding site, the E-site specific for deacylated tRNAs, resulted in the allosteric three-site model, the two major features of which are (1) the reciprocal relationship of A-site and E-site occupation, and (2) simultaneous codon-anticodon interactions of both tRNAs present at the elongating ribosome . However, structural studies do not support the three operationally defined sites in a simple fashion as three topographically fixed entities, thus leading to new concepts of tRNA binding and movement: (1) the hybrid-site model describes the tRNAs' movement through the ribosome in terms of changing binding sites on the 30S and 50S subunits in an alternating fashion . The tRNAs thereby pass through hybrid binding states . (2) The alpha-epsilon model introduces the concept of a movable tRNA-binding domain comprising two binding sites, termed alpha and epsilon . The translocation movement is seen as a result of a conformational change of the ribosome rather than as a diffusion process between fixed binding sites . The alpha-epsilon model reconciles most of the experimental data currently available.

Adv Exp Med Biol, 1998, 431, 221 - 6
Structure and functional relationships in human pur H; Beardsley GP et al.; 1 . The human pur H (ATIC) gene encoding a bifunctional protein, hPurH, which carries the penultimate and final enzymatic activities of the purine nucleotide synthesis pathway, AICARFT & IMPCH, has been cloned and sequenced . The gene product, hPurH has been overexpressed in E . coli, purified to homogeneity and crystallized . 2 . The human pur H gene lies on chromosome 2, between band q34 and q35 . There is at least one intron of 278 bp near the 5' end . 3 . Truncation mutant studies demonstrate two non-overlapping functional domains in the protein arranged as indicated in Figure 5 . The existence of a linker or interaction region between the catalytic domains remains to be established . 4 . Cleland-type kinetic inhibition experiments indicate that the AICARFT reaction is of the ordered, sequential type with the reduced folate cofactor binding first . 5 . The reaction has a broad pH optimum in the alkaline range, with a maximum at about pH 8.2 . 6 . Preliminary transient phase kinetic studies show the presence of a "burst" indicating that a late step in the reaction sequence is rate limiting . 7 . A PurH crystal structure is that of a dimer, with a putative single binding site for the reduced folate cofactor formed using elements from each of the monomer subunits . Probable binding sites for AICAR and FAICAR can be identified on each monomer . 8 . Equilibrium sedimentation studies show hPurH apoprotein to be a monomer:dimer equilibrium mixture with a kD of 0.55 uM . 9 . The crystal structure has permitted identification of a number of candidate amino acid residues likely to be involved in catalysis and/or substrate binding . Among these, we have thus far completed studies on two, Lysine 265 and Histidine 266 . These appear to be critically involved in the AICARFT reaction, although whether their role(s) are in catalysis or binding remains to be determined.

Int J Biochem Cell Biol, 1998 Jan, 30(1), 13 - 26
Structure, function and physiological role of glycine N-methyltransferase; Ogawa H et al.; Glycine N-methyltransferase (EC 2.1.1.20) catalyzes the transfer of the methyl group of S-adenosylmethionine (AdoMet) to glycine to form S-adenosylhomocysteine and sarcosine . Unlike most AdoMet-dependent methyltransferases, glycine N-methyltransferase is a tetramer of identical subunits . Crystallography of recombinant rat glycine N-methyltransferase indicates that four nearly spherical subunits are arranged to form a flat, square tetramer with a large hole in the centre . The enzyme occurs abundantly in the livers of rat, rabbit and mouse . Glycine N-methyltransferases from rat, rabbit, human and pig livers are shown to have similar amino acid sequences and, with the enzymes from rat and rabbit livers, it is demonstrated that the N-terminal valine is acetylated . Glycine N-methyltransferases from livers exhibit sigmoidal rate behaviour with respect to AdoMet and hyperbolic behaviour with respect to glycine at all pH tested . However, recombinant rat glycine N-methyltransferase which lacks the N-terminal acetyl group shows no cooperativity toward AdoMet at neutral pH, suggesting that elimination of the positive charge at the N-terminus is required for cooperative behaviour . Glycine N-methyltransferase binds 5-methyltetrahydropteroylpentaglutamate tightly, resulting in inhibition of the catalytic activity . The nature of these unique functional features is discussed in the light of the three-dimensional structure of the enzyme . The tissue and subcellular localization of the enzyme and its possible role in methionine metabolism are reviewed.

Can J Anaesth, 1998 Apr, 45(4), 352 - 9
Tumour necrosis factor-alpha, but not septic plasma depresses cardiac myofilament contraction; Gu M et al.; PURPOSE: In sepsis, myocardial depression may be caused by mediators released as part of the inflammatory reaction, lumour necrosis factor alpha (TNF alpha) is one mediator that may contribute to this depression . In the present study, we contrasted the effects of TNF alpha and septic plasma fraction (SP) obtained from an E . coli model on contractile tension in intact and skinned canine ventricular trabecular (VT) preparations . The objectives were to determine whether SP or TNF alpha could impair contractile tension at the level of the myofilaments, and to determine the extent to which TNF alpha may account for myocardial depression found in E . coli sepsis . METHODS: Measurements of isometric tensions were made after TNF alpha and SP (10,000 to 30,000 MW fraction) were added to respective intact or skinned canine VT preparations . In the skinned preparation, trabeculae were chemically skinned with Triton X-100 . RESULTS: Septic plasma caused a decrease in contraction in the intact preparation compared with preseptic plasma (50 +/- 7 vs 33 +/- 7%, P < 0.05), but had no effect in the skinned preparation . On the other hand, TNF alpha (30 ng.ml-1) caused an approximately 50% reduction in tension (29 +/- 2 mg vs 16 +/- 5 mg) in the skinned preparation (P < 0.05), but had no effect in the intact preparation . CONCLUSION: These results suggest that TNF alpha and SP act through different mechanisms . While SP requires an intact membrane, TNF alpha impairs function by a direct effect on the myofilaments.

Zhonghua Yi Xue Za Zhi, 1997 Apr, 77(4), 274 - 7
{The inhibitory effect of recombinant human C3 fragment on murine endotoxic shock}; Liang H et al.; OBJECTIVE: To study the potentially practical use of C3 inactive fragment in anti-inflammation . METHODS: A vector expressing RGD polypeptide derived from the alpha chain segment of human C3 was constructed by using PCR and genetic engineering methods and a recombinant protein (namely C 33) was expressed with high efficiency in E . coli . RESULTS: The analysis of SDS-PAGE showed the molecular weight of C 33 was about 15 KD . Its purity was above 95% after purification . The amino acid composition was inconsistent with the theoretical values . U937 cells stimulated by low dosage PMA adhered with coated C 33, and the adhesion was blocked by anti-CD 11b monoclonal antibody . After injection of purified C 33 into mice which were consequently challenged by dead E . coli, the mortality of the endotoxic shock was significantly reduced . CONCLUSION: C 33 can specifically bind to CD 11b/CD 18 . C 33 as a ligand for CD 11b/CD 18 might be potentially used as an anti-inflammatory agent.

Zhonghua Jie He He Hu Xi Za Zhi, 1996 Aug, 19(4), 202 - 5
{Effect of inhaled nitric oxide on endotoxin induced acute lung injury in rabbit}; Liao J et al.; OBJECTIVE: In order to observe the effects of inhaling nitric oxide (NO) on acute lung injury (ALI) . METHODS: 24 rabbits divided into 4 groups . Six rabbits injured with intravenous E . Coli endotoxin, then followed by treatment of inhaling 80 ppm NO in inspired gas . Before and after the infusion of endotoxin, the mean pulmonary arterial pressure (mPAP), mean systemic arterial pressure (mPSA) and the PaO2 were examined . The venous methemoglobin (MHb) was measured by using spectrophometer colorimitry . The extravasculur lung water was evaluated with rate of dried to wet lung weight at the end of study . RESULTS: The rabbits injured with endotoxin inhaling 80 ppm NO could rapidly reduce the mPAP, increase the PaO2 and without inducing significant change of mPSA, MHb and extravasculur lung water . CONCLUSIONS: Inhalation of 80 ppm NO can selectively cause pulmonary artery dilatation, reduce mPAP, improve pulmonary gas exchange, without producing system vasodilation and toxic effects to the rabbits.

Biochim Biophys Acta, 1998 Apr 10, 1380(2), 209 - 22
Six isoforms of cardiotoxin in malayan spitting cobra (Naja naja sputatrix) venom: cloning and characterization of cDNAs; Jeyaseelan K et al.; Cardiotoxins are the most abundant toxin components of cobra venom . Although many cardiotoxins have been purified and characterized by amino acid sequencing and other pharmacological and biochemical studies, to date only five cardiotoxin cDNAs from Taiwan cobra (Naja naja atra), three cDNAs from Chinese cobra (Naja atra) and two more of uncertain origin (either Chinese or Taiwan cobra) have been reported . In this paper we show the existence of four isoforms of cardiotoxin by protein analysis and nine cDNA sequences encoding six isoforms of cardiotoxins (CTX 1-3, 4a, 4b and 5) from N . n . sputatrix by cDNA cloning . This forms the first report on the cloning and characterization of several cardiotoxin genes from a single species of a spitting cobra . The cDNAs encoding these isoforms, obtained by reverse transcription-polymerase chain reaction (RT-PCR), were subsequently expressed in Escherichia coli . The native and recombinant cardiotoxins were first characterized by Western blotting and N-terminal protein sequencing . These proteins were also found to have different levels of cytolytic activity on cultured baby hamster kidney cells . Four of the isoforms (CTX 1, 2, 4 and 5) are unique to N . n . sputatrix, with CTX 2 being the most abundant species constituting about 50% of the total cardiotoxins . The isoform CTX 3 (20% constitution) is highly homologous to the cardiotoxins of N . n . atra and N . n . naja, indicating that it may be universally present in all Naja naja subspecies . Our studies suggest that the most hydrophilic isoform (CTX 5) could have evolved first followed by the hydrophobic isoforms (CTX 1, 2, 3 and 4) . We also speculate that Asiatic cobras could be the modern descendants of the African and Egyptian counterparts .

Biochemistry, 1998 Apr 28, 37(17), 6199 - 204
Inhibition of type I and type II phospholipase A2 by phosphatidyl-ethanolamine linked to polymeric carriers; Dan P et al.; We have previously shown that cell surface proteoglycans protect the cell membrane from the action of extracellular phospholipase A2 (PLA2) enzymes {Dan, P., Nitzan, D . W., Dagan, A., Ginsburg, I., and Yedgar, S . (1996) FEBS Lett . 383, 75-78} . Cell-impermeable PLA2 inhibitors (ExPLIs) were prepared by linking phosphatidylethanolamine (PE) to polymeric carriers, specifically, carboxymethylcellulose, heparin, or hyaluronic acid . The structure of these inhibitors enables the incorporation of their PE moiety into the membrane while the polymer remains at the membrane surface . In the present study, we show that the ExPLIs are effective inhibitors of the hydrolysis of different phospholipids in biological (Escherichia coli) and model (phospholipid vesicle) membranes, by diverse types of PLA2 enzymes, specifically human recombinant synovial fluid and C . atrox (type II), as well as Naja mocambique and porcine pancreatic (type I) PLA2 . It is proposed that the external polymers of the ExPLIs, which are anchored to the membrane by the PE, mimic the naturally occurring cell surface proteoglycans and similarly protect membranes from the action of exogenous PLA2.

Biochemistry, 1998 Apr 28, 37(17), 6114 - 23
The flavoprotein component of the Escherichia coli sulfite reductase: expression, purification, and spectral and catalytic properties of a monomeric form containing both the flavin adenine dinucleotide and the flavin mononucleotide cofactors; Zeghouf M et al.; The flavoprotein component (SiR-FP) of the sulfite reductase from Escherichia coli is an octamer containing one FAD and one FMN per polypeptide chain . SiR-FP60, a SiR-FP fragment starting with alanine-52, was overexpressed in E . coli and purified as a monomer . The N-terminal part of the native protein contains thus all the determinants required for the polymerization . SiR-FP60 retains both FAD and FMN with comparable contributions of the two flavins and the catalytic properties of SiR-FP . Thus, SiR-FP60 can be considered as a reliable simplified model of the sulfite reductase flavoprotein component . The formation and the stabilization of the neutral FMN semiquinone is thermodynamically favorable in SiR-FP60 upon reduction with photoreduced deazaflavin, dithionite, or NADPH . Generation of FMNH* is explained from a disproportionation of electrons between the reduced and oxidized FMN moieties during an intermolecular reaction, as shown with SiR-FP23, the FMN-binding domain of SiR-FP . The neutral FAD semiquinone can be observed only within SiR-FP43, the isolated FAD-binding domain . NADPH was used as a titrant or in excess to demonstrate that electron transfer is possible only because the FMN cofactor is coupled to FAD as an electron acceptor in the protein . The electron distribution within the various reduced forms of SiR-FP60 has been compared with that of the reduced forms of cytochrome P450 reductase, bacterial cytochrome P450, and nitric-oxide synthase . Despite the conservation of the bi-flavin-domain structure between these proteins over evolutionary time, each of them provides significantly different flavin reactivities.

Biochemistry, 1998 Apr 28, 37(17), 6106 - 13
NADPH-flavodoxin reductase and flavodoxin from Escherichia coli: characteristics as a soluble microsomal P450 reductase; Jenkins CM et al.; In addition to their endogenous roles as an activation system for various Escherichia coli metabolic pathways, the soluble flavoproteins flavodoxin (Fld) and NADPH-flavodoxin (ferredoxin) reductase (Fpr) can serve as an electron-transfer system for microsomal cytochrome P450s . Furthermore, since Fld and Fpr are structurally similar to the functional domains (FMN binding and NADPH/FAD binding domains, respectively) of NADPH-cytochrome P450 reductases (P450 reductases), these bacterial proteins represent a potentially useful model system for eukaryotic P450 reductases . Here we delineate similarities and differences between the E . coli Fpr-Fld system and rat P450 reductase as electron donors to bovine 17alpha-hydroxylase/17,20-lyase P450 (P450c17) . Importantly, recombinant Fpr, in combination with recombinant Fld, supports both the hydroxylase and lyase activities of P450c17 to the same proportional extent (hydroxylase-to-lyase ratio) as does P450 reductase . Maximum P450c17 turnover {5-6 mol of 17alpha-OH-progesterone (mol of P450c17)-1 min-1} was achieved using a large molar excess (50-100-fold over P450c17) of a 1:1 ratio of Fpr-Fld, although this rate was an order of magnitude less than the maximal P450 reductase-supported activity . Using these conditions, we have examined the effects of increasing ionic strength and the presence of cytochrome b5 (b5) on these two systems . Critical Fld-P450c17 electrostatic interactions are disrupted at moderate ionic strength (>100 mM NaCl) as evidenced by significant inhibition (>50%) of Fpr-Fld-supported P450c17 activity while much higher ionic strength (300 mM NaCl) is required to disrupt P450 reductase-P450c17 interactions to the same extent . Interestingly, cytochrome b5 was found to dramatically inhibit both P450 reductase- and Fpr-Fld-supported P450c17 progesterone 17alpha-hydroxylase activity while in contrast 17alpha-OH-pregnenolone lyase activity was stimulated by b5 . Investigation of the fate of reducing equivalents from NADPH added to Fpr under aerobic conditions revealed that the majority of the protein-bound FAD of Fpr is converted to the hydroquinone form . In constrast, the FMN of Fld is reduced by Fpr to a stable blue, neutral semiquinone which serves as the predominant electron donor to P450c17 in reconstitution assays . Thus, while the Fpr-Fld system and P450 reductase are fundamentally different with respect to their electrostatic interactions with P450c17, their ability to support maximal P450c17 turnover, and the FMN redox states (one-electron-reduced for Fld and two-electron-reduced for P450 reductase) capable of transferring electrons to microsomal cytochrome P450s, these differences do not appear to influence the relative catalytic efficiency of the P450c17 hydroxylase and lyase reactions.

Biochemistry, 1998 Apr 28, 37(17), 6041 - 9
Role of base G-2 of pre-tRNAfMet in cleavage site selection by Escherichia coli RNase P in vitro; Lazard M et al.; In this study, a protocol for the purification of fully active Escherichia coli RNase P holoenzyme from a strain overproducing both the C5 protein and the M1 RNA components is described . A total of 0 . 8 mg of homogeneous enzyme, with a 1:1 protein/RNA subunit stoichiometry, was recovered from a 1 L bacterial culture . In addition, a convenient and reliable method based on capillary gel electrophoresis was developed to measure initial rates of pre-tRNA maturation by RNase P . Using these tools, the kinetic parameters of cleavage by RNase P of various mutants of pre-tRNAfMet showing maturation defects in vivo {Meinnel and Blanquet (1995) J . Biol . Chem . 270, 15906-15914} were investigated in vitro and the locations of cleavage sites were determined from the length of the various products of the reaction . The nucleotide at position -2 of pre-tRNAfMet is shown to be important only in the selection of the cleavage site, whereas it has no role in the efficiency of the reaction . It is concluded that base G-2 acts as an antideterminant by preventing an alternative cleavage by RNase P . In addition, the presence of G-2 alone is enough to fully compensate for the lack of a G at position +1 of pre-tRNAfMet.

Biochemistry, 1998 Apr 28, 37(17), 5953 - 60
Selective inactivation of parvulin-like peptidyl-prolyl cis/trans isomerases by juglone; Hennig L et al.; In contrast to FK506 binding proteins and cyclophilins, the parvulin family of peptidyl-prolyl cis/trans isomerases (PPIases; E.C . 5.2.1.8) cannot be inhibited by either FK506 or cyclosporin A . We have found that juglone, 5-hydroxy-1,4-naphthoquinone, irreversibly inhibits the enzymatic activity of several parvulins, like the E . coli parvulin, the yeast Ess1/Ptf1, and human Pin1, in a specific manner, thus allowing selective inactivation of these enzymes in the presence of other PPIases . The mode of action was studied by analyzing the inactivation kinetics and the nature of products of the reaction of E . coli parvulin and its Cys69Ala variant with juglone . For all parvulins investigated, complete inactivation was obtained by a slow process that is characterized by pseudo-first-order rate constants in the range of 5.3 x 10(-)4 to 4 . 5 x 10(-)3 s-1 . The inactivated parvulin contains two juglone molecules that are covalently bound to the side chains of Cys41 and Cys69 because of a Michael addition of the thiol groups to juglone . Redox reactions did not contribute to the inactivation process . Because thiol group modification was shown to proceed 5-fold faster than the rate of enzyme inactivation, it was considered as a necessary but not sufficient condition for inactivation . When measured by far-UV circular dichroism (CD), the rate of structural alterations following thiol group modification parallels exactly the rate of inactivation . Thus, partial unfolding of the active site of the parvulins was thought to be the cause of the deterioration of PPIase activity.

Biochemistry, 1998 Apr 28, 37(17), 5939 - 46
Electrostatic stabilization in methionine aminopeptidase from hyperthermophile Pyrococcus furiosus; Ogasahara K et al.; The thermostability of methionine aminopeptidase from a hyperthermophile P . furiosus (PfMAP) was extremely high: the denaturation temperature was 106.2 degreesC at pH 10.2 . To explore the contribution of electrostatic interaction to the superior thermostability of PfMAP, the thermostability of PfMAP was examined by differential scanning calorimetry (DSC) in various salt concentrations in the acidic region far from the isoelectric point of PfMAP . (1) In 20 mM glycine buffer, the DSC curve of PfMAP exhibited a single peak . Transition temperatures (Tm) were lowered with decreasing pH from 4 to 3 . The heat denaturation of PfMAP was not reversible . (2) Denaturation enthalpy (DeltaH) measured at different pHs linearly correlated with Tm up to 102 degreesC, suggesting that the denaturation heat capacity (DeltaCp) for PfMAP is constant up to 100 degreesC . DeltaCp was estimated to be 0.82 J K-1 g-1 . (3) In the presence of 10-100 mM KCl at pH 3.2, two peaks appeared on the DSC curves . The first peak shifted to lower temperatures with increasing concentration of KCl and, oppositely, the second one to higher temperatures . It was found that the first and second peaks originated from the heat denaturation of the native form of PfMAP and the melting of the non-native associated form having molten globule-like structure, respectively, judged from the CD spectra and ultracentrifugation analyses . This indicates the following: first, the attractive electrostatic interaction is an important factor in stabilizing the native form of PfMAP; second, the presence of KCl stimulates the formation of the molten globule-like state of PfMAP and stabilizes it . (4) In a comparison of the sequence and crystal structure of PfMAP, which has been recently determined (1xgs.pdb), with those of MAP from Escherichia coli (EcMAP), it was predicted that the extra four short-range ion pairs less than 3 A involved in PfMAP are crucial candidates as determinants for the superior thermostability of PfMAP.

Biochemistry, 1998 Apr 28, 37(17), 5849 - 57
Preparation, characterization, and complete heteronuclear NMR resonance assignments of the glutaredoxin (C14S)-ribonucleotide reductase B1 737-761 (C754S) mixed disulfide; Berardi MJ et al.; The first committed step in de novo DNA biosynthesis involves the conversion of ribonucleotides to the corresponding deoxyribonucleotides catalyzed by the enzyme ribonucleotide reductase . Reduction of disulfides in ribonucleotide reductase is essential and is catalyzed by the protein disulfide reductants glutaredoxin or thioredoxin . The interaction region between Escherichia coli glutaredoxin-1 and E . coli ribonucleotide reductase has been localized to the C-terminal end of the B1 subunit of ribonucleotide reductase . We have demonstrated that a 25-residue peptide corresponding to this C-terminal sequence is a very good substrate for glutaredoxin via a fluorescence assay and that this peptide binds in a specific manner via isothermal titration calorimetric measurements . By selectively mutating the two cysteines in the peptide, we have identified the electrophilic cysteine as C759 (B1 numbering) and prepared a mixed disulfide between E . coli glutaredoxin-1 (C14 --> S) and the C759 monothiol form of the peptide . The peptide and the protein have been labeled with 13C and 15N, and complete heteronuclear NMR resonance assignments have been completed for both the peptide and the protein in the complex . By using half-filtered NOESY spectra, intermolecular NOEs between the protein and the peptide have been identified and the binding site on glutaredoxin has been mapped . The electrostatic charge distribution of the protein in this region is very positive, thus providing an excellent match for the highly negatively charged peptide . In addition, the electrostatic potential of the peptide provides a rationale for the observed cysteine selectivity in the reaction between glutaredoxin and the B1 peptide.

Biochemistry, 1998 Apr 28, 37(17), 5840 - 8
Characterization of Y122F R2 of Escherichia coli ribonucleotide reductase by time-resolved physical biochemical methods and X-ray crystallography; Tong W et al.; Ribonucleotide reductase (RNR) from Escherichia coli catalyzes the conversion of ribonucleotides to deoxyribonucleotides . It is composed of two homodimeric subunits, R1 and R2 . R2 contains the diferric-tyrosyl radical cofactor essential for the nucleotide reduction process . The in vitro mechanism of assembly of this cluster starting with apo R2 or with a diferrous form of R2 has been examined by time-resolved physical biochemical methods . An intermediate, Fe3+/Fe4+ cluster (intermediate X), has been identified that is thought to be directly involved in the oxidation of Y122 to the tyrosyl radical (*Y122) . An R2 mutant in which phenylalanine has replaced Y122 has been used to accumulate intermediate X at sufficient levels that it can be studied using a variety of spectroscopic methods . The details of the reconstitution of the apo and diferrous forms of Y122F R2 have been examined by stopped-flow UV/vis spectroscopy and by rapid freeze quench electron paramagnetic resonance, and Mossbauer spectroscopies . In addition the structure of this mutant, crystallized at pH 7.6 in the absence of mercury, at 2.46 A resolution has been determined . These studies suggest that Y122F R2 is an appropriate model for the examination of intermediate X in the assembly process . Studies with two mutants, Y356F and double mutant Y356F and Y122F R2, are interpreted in terms of the possible role of Y356 in the putative electron transfer reaction between the R1 and R2 subunits of this RNR.

Biochemistry, 1998 Apr 28, 37(17), 5831 - 9
Fidelity of mutant HIV-1 reverse transcriptases: interaction with the single-stranded template influences the accuracy of DNA synthesis; Kim B et al.; We have used random sequence mutagenesis and complementation in a bacterial selection system to establish a large library of immunodeficiency virus type 1 (HIV-1) reverse transcriptase (RT) mutants with amino acid substitutions in the beta3-beta4 region of the fingers subdomain {Kim, B., Hathaway, T . R., and Loeb, L . A . (1996) J . Biol . Chem . 271, 4872-4878} . We show here that one of these mutants, D76V, exhibits increased accuracy of copying both DNA and RNA templates in a primer extension assay with biased dNTP pools . More detailed analysis of DNA-dependent polymerization showed that the D76V mutation conferred an up to 14-fold increase in fidelity of nucleotide insertion and a 9-fold reduced mutation rate in an M13mp2 lacZalpha forward mutation assay . Substitution at D76 with positively charged (D76R) and nonpolar (D76V and D76I) residues increased replicational accuracy, while substitutions with negatively charged (D76E) and polar residues (D76S and D76C) had little effect on fidelity . We propose that D76 affects replicational accuracy by mediating interaction between the fingers subdomain and the single-stranded template . Our work shows that the Escherichia coli complementation system can yield HIV RT mutants with increased fidelity that have not been isolated from the natural host and that are valuable in understanding the molecular bases of replicational accuracy.

J Biol Chem, 1998 Apr 17, 273(16), 10026 - 35
Enzymatic processing of uracil glycol, a major oxidative product of DNA cytosine; Purmal AA et al.; A major stable oxidation product of DNA cytosine is uracil glycol (Ug) . Because of the potential of Ug to be a strong premutagenic lesion, it is important to assess whether it is a blocking lesion to DNA polymerase as is its structural counterpart, thymine glycol (Tg), and to evaluate its pairing properties . Here, a series of oligonucleotides containing Ug or Tg were prepared and used as templates for a model enzyme, Escherichia coli DNA polymerase I Klenow fragment (exo-) . During translesion DNA synthesis, Ug was bypassed more efficiently than Tg in all sequence contexts examined . Furthermore, only dAMP was incorporated opposite template Ug and Tg and the kinetic parameters of incorporation showed that dAMP was inserted opposite Ug more efficiently than opposite Tg . Ug opposite G and A was also recognized and removed in vitro by the E . coli DNA repair glycosylases, endonuclease III (endo III), endonuclease VIII (endo VIII), and formamidopyrimidine DNA glycosylase . The steady state kinetic parameters indicated that Ug was a better substrate for endo III and formamidopyrimidine DNA glycosylase than Tg; for endonuclease VIII, however, Tg was a better substrate.

J Biol Chem, 1998 Apr 17, 273(16), 9927 - 34
The proton motive force, acting on acidic residues, promotes translocation of amino-terminal domains of membrane proteins when the hydrophobicity of the translocation signal is low; Delgado-Partin VM et al.; We have shown previously that the first transmembrane segment of leader peptidase can function to translocate the polar amino-terminal Pf3 domain across the membrane into the periplasm independently of the proton motive force (pmf) (Lee, J . I., Kuhn, A., and Dalbey, R . E . (1992) J . Biol . Chem . 267, 938-943) . We now show that when the first transmembrane segment lacks a strong hydrophobic character, the pmf is required for translocation . In addition, we find that the amino-terminal acidic residue proximal to the transmembrane domain plays a critical role in pmf-dependent amino-terminal translocation . Moreover, the pmf is required to hold the amino-terminal domain in the periplasm to prevent it from slipping such that the amino terminus is no longer exposed to the periplasm . In all cases, translocation occurs under conditions in which the function of the Sec machinery is impaired . These studies show that the low hydrophobicity of the first apolar domain (the translocation signal) can be compensated for by a negative charge in the amino-terminal region, upon which the pmf acts.

J Biol Chem, 1998 Apr 17, 273(16), 9872 - 7
Reduction in abortive transcription from the lambdaPR promoter by mutations in region 3 of the sigma70 subunit of Escherichia coli RNA polymerase; Sen R et al.; Transcription initiation by Escherichia coli RNA polymerase at most promoters is associated with a reiterative synthesis and release of short abortive RNA products . We have investigated the mechanism of abortive RNA synthesis by using holoenzymes containing mutant sigma70 subunits with changes in region 3 (S506F and P504L), which reduce the ratio of abortive to full-length products . Binary complexes formed by these mutant enzymes at a modified lambdaPR promoter contained a smaller fraction of open complexes than for normal polymerase, suggesting an involvement of region 3 in melting duplex DNA or in maintenance of the open complex . The half-lives of the majority of binary complexes formed by the mutant enzymes were less than 1 min, in contrast to 30 min for the wild-type complexes . The time courses of transcription and pulse-labeling assays showed that moribund complexes, which generate only abortive products (Kubori, T., and Shimamoto, N . (1996) J . Mol . Biol . 256, 449-457), were formed by the mutant enzymes . However, they accumulated to a lesser extent than for the wild-type enzyme, due both to faster dissociation and conversion into inactive complexes . This is the main cause of the low degree of abortive transcription displayed by the mutant enzymes on this promoter.

J Biol Chem, 1998 Apr 17, 273(16), 9695 - 702
Characterization and functional analysis of the cis-autoproteolysis active center of glycosylasparaginase; Guan C et al.; Glycosylasparaginase is an N-terminal nucleophile hydrolase and is activated by intramolecular autoproteolytic processing . This cis-autoproteolysis possesses unique kinetics characterized by a reversible N-O acyl rearrangement step in the processing . Arg-180 and Asp-183, involved in binding of the substrate in the mature enzyme, are also involved in binding of free amino acids in the partially formed substrate pocket on certain mutant precursors . This binding site is sequestered in the wild-type precursor . Binding of free amino acids on mutant precursors can either inhibit or accelerate their processing, depending on the individual mutants and amino acids . The polypeptide sequence at the processing site, which is highly conserved, adopts a special conformation . Asp-151 is essential for maintaining this conformation, possibly by anchoring its side chain into the partially formed substrate pocket through interaction with Arg-180 . The reactive nucleophile Thr-152 is activated not only by deprotonation by His-150 but also by interaction with Thr-170, suggesting a His-Thr-Thr active triad for the autoproteolysis.

J Biol Chem, 1998 Apr 17, 273(16), 9602 - 7
Re-evaluating the role of His-143 in the mechanism of type I dehydroquinase from Escherichia coli using two-dimensional 1H,13C NMR; Leech AP et al.; Type I dehydroquinase from the shikimate pathway of Escherichia coli dehydrates dehydroquinate to dehydroshikimate . pH/log Vmax profiles of the enzyme indicate the presence of a single ionizing group with a pKa of 6.2 . Chemical modification experiments with diethyl pyrocarbonate have identified the conserved residue His-143 as essential for catalysis in this enzyme and the pKa for this modification is also 6.2, implying that this is the single ionizing residue in dehydroquinase that may be acting as a general base in the catalytic mechanism . Subsequent mutagenesis of this residue (Leech, A . P., James, R., Coggins, J . R., and Kleanthous, C . (1995) J . Biol . Chem . 270, 25827-25836) further suggested that His-143 may be involved in Schiff base formation/breakdown as well as being the proton abstracting general base . The importance of this residue was confirmed by recent x-ray crystallographic data showing His-143 to be at the center of a hydrogen-bonded triad, flanked by the essential Schiff base forming residue Lys-170 and Glu-86 . In the present study, we have used mutagenesis and 1H and 13C NMR to assign the resonance of His-143 and probe its ionization state to define more precisely its role in the mechanism of type I dehydroquinase . Following isotopic enrichment of wild-type and H143A dehydroquinase enzymes with {2-13C}histidine, the resonance for His-143 was assigned by comparing their 1H,13C heteronuclear single quantum correlation NMR spectra . pH titrations revealed that whether in the liganded or unliganded state, His-143 does not ionize over the pH range 6-9.5 and so cannot possess a pKa of 6.2 . The NMR data are consistent with this residue remaining unprotonated at pH values optimal for the activity of this enzyme (pH > 7) . The role of His-143 is re-evaluated in light of these and the recent structural data, and an alternative candidate for the pKa of 6.2 is discussed.

J Biol Chem, 1998 Apr 17, 273(16), 9501 - 9
Proteasome activation by REG molecules lacking homolog-specific inserts; Zhang Z et al.; The peptidase activities of eukaryotic proteasomes are markedly activated by the 11 S REG or PA28 . The three identified REG subunits, designated alpha, beta, and gamma, differ significantly in sequence over a short span of 15-30 amino acids that we call homolog-specific inserts . These inserts were deleted from each REG to produce the mutant proteins REGalphaDeltai, REGbetaDeltai, and REGgammaDeltai . The purified recombinant proteins were then tested for their ability to oligomerize and activate the proteasome . Both REGalphaDeltai and REGgammaDeltai formed apparent heptamers and activated human red cell proteasomes to the same extent as their full-length counterparts . By contrast, REGbetaDeltai exhibited, at low protein concentrations, reduced proteasome activation when compared with the wild-type REGbeta protein . REGbetaDeltai was able to form hetero-oligomers with a single site, monomeric REGalpha mutant and with REGalphaDeltai . At low concentrations, the REGalphaDeltai/REGbetaDeltai hetero-oligomers stimulated the proteasome less than REGalpha/REGbeta oligomers formed from wild-type subunits, and the reduced activation by REGalphaDeltai/REGbetaDeltai was due to removal of the REGbeta insert, not the REGalpha insert . These studies demonstrate that the REGalpha and REGgamma inserts play virtually no role in oligomerization or in proteasome activation . By contrast, removal of REGbeta insert reduces binding of this subunit and REGalpha/REGbeta oligomers to proteasomes . On the whole, however, our findings show that REG inserts are not required for binding and activating the proteasome . We speculate that they serve to localize REG-proteasome complexes within cells, possibly by binding components in endoplasmic reticulum membranes.

Protein Eng, 1997 Dec, 10(12), 1461 - 3
Purification and crystallization of complexes modeling the active state of the fragile histidine triad protein; Brenner C et al.; Fragile histidine triad protein (Fhit) is a diadenosine triphosphate (ApppA) hydrolase encoded at the human chromosome 3 fragile site which is frequently disrupted in tumors . Reintroduction of FHIT coding sequences to cancer cell lines with FHIT deletions suppressed the ability of these cell lines to form tumors in nude mice even when the reintroduced FHIT gene had been mutated to allow ApppA binding but not hydrolysis . Because this suggested that the tumor suppressor activity of Fhit protein depends on substrate-dependent signaling rather than ApppA catabolism, we prepared two crystalline forms of Fhit protein that are expected to model its biologically active, substrate-bound state . Wild-type and the His96Asn forms of Fhit were overexpressed in Escherichia coli, purified to homogeneity and crystallized in the presence and absence of ApppA and an ApppA analog . Single crystals obtained by vapor diffusion against ammonium sulfate diffracted X-rays to beyond 2.75 A resolution . High quality native synchrotron X-ray data were collected for an orthorhombic and a hexagonal crystal form.

Protein Eng, 1997 Dec, 10(12), 1453 - 9
A form of anti-Tac(Fv) which is both single-chain and disulfide stabilized: comparison with its single-chain and disulfide-stabilized homologs; Rajagopal V et al.; Disulfide-stabilized Fvs (dsFvs) are recombinant proteins composed of a heavy-chain variable domain (VH) of an antibody connected via a disulfide bond to the light-chain variable domain (VL) . In single-chain Fvs (scFvs), a peptide connector links VH and VL . The dsFv form of the anti-Tac monoclonal antibody which reacts with the alpha subunit of the IL2 receptor was recently reported to be more stable and to aggregate less during renaturation than anti-Tac(scFv) . In addition, it could be produced in a better yield owing to less aggregation . However, the yields are still too low to permit the production of material for clinical trials in which the dsFv will be used to image or treat IL2 receptor (CD25)-containing tumors . To increase the efficiency by which VH and VL associate and form a disulfide bond during renaturation, we have prepared an Fv form of anti-Tac which is both single chain and disulfide stabilized (scdsFv) . The recombinant protein is expressed in Escherichia coli, where it accumulates in inclusion bodies . Using inclusion body protein as the reference point, the yield of purified anti-Tac(scdsFv) was 13% compared with 2% for anti-Tac(dsFv) . Anti-Tac(scdsFv) has equivalent binding affinity, immunoreactivity after radiolabeling and stability . The results show that a linker between VH and VL facilitates heterodimer formation and leads to disulfide bond formation in a higher percentage of the molecules renatured . Thus anti-Tac(scdsFv) is the preferred form of anti-Tac(Fv) to be used for clinical studies . We anticipate that scdsFvs will be the optimum recombinant form of Fv to produce from bacteria.

Protein Eng, 1997 Dec, 10(12), 1425 - 32
Structural and functional roles of a conserved proline residue in the alpha2 helix of Escherichia coli thioredoxin; de Lamotte-Guery F et al.; Proline 40 in Escherichia coli thioredoxin is located close to the redox active site (Cys32-Cys35) within the alpha2 helix . The conservation of this residue among most of the thioredoxins suggests that it could play an important role in the structure and/or function of this protein . We have substituted Pro40 for Ala by using site-directed mutagenesis and expressed the mutant P40A in E.coli . The effects of the mutation on the biophysical and biological properties of thioredoxin have been analyzed and compared with molecular dynamics simulations . Modeling predicted that the replacement of Pro40 by Ala induced a displacement of the active site which exposes Trp31 to the solvent and opens a cleft located between helices alpha2 and alpha3 . The solvation free energy (SFE) calculation also indicated that P40A became more hydrophobic as W31 became more accessible . These predictions were totally in agreement with the experimental results . The mutant P40A exhibited chromatographic behavior and fluorescence properties very different from those of the wild-type (WT) protein, in relationship with the displacement of W31 . The determination of the free energy of unfolding of P40A showed that the mutant was globally destabilized by 2.9 kcal/mol . However, the effect of the mutation on the transition curve was highly unusual as the midpoint of the unfolding transition increased, indicating that some local structures were actually stabilized by the mutation . Despite these structural modifications, neither the ability of the protein to reduce a chloroplastic enzyme nor its reactivity with the bacterial reductase decreased . The only functional difference was the higher stability of P40A in light activation of NADP-malate dehydrogenase under air, which suggests that the mutant was less rapidly re-oxidized than WT . Therefore, it can be concluded that Pro40 is not essential for maintaining the redox function of thioredoxin but rather is required for the stability of the protein.

J Clin Microbiol, 1998 Apr, 36(4), 857 - 61
Enzyme-linked immunosorbent assay using recombinant OspC and the internal 14-kDa flagellin fragment for serodiagnosis of early Lyme disease; Rauer S et al.; The outer surface protein C (OspC) and the internal 14-kDa flagellin fragment of strain GeHo of Borrelia burgdorferi sensu stricto were expressed as recombinant proteins in Escherichia coli and were purified for use in an immunoglobulin M (IgM) enzyme-linked immunosorbent assay (OspC-14-kDa antigen ELISA) . No hint at disturbing protein-protein interferences, which might influence the availability of immunoreactive epitopes, was found when the recombinant antigens were combined in the ELISA . The recombinant OspC-14-kDa antigen ELISA was compared to a commercial IgM ELISA that used a detergent cell extract from Borrelia afzelii PKo as the antigen . According to the manufacturer's information, the cell extract contains, in addition to other antigens, the following diagnostically relevant antigens: the 100-kDa (synonyms, 93- and 83-kDa antigens), 41-kDa, OspA, OspC, and 17-kDa antigens . The specificity was adjusted to 95% on the basis of data for 154 healthy controls . On testing of 104 serum samples from patients with erythema migrans (EM), the sensitivity of the recombinant ELISA (46%) for IgM antibodies was similar to that of the commercial ELISA (45%) . However, when 42 serum samples from patients with polyclonal B-cell stimulation due to an Epstein-Barr virus infection were tested, false-positive reactions were significantly less frequent in the recombinant ELISA (10%) than in the whole-cell-extract ELISA (23%) . OspC displays sequence heterogeneity of up to 40% according to the genomospecies . However, when the reactions of serum specimens from controls and EM patients with OspC from representative strains of B . burgdorferi sensu stricto (strain GeHo) and B . afzelii (strain PKo) were compared in an ELISA, almost no differences in specificity and sensitivity were seen . This demonstrates that the sera predominantly recognize the common epitopes of OspC tested in this study . In conclusion, we suggest that the OspC-14-kDa antigens ELISA is a suitable test for the detection of an IgM response in early Lyme disease.

Plant Physiol, 1998 Apr, 116(4), 1339 - 50
Cloning and characterization of AtRGP1 . A reversibly autoglycosylated arabidopsis protein implicated in cell wall biosynthesis; Delgado IJ et al.; A reversibly glycosylated polypeptide from pea (Pisum sativum) is thought to have a role in the biosynthesis of hemicellulosic polysaccharides . We have investigated this hypothesis by isolating a cDNA clone encoding a homolog of Arabidopsis thaliana, Reversibly Glycosylated Polypeptide-1 (AtRGP1), and preparing antibodies against the protein encoded by this gene . Polyclonal antibodies detect homologs in both dicot and monocot species . The patterns of expression and intracellular localization of the protein were examined . AtRGP1 protein and RNA concentration are highest in roots and suspension-cultured cells . Localization of the protein shows it to be mostly soluble but also peripherally associated with membranes . We confirmed that AtRGP1 produced in Escherichia coli could be reversibly glycosylated using UDP-glucose and UDP-galactose as substrates . Possible sites for UDP-sugar binding and glycosylation are discussed . Our results are consistent with a role for this reversibly glycosylated polypeptide in cell wall biosynthesis, although its precise role is still unknown.

Plant Physiol, 1998 Apr, 116(4), 1271 - 8
The GA2 locus of Arabidopsis thaliana encodes ent-kaurene synthase of gibberellin biosynthesis; Yamaguchi S et al.; The ga2 mutant of Arabidopsis thaliana is a gibberellin-deficient dwarf . Previous biochemical studies have suggested that the ga2 mutant is impaired in the conversion of copalyl diphosphate to ent-kaurene, which is catalyzed by ent-kaurene synthase (KS) . Overexpression of the previously isolated KS cDNA from pumpkin (Cucurbita maxima) (CmKS) in the ga2 mutant was able to complement the mutant phenotype . A genomic clone coding for KS, AtKS, was isolated from A . thaliana using CmKS cDNA as a heterologous probe . The corresponding A . thaliana cDNA was isolated and expressed in Escherichia coli as a fusion protein . The fusion protein showed enzymatic activity that converted {3H}copalyl diphosphate to {3H}ent-kaurene . The recombinant AtKS protein derived from the ga2-1 mutant is truncated by 14 kD at the C-terminal end and does not contain significant KS activity in vitro . Sequence analysis revealed that a C-2099 to T base substitution, which converts Gln-678 codon to a stop codon, is present in the AtKS cDNA from the ga2-1 mutant . Taken together, our results show that the GA2 locus encodes KS.

J Mol Biol, 1998 Apr 3, 277(3), 723 - 32
Recognition of protein substrates by the prolyl isomerase trigger factor is independent of proline residues; Scholz C et al.; The trigger factor is associated with bacterial ribosomes and catalyzes proline-limited protein folding reactions . Its folding activity is very high and conserved in evolution, as shown for the homologous enzymes from Escherichia coli and Mycoplasma genitalium . The folding protein substrate (a variant of ribonuclease T1) binds with high affinity to the trigger factors, and permanently unfolded proteins are strong, competitive inhibitors . We used this inhibition to characterize the substrate binding sites of the trigger factors . Unfolded alpha-lactalbumin binds very tightly and inhibits the trigger factor from M . genitalium with a KI value of 50 nM . The binding of inhibitory proteins is independent of proline residues, as shown for unfolded tendamistat, which binds to the trigger factor with equal affinity in the presence and in the absence of its three proline residues . The good inhibition by a non-folding variant of ribonuclease T1 that lacks Pro39 showed that this proline, at which the catalysis of folding occurs, is dispensable for substrate binding . The trigger factors cannot catalyze prolyl isomerization when proteins are partially folded already . They preferentially recognize unstructured protein chains, which bind with high affinity to a site distinct from the catalytic prolyl isomerase center in the FKBP domain .

J Mol Biol, 1998 Apr 3, 277(3), 707 - 22
Structural stability and internal motions of Escherichia coli ribonuclease HI: 15N relaxation and hydrogen-deuterium exchange analyses; Yamasaki K et al.; The relationship between the structural stability and the internal motions of proteins was investigated through measurements of 15N relaxation and hydrogen-deuterium exchange rates of ribonuclease HI from Escherichia coli and its thermostable quintuple mutant (Gly23-->Ala, His62-->Pro, Val74-->Leu, Lys95-->Gly, and Asp134-->His), which has a higher melting temperature by 20.2 degreesC . For most of the residues, the generalized order parameters (S2) obtained from 15N relaxation analyses as well as the localized hydrogen-bond-breaking motions (local breathing) observed as fast H-D exchange rates were largely unaffected by the mutations, indicating no global mutational effect on the internal motions . Several local mutational effects were observed for residues close to the mutation sites as follows . The S2 value significantly increased for Lys96 and Val98, which indicated that motions on the pico- to nanosecond time-scale became restricted within a protruding region including the Lys95-->Gly mutation site . In contrast, slight decreases in S2, and drastic increases in the chemical exchange motion on the micro- to millisecond time-scale (Deltaex), were observed for residues located in the joining region between the protrusion and the major domain of the protein . These changes may be caused by the elimination of the bulky Lys95 side-chain at the center of the protrusion . Deltaex observed for residues in alpha-helix I of the wild-type protein was reduced for the mutant, probably because a cavity in the hydrophobic core is filled by the Val74-->Leu mutation . The local breathing at position 134 was restricted by the Asp134-->His mutation, probably because the reduction of the negative charge repulsion contributes to the stability of the native major conformation relative to the breathing conformations around position 134 .

Zhonghua Yi Xue Yi Chuan Xue Za Zhi, 1998 Apr 10, 15(2), 69 - 71
{Expression of human ciliary neurotrophic factor gene in Escherichia coli}; Huang X et al.; OBJECTIVE: To express biologically active human ciliary neurotrophic factor(hCNTF) gene in Escherichia coli . METHODS: Total RNA was extracted from human peripheral nerves and cDNA was synthesized by superscript reverse-transcriptase, a polymerase chain reaction(PCR) was conducted to obtain a full length cDNA fragment encode for hCNTF gene . After recovery from gel and purification, the PCR product was cloned into the pGEM-5Zf(+) vector and DNA sequence analysis was performed to verify hCNTF gene . The hCNTF gene was then placed under control of T7 promoter in the expression vector pET-11d and transformed into Escherichia coli strain BL21(DE3) . Cultures of this transformat were induced by IPTG for the expression of recombinant protein . The bioactivity of recombinant protein was evaluated by its ability to support the survival of embryonic chick dorsal root ganglion neurons in culture . RESULTS: The human CNTF gene was cloned and biologically active hCNTF was expressed efficiently . Based on densitometry of stained gel,the recombinant hCNTF accounted for more than 25% of the total bacterial protein . CONCLUSION: The cloning and expression at high level of hCNTF gene in E.coli provides a basis for understanding the structure-activity relationship of CNTF and its potential clinical application.

Gene, 1998 Mar 27, 210(1), 103 - 8
Cloning and expression of caprine interferon-gamma; Beyer JC et al.; Caprine interferon-gamma (IFN-gamma) cDNA was cloned from mitogen stimulated peripheral blood mononuclear cell (PBMC) RNA utilizing the reverse transcription-polymerase chain reaction (RT-PCR) . The cDNA open reading frame (ORF) is 498bp, encoding a putative 166 amino acid (aa) protein (19327Da) . The predicted aa sequence homology of caprine IFN-gamma and the corresponding ovine, bovine and cervine cytokine is 98.8%, 95.2% and 92.8%, respectively . IFN-gamma cDNA was subcloned and expressed in two different plasmids under the control of either the human cytomegalovirus (CMV) immediate early promoter or the caprine arthritis-encephalitis virus long terminal repeat (CAEV LTR) . Recombinant caprine IFN-gamma (rCaIFN-gamma) secreted by transfected COS-7 cells shared at least two antigenic epitopes with recombinant bovine IFN-gamma (rBoIFN-gamma) and exhibited biological activity in the vesicular stomatitis virus (VSV) cytopathic effect reduction assay . In-vivo expression of IFN-gamma cDNA promoted by the CAEV LTR was confirmed by the intramuscular (IM) injection of Balb/C mice with plasmid followed by Western blot analysis of mouse serum against purified rCaIFN-gamma produced in E . coli.

Curr Genet, 1998 Feb, 33(2), 157 - 63
Integrative transformation of the dimorphic yeast arxula adeninivorans LS3 based on hygromycin B resistance; Rosel H et al.; A transformation system has been developed for the dimorphic yeast Arxula adeninivorans based on a stable integration of the donor DNA into ribosomal DNA . For this purpose a cassette was constructed which contains the E . coli hph gene, conferring hygromycin B resistance, fused to the 5' expression signals of the A . adeninivorans TEF1 gene, encoding the translation elongation factor EF-1alpha, and the transcription termination region of the Saccharomyces cerevisiae PHO5 gene . This cassette was fused into the 25S rDNA of A . adeninivorans . Linearization of this vector was required for high transformation frequencies . The vector was integrated in multiple copies into the 25S rDNA by homologous recombination . Copy number was not altered even after the growth of transformants for 15 generations under non-selective growth conditions . Microscopical analyses revealed that integration of the transformed plasmid did not influence the dimorphism, which is triggered at 42 degrees C for both transformed and non-transformed cells.

Curr Genet, 1998 Feb, 33(2), 124 - 30
Small insertions at a shared position in the SSU rDNA of lecanorales (lichen-forming ascomycetes); Stenroos S et al.; Small insertions are reported from the SSU rDNA of the genera Cladonia, Cladina, Stereocaulon, Pertusaria and Physcia (lichen-forming Lecanorales, Ascomycetes) . The insertions range in length from 56 to 81 nucleotides, and occur at a shared position, 330 (relative to Escherichia coli), in a semi-conserved region of the SSU rDNA . These small insertions have a simple secondary structure with two stem-loops, and may represent degenerate group-I introns . Species of the same genus often have small insertions of similar lengths, secondary structures and sequences . These similarities suggest that the insertions are homologous and can provide phylogenetic information to support current classifications . Variable sequences of rDNA, such as the small insertions and group-I introns, may be powerful tools for resolving evolutionary relationships among genera and species.

Biochem J, 1998 Mar 15, 330 ( Pt 3), 1325 - 32
The inhibitory effect of novel triterpenoid compounds, fomitellic acids, on DNA polymerase beta; Mizushina Y et al.; We previously found new triterpenoid compounds, designated fomitellic acid A and B, which selectively inhibit the activities of mammalian DNA polymerase alpha and beta in vitro . On DNA polymerase beta, the fomitellic acids acted by competing with both the substrate and the template primer, but on DNA polymerase alpha, they acted non-competitively . At least on DNA polymerase beta, the evidence suggests that each of the fomitellic acids bind to the active region competing with the substrate and/or template primer, and subsequently inhibits the catalytic activity . We therefore further investigated the enzyme-binding properties by using DNA polymerase beta and its proteolytic fragments . The 39 kDa enzyme was proteolytically separated into two fragments of the template-primer-binding domain (8 kDa) and the catalytic domain (31 kDa) . The fomitellic acids bound tightly to the 8 kDa fragment, but not to the 31 kDa fragment . The immuno-precipitation by antibodies against the enzyme or each of the fragments also proved the binding . These results suggest that the fomitellic acid molecule competes with the template-primer molecule on its 8 kDa binding site, binds to the site, and the fomitellic acid molecule simultaneously disturbs the substrate incorporation into the template primer.

Biochem J, 1998 Mar 15, 330 ( Pt 3), 1229 - 34
Identification of the major allergens in wheat flour responsible for baker's asthma; Amano M et al.; Baker's asthma, a typical occupational allergic disease, is a serious problem in the food industries . In this study, purification and identification of major allergens recognized by IgEs in sera of allergic patients were performed . Major immunoreactive proteins were purified from the albumin fraction by gel filtration on a Toyopearl HW-50 column followed by reverse-phase HPLC . The N-terminal amino acid sequences and molecular masses measured by MS indicated that the major immunoreactive proteins are members of the alpha-amylase inhibitor family, 0.19 and 0.28 . Significant leukotriene release by each purified protein was observed in cell-associated stimulation tests, suggesting in vivo activity of these antigens . Carbohydrate analyses of major allergens indicated that they are monoglycosylated but not N-glycosylated in spite of the presence of a potential N-glycosylation site . Recombinant 0.19 expressed in Escherichia coli showed the same reactivity with IgE as native wheat 0.19 in Western blotting and ELISA using methyl vinyl ether maleic anhydride co-polymer as an immobilizing reagent, suggesting that the allergenic epitopes are located in the peptide portions.

Biochem J, 1998 Mar 15, 330 ( Pt 3), 1217 - 21
Recombinant expression and biochemical characterization of an NADPH:flavin oxidoreductase from Entamoeba histolytica; Bruchhaus I et al.; The gene encoding a putative NADPH:flavin oxidoreductase of the protozoan parasite Entamoeba histolytica (Eh34) was recombinantly expressed in Escherichia coli . The purified recombinant protein (recEh34) has a molecular mass of about 35 kDa upon SDS/PAGE analysis, exhibits a flavoprotein-like absorption spectrum and contains 1 mol of non-covalently bound FMN per mol of protein . RecEh34 reveals two different enzymic activities . It catalyses the NADPH-dependent reduction of oxygen to hydrogen peroxide (H2O2), as well as of disulphides such as 5,5'-dithiobis-(2-nitrobenzoic acid) (DTNB) and cystine . The disulphide reductase but not the H2O2-forming NADPH oxidase activity is inhibitable by sulphydryl-active compounds, indicating that a thiol component is part of the active site for the disulphide reductase activity, whereas for the H2O2-forming NADPH oxidase activity only the flavin is required . Compared with the recombinant protein, similar activities are present in amoebic extracts . Native Eh34 is active in a monomeric as well as in a dimeric state . In contrast to recEh34, no flavin was associated with the native protein . However, both NADPH oxidase as well as DTNB reductase activity were found to be dependent on the addition of FAD or FMN.

Biochem J, 1998 Mar 15, 330 ( Pt 3), 1079 - 85
Biotin synthase from Escherichia coli: isolation of an enzyme-generated intermediate and stoichiometry of S-adenosylmethionine use; Shaw NM et al.; A cell-free extract from Escherichia coli containing an E . coli biotin synthase that was expressed to approx . 1% of soluble cell protein by cloning the E . coli bioB gene was used to investigate the biotin synthase reaction . The pH optimum was between 8 and 8.5, and the reaction velocity was dependent on the concentrations of dethiobiotin, cysteine, S-adenosylmethionine and asparagine . The catalytic-centre activity of the enzyme in vitro was estimated to be 0.95 h-1, and each molecule of enzyme turned over less than one molecule of dethiobiotin, i.e . the enzyme was not acting catalytically . HPLC analysis of reaction mixtures revealed the presence of a compound with the characteristics of an intermediate: (1) it was labelled with 14C, and therefore derived from the {14C}dethiobiotin substrate; (2) it was present only in reaction mixtures containing biotin synthase; (3) it was not derived from {14C}biotin; (4) 35S from {35S}cystine was incorporated into the intermediate during the reaction; (5) its synthesis was dependent on the presence of S-adenosylmethionine, and was decreased when free cysteine was omitted from the reaction; (6) it could be isolated from the reaction mixture by chromatography and then re-introduced into an assay as the substrate, whereupon it was converted to biotin; (7) this conversion to biotin was S-adenosylmethionine-dependent . During the reaction S-adenosylmethionine was cleaved to methionine and presumably 5'-deoxyadenosine . Observation of the intermediate allowed us to perform experiments to determine the stoichiometry of S-adenosylmethionine use . We propose that two molecules of S-adenosylmethionine are used to synthesize one molecule of biotin, i.e . one from dethiobiotin to the intermediate, and a second from the intermediate to biotin.

FEMS Microbiol Lett, 1998 May 1, 162(1), 135 - 41
The leuX-encoded tRNA5(Leu) but not the pathogenicity islands I and II influence the survival of the uropathogenic Escherichia coli strain 536 in CD-1 mouse bladder mucus in the stationary phase; Dobrindt U et al.; The uropathogenic Escherichia coli strain 536 carries two pathogenicity islands, each of which is associated with either of the tRNA genes selC or leuX, respectively . Growth competition in CD-1 mouse mucus between the wild-type strain E . coli 536, its leuX mutant 536 delta 102 and its mutant 536R3, lacking both pathogenicity islands but expressing a functional tRNA5(Leu), revealed a major impact of leuX on E . coli survival in bladder mucus . The impaired survival in CD-1 mouse mucus observed upon deletion of the leuX gene was abolished after complementation with the leuX gene . The survival of bacteria in bladder mucus was not influenced by the presence of pathogenicity islands I and II.

FEMS Microbiol Lett, 1998 May 1, 162(1), 17 - 23
Differential temperature modulation by H-NS of the fimB and fimE recombinase genes which control the orientation of the type 1 fimbrial phase switch; Olsen PB et al.; Phase variation of type 1 fimbriation in Escherichia coli is associated with the inversion of a 314-bp DNA element . This DNA switch directs transcription of fimA, encoding the major type 1 fimbrial subunit, in the on orientation but not in the off orientation . Inversion of the DNA element requires either FimB (confers both on and off orientations) or FimE (confers off orientation) . Here we show, by chromosomally located fimB- and fimE-lacZ cassettes in isogenic strain sets differing only in the hns locus, how the global regulator H-NS affects the expression of type 1 fimbriae . H-NS was found to downregulate fimB and fimE in a temperature-dependent manner which affected the genes inversely at 30 degrees C and 37 degrees C . By gel-retardation assays H-NS binding was demonstrated to the regions containing the fimB promoter and the fimE promoter, respectively . In vitro recombination analysis suggested no direct involvement of H-NS in the inversion of the phase switch . Rather than directly affecting the switching process per se, it appeared that the orientation of this element was affected by the differential temperature modulation of H-NS of the fimB and fimE genes . Taken together the results suggest that H-NS modulates expression of type 1 fimbriae in a way which seems to favor a fimbriate state at the mammalian body temperature.

Mol Membr Biol, 1998 Jan-Mar, 15(1), 15 - 20
In vitro folding of a membrane protein: effect of denaturation and renaturation on substrate binding by the lactose permease of Escherichia coli; He MM et al.; Site-directed mutagenesis and site-directed fluorescence spectroscopy demonstrate that Cys148 interacts hydrophobically with the galactosyl moiety of substrates of the lactose permease of Escherichia coli . By taking advantage of the finding that labelling of single-Cys148 permease with the thiol-specific fluorophore 2-(4'-maleimidylanilino)naphthalene-6-sulfonic acid (MIANS) is blocked specifically by substrates of the permease, it is demonstrated that the high-affinity ligand beta,D-galactopyranosyl 1-thio-beta,D-galactopyranoside (TDG) stabilizes solubilized, purified permease against heat denaturation . Furthermore, TDG protection against MIANS labelling of single-Cys148 permease is abolished by guanidinium hydrochloride . After dialysis of the denaturant, TDG protection against MIANS labelling is recovered, indicating that the permease has been refolded . The conclusion is confirmed and extended by studying site-directed fluorescence of purified single-Cys331 permease, where the emission spectrum of the MIANS-labelled protein is differentially altered by low or high concentrations of TDG . The results demonstrate that both low- and high-affinity binding, as well as ligand-induced conformational changes in the permease, can be denatured reversibly in vitro.

J Cardiovasc Pharmacol, 1998, 31 Suppl 1, S548 - 50
In vivo role of endothelin-converting enzyme-1 as examined by adenovirus-mediated overexpression in rats; Telemaque S et al.; We previously reported that adenovirus-mediated overexpression of endothelin-1 (ET-1) elevates systemic blood pressure in rats . In this model, plasma big ET-1: ET-1 ratios were almost 30, whereas they were only 5 in the control group, suggesting that endothelin-converting enzyme (ECE) may be a rate-limiting step in the production of ET-1 under these conditions . To further investigate the role of ECE in vivo, we prepared recombinant adenovirus strains carrying a soluble, secretory form of bovine ECE-1 cDNA (Ad.CMV . secECE), human ET-1 cDNA (Ad.CMV.ET-1), and, as a control, E . coli lacZ (Ad.CMV.beta-gal) . Ad.CMV.secECE (1-10 x 10(9) pfu/ml) was injected into the caudal vein of male Wistar rats and the animals were studied 96 h later . Immunoblot analysis of circulating plasma confirmed the expression of the soluble ECE-1 . The plasma levels of big ET-1 and mature ET-1 were similar in Ad.CMV.secECE and Ad.CMV.beta-gal groups (0.3-0.5 pM) . When Ad.CMV.secECE was co-injected with Ad.CMV.ET-1 (2.5 x 10(9) pfu/ml each), plasma ET-1 levels were significantly elevated compared to the control group co-injected with Ad.CMV.secECE and Ad.CMV.beta-gal (10.2 +/- 2.4 vs . 1.1 +/- 0.2 pM) . Big ET-1 levels were threefold higher (3.7 +/- 1.1 vs . 1.2 +/- 0.4 pM), and systemic blood pressure was significantly elevated (132 +/- 3 vs . 90 +/- 3 mm Hg) in the Ad.CMV.secECE + Ad.CMV.ET-1 group . Administration of an ECE inhibitor (CGS 26303, 30 mg/kg) significantly reduced the blood pressure in the Ad.CMV.secECE + Ad.CMV.ET-1 group (from 125 +/- 5 to 74 +/- 6 mm Hg) but not in the control group (from 85 +/- 2 to 75 +/- 3 mm Hg) . Infusion of an ETA antagonist (FR 139317; 0.2 mg/kg/min for 30 min) also significantly reduced the blood pressure only in the Ad.CMV.secECE + Ad.CMV.ET-1 group, without any significant effect in the control group . This study demonstrates that even though overexpression of ECE-1 in itself does not lead to systemic hypertension, the enzyme can be a crucial rate-limiting factor in the production of mature ET-1 in vivo . Furthermore, this model may prove to be useful for in vivo screening of ECE inhibitors.

J Cardiovasc Pharmacol, 1998, 31 Suppl 1, S518 - 20
Effects of endothelin-1 on inflammatory incapacitation of the rat knee joint; De-Melo JD et al.; This study assessed the possible local nociceptive and hyperalgesic properties of endothelin-1 (ET-1) in the rat knee-joint incapacitation test, in which animals are placed for 1 min/h on a revolving (3 rpm) metal cylinder and nociception is measured as the time the hindpaw of the injected limb was off the cylinder (i.e., paw elevation time, PET) . Carrageenan (Cg; 150 micrograms/joint), E . coli LPS (1 microgram/joint), and ET-1 (120 or 240 pmol/joint) each increased PET persistently, unlike sarafotoxin S6c (120-240 pmol/joint) or PBS . ET-1 (15 and 30 pmol/joint, 30 min before) did not cause incapacitation per se but potentiated PET induced by Cg, increasing the area under the curve (AUC in arbitrary units, 0-6 h) from 105 +/- 9 to 165 +/- 10 and 169 +/- 25, respectively . Prior Cg injection (300 micrograms/joint, 72 h before) sensitized the joint to incapacitation triggered by restimulation with either Cg (300 micrograms/joint), LPS (1 microgram/joint), or ET-1 (30 pmol/joint) . Treatment with bosentan (10 mg/kg i.v., 15 min before joint stimulation) did not affect PET values in naive animals to Cg or LPS, but significantly reduced the upregulated response evoked by restimulation with LPS (but not Cg), from 465 +/- 24 to 290 +/- 49 (AUC 0-12 h) . Therefore, ET-1 triggers nociception and hyperalgesia in the naive knee joint of the rat, perhaps via ETA receptors . Although local endogenous ETs may not have a role in inflammatory nociception in the naive joint, they may participate in articular incapacitation induced by restimulation with LPS . This latter finding could be relevant to the etiology of pain associated with chronic arthritic diseases.

J Cardiovasc Pharmacol, 1998, 31 Suppl 1, S233 - 5
Pathologic role of endothelin-1 in septic shock; Mitaka C et al.; To elucidate the pathologic role of endothelin-1 (ET-1) in septic shock, we measured plasma ET-1 concentrations after bacterial lipopolysaccharide (LPS) administration in dogs and determined systemic, pulmonary, and renal hemodynamics and blood gas parameters with or without the nonselective ET receptor antagonist TAK-044 . Plasma ET-1 concentrations increased significantly after LPS administration, which correlated positively with mean arterial pressure, mean pulmonary arterial pressure, pulmonary capillary wedge pressure, and central venous pressure . LPS infusion induced hypotension, metabolic acidosis, hypoxemia, and renal dysfunction . TAK-044 prevented LPS-induced metabolic acidosis, hypoxemia, and renal dysfunction, but not hypotension . These findings suggest that increased circulating ET-1 plays a compensatory role in the reversal of systemic vasodilatation in septic shock, but exerts deleterious effects on renal and pulmonary circulation.

Rinsho Byori, 1998 Apr, 46(4), 303 - 10
{Analysis of autoantigens and clinical significance of antinuclear antibodies}; Mimori T et al.; A highly sensitive RNA-immunoprecipitation assay (Lerner-Steitz assay) is a unique and useful method of identifying autoantibodies to RNA-associated antigens . In this study, we identified novel autoantibodies to tRNAs using RNA-immunoprecipitation assay . In screening of 1472 sera by RNA-immunoprecipitation using HeLa cell extracts as an antigen source, 41 sera were found to immunoprecipitate tRNAs . Fifteen of these 41 sera also immunoprecipitated deproteinized tRNAs, indicating that these 15 sera contained anti-tRNA antibodies . Three sera immunoprecipitated naked tRNA from E . coli . When in vitro transcripts from cDNAs encoding E . coli tRNAs and their synthesized mutants were used as antigens, it was demonstrated that the representative serum recognized the conformational epitope of the "L"-shape structure which was conserved in all tRNAs of all species . This finding suggests the role of bacterial infection in the development of autoantibodies and autoimmune diseases . Two of 15 sera containing anti-tRNA antibodies were identified as anti-PL-12 (alanyl-tRNA synthetase) antibodies . Eleven of the remaining 13 patients were diagnosed as either SLE, Sjogren's syndrome or their overlap . In addition, fever, Raynaud's phenomenon, polyarthritis, leukocytopenia and characteristic annular erythema were frequently found in these patients . Novel autoantibodies to tRNAs appeared to be associated with common clinical features but were distinct from previously described anti-aminoacyl-tRNA synthetases.

Plant Physiol, 1998 May, 117(1), 245 - 54
Cloning, expression in Escherichia coli, and characterization of Arabidopsis thaliana UMP/CMP kinase; Zhou L et al.; A cDNA encoding the Arabidopsis thaliana uridine 5'-monophosphate (UMP)/cytidine 5'-monophosphate (CMP) kinase was isolated by complementation of a Saccharomyces cerevisiae ura6 mutant . The deduced amino acid sequence of the plant UMP/CMP kinase has 50% identity with other eukaryotic UMP/CMP kinase proteins . The cDNA was subcloned into pGEX-4T-3 and expressed as a glutathione S-transferase fusion protein in Escherichia coli . Following proteolytic digestion, the plant UMP/CMP kinase was purified and analyzed for its structural and kinetic properties . The mass, N-terminal sequence, and total amino acid composition agreed with the sequence and composition predicted from the cDNA sequence . Kinetic analysis revealed that the UMP/CMP kinase preferentially uses ATP (Michaelis constant {Km} = 29 microM when UMP is the other substrate and Km = 292 microM when CMP is the other substrate) as a phosphate donor . However, both UMP (Km = 153 microM) and CMP (Km = 266 microM) were equally acceptable as the phosphate acceptor . The optimal pH for the enzyme is 6.5 . P1, P5-di(adenosine-5') pentaphosphate was found to be a competitive inhibitor of both ATP and UMP.

Proteins, 1998 May 1, 31(2), 214 - 23
Circular permutants in beta-glucosidases (family 3) within a predicted double-domain topology that includes a (beta/alpha)8-barrel; Garcia-Vallve S et al.; By predicting the general secondary structure for beta-glucosidases (family 3), in conjunction with existing knowledge of the circular permutants present in B . fibrisolvens and R . albus, we were able to find the canonical elements of the secondary structure . The way these elements are linked suggests that there is a double-domain topology made up of a (beta/alpha)8-barrel domain and a "mainly all-beta" domain . A number of already known conserved motifs are located within (or near) the C-terminal part of the putative parallel beta-strands of the (bet/alpha)8-barrel, which is consistent with what is known about the location of catalytical sites for enzymes that have this domain topology . Within the circular permutants, two beta/alpha units are located at the N-terminal part of the molecule, whereas the other six beta/alpha units are located at the C-terminal end . In this way, the circular permutants can be seen to have a putative discontinuous double-domain topology.

Nucleic Acids Res, 1998 Jun 1, 26(11), 2771 - 8
Elements in abasic site recognition by the major human and Escherichia coli apurinic/apyrimidinic endonucleases; Erzberger JP et al.; Sites of base loss in DNA arise spontaneously, are induced by damaging agents or are generated by DNA glycosylases . Repair of these potentially mutagenic or lethal lesions is carried out by apurinic/apyrimidinic (AP) endonucleases . To test current models of AP site recognition, we examined the effects of site-specific DNA structural modifications and an F266A mutation on incision and protein-DNA complex formation by the major human AP endonuclease, Ape . Changing the ring component of the abasic site from a neutral tetrahydrofuran (F) to a positively charged pyrrolidine had only a 4-fold effect on the binding capacity of Ape . A non-polar 4-methylindole base analog opposite F had a <2-fold effect on the incision activity of Ape and the human protein was unable to incise or specifically bind 'bulged' DNA substrates . Mutant Ape F266A protein complexed with F-containing DNA with only a 6-fold reduced affinity relative to wild-type protein . Similar studies are described using Escherichia coli AP endonucleases, exonuclease III and endonuclease IV . The results, in combination with previous findings, indicate that the ring structure of an AP site, the base opposite an AP site, the conformation of AP-DNA prior to protein binding and the F266 residue of Ape are not critical elements in targeted recognition by AP endonucleases.

Nucleic Acids Res, 1998 Jun 1, 26(11), 2715 - 22
Selection and characterization of RNAs that relieve transcriptional interference in Escherichia coli; Soukup GA et al.; Oligonucleotide-directed triple helix formation offers a method for duplex DNA recognition, and has been proposed as an approach to the rational design of gene-specific repressors . Indeed, certain RNA and DNA oligonucleotides have previously been shown to bind duplex DNA and repress in vitro transcription by occluding the binding of transcription factors or RNA polymerase at target genes . While similar oligonucleotides have reportedly caused repression of target genes in cultured cells, physical evidence of triple helix formation in vivo is generally lacking . In the present study we wished to determine whether RNA transcripts could repress the activity of an Escherichia coli promoter in vivo by binding to the duplex promoter DNA . An in vivo genetic selection previously developed to identify DNA binding proteins was modified for this purpose . Using expression libraries encoding RNAs predisposed to forming triple helices with a DNA target site, we have selected RNA transcripts that confer survival to E.coli by disrupting transcriptional interference . Surprisingly, genetic and biochemical evidence shows that these RNAs do not form triple helices at the target promoter in vivo , despite the fact that they contain sequences capable of forming triple helices at the duplex DNA target in vitro . Rather, the selected RNAs appear to disrupt transcriptional interference via an antisense mechanism.

Leuk Res, 1998 Feb, 22(2), 115 - 7
Chronic myelogenous leukemia: effect of interferon-alpha treatment on phagocytic activity and capacity of circulating neutrophils; Kasimir-Bauer S et al.; This study focuses on the effect of interferon (IFN)-alpha on phagocytosis of FITC-labeled Escherichia coli by polymorphonuclear neutrophils (PMNs) in chronic myelogenous leukemia (CML) . The phagocytic activity and capacity of PMNs from IFN-alpha treated patients (n = 17), untreated CML patients (n = 9) and from healthy donors (n = 20) were compared using flow cytometry . Both parameters of PMN phagocytosis were reduced in untreated CML and in IFN-alpha treated CML with Ph1 chromosome persistence but normal in IFN-alpha treated CML with Ph1 conversion . Thus, the phagocytic performance of PMNs in patients with chronic phase CML is significantly improved after successful treatment with IFN-alpha.

Mol Microbiol, 1998 Apr, 28(1), 205 - 16
Involvement of products of the nrfEFG genes in the covalent attachment of haem c to a novel cysteine-lysine motif in the cytochrome c552 nitrite reductase from Escherichia coli; Eaves DJ et al.; Cytochrome c552 is the terminal component of the formate-dependent nitrite reduction pathway of Escherichia coli . In addition to four 'typical' haem-binding motifs, CXXCH-, characteristic of c-type cytochromes, the N-terminal region of NrfA includes a motif, CWSCK . Peptides generated by digesting the cytochrome from wild-type bacteria with cyanogen bromide followed by trypsin were analysed by on-line HPLC MS/MS in parent scanning mode . A strong signal at mass 619, corresponding to haem, was generated by fragmentation of a peptide of mass 1312 that included the sequence CWSCK . Neither this signal nor the haem-containing peptide of mass 1312 was detected in parallel experiments with cytochrome that had been purified from a transformant unable to synthesize NrfE, NrfF and NrfG: this is consistent with our previous report that NrfE and NrfG (but not NrfF) are essential for formate-dependent nitrite reduction . Redox titrations clearly revealed the presence of high and low mid-point potential redox centres . The best fit to the experimental data is for three n=1 components with mid-point redox potentials (pH 7.0) of +45 mV (21% of the total absorbance change), -90 mV (36% of the total) and -210mV (43% of the total) . Plasmids in which the lysine codon of the cysteine-lysine motif, AAA, was changed to the histidine codon CAT (to create a fifth 'typical' haem c-binding motif), or to the isoleucine and leucine codons, ATT and CTT, were unable to transform a Nrf deletion mutant to Nrf+ or to restore formate-dependent nitrite reduction to the transformants . The presence of a 50 kDa periplasmic c-type cytochrome was confirmed by staining proteins separated by SDS-PAGE for covalently bound haem, but the methyl-viologen-dependent nitrite reductase activities associated with the mutated proteins, although still detectable, were far lower than that of the native protein . The combined data establish not only that there is a haem group bound covalently to the cysteine-lysine motif of cytochrome c552 but also that one or more products of the last three genes of the nrf operon are essential for the haem ligation to this motif.

Mol Microbiol, 1998 Apr, 28(1), 143 - 55
Protein translocation into host epithelial cells by infecting enteropathogenic Escherichia coli; Wolff C et al.; Enteropathogenic Escherichia coli (EPEC) causes diarrhoea in young children . EPEC induces the formation of actin pedestal in infected epithelial cells . A type III protein secretion system and several proteins that are secreted by this system, including EspB, are involved in inducing the formation of the actin pedestals . We have demonstrated that contact of EPEC with HeLa cells is associated with the induction of production and secretion of EspB . Shortly after infection, EPEC initiates translocation of EspB, and EspB fused to the CyaA reporter protein (EspB-CyaA), into the host cell . The translocated EspB was distributed between the membrane and the cytoplasm of the host cell . Translocation was strongly promoted by attachment of EPEC to the host cell, and both attachment factors of EPEC, intimin and the bundle-forming pili, were needed for full translocation efficiency . Translocation and secretion of EspB and EspB-CyaA were abolished in mutants deficient in components of the type III protein secretion system, including sepA and sepB mutants . EspB-CyaA was secreted but not translocated by an espB mutant . These results indicate that EspB is both translocated and required for protein translocation by EPEC.

Mol Microbiol, 1998 Apr, 28(1), 95 - 102
Alteration in the contents of unsaturated fatty acids in dnaA mutants of Escherichia coli; Suzuki E et al.; DnaA protein, the initiator of chromosomal DNA replication in Escherichia coli, has a high affinity for acidic phospholipids containing unsaturated fatty acids . We have examined here the fatty acid composition of phospholipids in dnaA mutants . A temperature-sensitive dnaA46 mutant showed a lower level of unsaturation of fatty acids (ratio of unsaturated to saturated fatty acids) at 42 degrees C (non-permissive temperature) and at 37 degrees C (semi-permissive temperature), but not at 28 degrees C (permissive temperature), compared with the wild-type strain . Plasmid complementation analysis revealed that the dnaA46 mutation is responsible for the phenotype . Other temperature-sensitive dnaA mutants showed similar results . On the other hand, a cold-sensitive dnaAcos mutant, in which over-initiation of DNA replication occurs at low temperature (28 degrees C), showed a higher level of unsaturation of fatty acids at 28 degrees C . Based on these observations, we discuss the role of phospholipids in the regulation of the activity of DnaA protein.

Mol Microbiol, 1998 Apr, 28(1), 5 - 13
The Escherichia coli ATP-binding cassette (ABC) proteins; Linton KJ et al.; The recent completion of the Escherichia coli genome sequence (Blattner et al., 1997) has permitted an analysis of the complement of genomically encoded ATP-binding cassette (ABC) proteins . A total of 79 ABC proteins makes this the largest paralogous family of proteins in E . coli . These 79 proteins include 97 ABC domains (as some proteins include more than one ABC domain) and are components of 69 independent functional systems (as many systems involve more than one ABC domain) . The ABC domains are often, but not exclusively, the energy-generating domains of multicomponent membrane-bound transporters . Thus, 57 of the 69 systems are ABC transporters, of which 44 are periplasmic-binding protein-dependent uptake systems and 13 are presumed exporters . The genes encoding these ABC transporters occupy almost 5% of the genome . Of the 12 systems that are not obviously transport related, the function of only one, the excision repair protein UvrA, is known . A phylogenetic analysis suggests that the majority of ABC proteins can be assigned to 10 subfamilies . Together with statistical and, importantly, biological evidence, this analysis provides insight into the evolution and function of the ABC proteins.

J Infect Dis, 1998 May, 177(5), 1398 - 401
In vivo expression of interleukin-1 receptors during various experimentally induced inflammatory conditions; van der Laken CJ et al.; Systemically administered interleukin-1 (IL-1) has been shown to preferentially bind to IL-1 receptors (IL-1Rs) in inflammation . Using radiolabeled IL-1alpha and molecular methods to assess gene expression for these receptors, the in vivo behavior of these receptors was investigated in a number of experimental inflammatory conditions . The uptake of 125I-labeled IL-1alpha in inflammatory foci significantly correlated with the mRNA expression for the type I and type II IL-1Rs (P < .05) . Type II IL-1R mRNA showed a greater increase in expression than type I IL-1R mRNA . In neutropenic mice, inflammatory lesions, which are devoid of granulocytes, significantly lower 125I-labeled IL-1alpha uptake (P < .001), and type II IL-1R mRNA expression (P < .005) was found . Thus, there is strong up-regulation of IL-1Rs at sites of focal inflammation . Of interest, this mainly involved the type II IL-1R on granulocytes, which is not involved in signal transduction.

Rheumatol Int, 1998, 17(6), 237 - 43
Pathological significance of elevated soluble CD14 production in rheumatoid arthritis: in the presence of soluble CD14, lipopolysaccharides at low concentrations activate RA synovial fibroblasts; Yu S et al.; In order to establish what contributes to elevated levels of soluble CD14 (sCD14) in rheumatoid arthritis (RA) plasma, levels of sCD14 were compared in RA-paired plasma and synovial fluids and, further, in the culture supernatants of monocyte-rich fractions from patients with RA and healthy donors, and macrophage-rich fractions from RA synovial tissues . The results showed elevated sCD14 in RA synovial fluid in 9 of 16 paired samples and in RA macrophage-rich fractions, suggesting that elevated sCD14 in RA plasma might be due to the sCD14 production by RA synovial macrophages . From the molecular analysis of elevated sCD14, the proteolytic cleavage of membranous CD14 (mCD14) was important in accelerated sCD14 production . Lipopolysaccharides (LPS) at low concentrations and sCD14 increased the ICAM-1 expression on RA synovial fibroblasts . This result implies that in vivo RA synovial fibroblasts may be sensitive to LPS in the presence of sCD14 and LPS-binding protein (LBP).

Mol Biotechnol, 1998 Feb, 9(1), 17 - 24
Tightly controlled two-stage expression vectors employing the Flp/FRT-mediated inversion of cloned genes; Sektas M et al.; We have developed a tightly controlled, two-stage expression system . It is based on a single plasmid that carries the TetR repressor/Ptet promoter/Otet operator for the first-stage control, and the Flp recombinase/ FRT sites for the second-stage control . The gene to be expressed (GENE) is cloned in an inverted orientation (with respect to the stationary promoter) into a multiple-cloning site (MCS) located between two convergent FRT1 and FRT2 sites . In the OFF stage, no inadvertent transcription can enter the 5' end of cloned GENE because of four rrnBT1 terminators, located just outside the FRT1-MCS-FRT2 cassette and because the FRT2 construct was deprived of any promoter function . When using the lacZ reporter, it was shown that in their OFF stage our two-stage expression plasmids exhibit a significantly lower basal expression than the repressed single-stage tetR/PtetOtet-lacZ vectors . To enter the ON stage, the tetR/PtetOtet module is induced by adding autoclaved chlortetracycline (cTc), leading to synthesis of the Flp recombinase, which in turn, inverts the FRT1-MCS-FRT2 module together with the cloned GENE . This results in the massive GENE expression from one (pInvMS) or two (pImpMS) stationary promoters.

J Vet Med Sci, 1998 Apr, 60(4), 541 - 4
Expression of the glycoprotein E2 of the classical swine fever virus in Escherichia coli; Wong ML et al.; The glycoprotein E2 sequences of classical swine fever virus (strain p97) were cloned, sequenced and expressed in E . coli . Result from SDS-polyacrylamide gel electrophoresis analysis of expressed proteins revealed the presence of a prominently stained band corresponding to a molecular mass of 61 kDa, which is in agreement with the predicted size from the DNA sequence . The recombinant E2 protein contained an aminoterminal tag of six histidines that could be used for purification by the nickel chelate affinity chromatography . The elution fractions of the expressed protein also contain additional bands of 40 and 35 kDa proteins, indicating proteolytic cleavages might occur . Our Western blotting result also supported that the expression of the recombinant E2 protein of the classical swine fever virus were accomplished.

J Vet Med Sci, 1998 Apr, 60(4), 519 - 22
The effect of oligosaccharides on the production of tumor necrosis factor-alpha by macrophage-like cell line J774/JA-4; Momotani E et al.; Stimulation and modulation of tumor necrosis factor-alpha (TNF-alpha) production during treatment of the murine macrophage-like cell line J774/JA-4 with 25 oligosaccharides were studied . Direct stimulation of TNF-alpha production by oligosaccharides was measured with a cytotoxic assay using the L929 cell line . Twelve samples showed a significantly higher production (P < or = 0.01) of TNF-alpha than the controls . Modulation of TNF-alpha production by treatment with oligosaccharides, followed by stimulation with lipopolysaccharide (LPS) from E . coli, was examined using the L929 bioassay system . In three samples TNF-alpha production increased significantly, but in four samples, production was reduced significantly (P < or = 0.01) . No samples showed modulation of growth or viability of L929 cells within the first 26 hr . The present results are useful in the application of these oligosaccharides which is potentially applicable in medical and food technology.

Nat Biotechnol, 1998 May, 16(5), 473 - 7
Inhibition of a starch-granule-bound protein leads to modified starch and repression of cold sweetening; Lorberth R et al.; We have cloned a gene involved in starch metabolism that was identified by the ability of its product to bind to potato starch granules . Reduction in the protein level of transgenic potatoes leads to a reduction in the phosphate content of the starch . The complementary result is obtained when the protein is expressed in Escherichia coli, as this leads to an increased phosphate content of the glycogen . It is possible that this protein is responsible for the incorporation of phosphate into starch-like glucans, a process that is not understood at the biochemical level . The reduced phosphate content in potato starch has some secondary effects on its degradability, as the respective plants show a starch excess phenotype in leaves and a reduction in cold-sweetening in tubers.

Nucleic Acids Res, 1998 Jun 1, 26(11), 2747 - 53
Sequence and expression characteristics of a nuclear-encoded chloroplast sigma factor from mustard (Sinapis alba); Kestermann M et al.; Plant chloroplasts contain transcription factors that functionally resemble bacterial sigma factors . We have cloned the full-length cDNA from mustard (Sinapis alba) for a 53 kDa derived polypeptide that contains similarity to regions 1.2-4.2 of sigma70-type factors . The amino acid sequence at the N-terminus has characteristics of a chloroplast transit peptide . An in vitro synthesized polypeptide containing this region was shown to be imported into the chloroplast and processed . The recombinant factor lacking the N-terminal extension was expressed in Escherichia coli and purified . It confers the ability on E.coli core RNA polymerase to bind specifically to a DNA fragment that contains the chloroplast psbA promoter . Transcription of the psbA template by E.coli core enzyme in the presence of recombinant SIG1 results in enhanced formation of transcripts of the size expected for correct initiation at the in vivo start site . Together, these data suggest that the mature protein acts as one of the chloroplast transcription factors in mustard . RNA gel blot hybridization reveals a transcript at approximately 1.8 kb, which is more abundant in light-grown than in dark-grown mustard seedlings.

Nucleic Acids Res, 1998 Jun 1, 26(11), 2723 - 8
Mg2+ binding and structural stability of mature and in vitro synthesized unmodified Escherichia coli tRNAPhe; Serebrov V et al.; Mature tRNAPhe from Escherichia coli and the transcript of its gene lacking modified nucleotides were compared by a variety of physical techniques . Melting experiments revealed that at a low Mg2+level the transcript was partially denatured, while the mature tRNA possessed intact tertiary interactions . Mg2+binding to both tRNAs was studied by CD and UV techniques as well as by using the Mg2+-sensitive fluorescence indicator, 8-hydroxyquinoline 5-sulfonic acid . Both tRNA forms exhibited a single strong Mg2+-binding site, its dissociation constant was 10-fold higher for the transcript . Conformational changes in response to Mg2+ addition measured by CD and UV spectrometry revealed no difference for the estimated binding cooperativity and strong differences for affinities of Mg2+-binding sites for the two tRNA forms . Conformational transitions in mature and in in vitro synthesized tRNA required the binding of two Mg2+ ions per molecule and therefore should be associated not only with a single strong binding site . The Mg2+ dependence of Stokes radii measured by gel-filtration revealed insignificant differences between the overall sizes of the two tRNA forms at physiological Mg2+ levels (>1 mM) . Taken together, these results suggest that modified nucleotides stabilize tertiary interactions and increase the structure stability without affecting the mechanism of Mg2+binding and overall folding of the tRNA molecule . This conclusion is supported by the known biological activity of the E . coli tRNAPhe gene transcript.

Nucleic Acids Res, 1998 Jun 1, 26(11), 2611 - 7
The (6-4) photoproduct of thymine-thymine induces targeted substitution mutations in mammalian cells; Kamiya H et al.; Two major ultraviolet-induced photolesions of TpT, a (6-4) photoproduct {T(6-4)T} and a cis-syn cyclobutane TT dimer (T=T), were incorporated into a predetermined site of one of the leading and lagging template strands of a double-stranded vector, and the modified DNAs were transfected into simian COS-7 cells . The DNAs replicated in the cells were recovered and were transfected again into Escherichia coli . The DNA replication efficiencies of plasmids containing T(6-4)T and T=T in the template strand for lagging strand synthesis were 93 and 79%, respectively, as compared with the unmodified DNA . Similar inhibitory effects were observed in the cases of the photoproducts in the template strand for leading strand synthesis (71 and 58%, respectively) . These results indicated that T(6-4)T blocked DNA replication more weakly than T=T during leading and lagging strand syntheses in mammalian cells . The mutation frequencies of T(6-4)T were 2.3 and 4.7% in the leading and lagging template strands, respectively . The T=T lesion was less mutagenic and induced mutations with 0.2-0.7% frequencies . The T(6-4)T lesion primarily elicited 3'-T-->C substitutions, and T=T induced various types of mutations . These results indicate that T(6-4)T is more mutagenic than T=T during leading and lagging strand syntheses in simian cells . Moreover, this is the first evidence that shows T(6-4)T mainly elicits targeted substitutions at its 3'-T site in mammalian cells.

Nucleic Acids Res, 1998 Jun 1, 26(11), 2606 - 10
Identification of a gene involved in the generation of 4-thiouridine in tRNA; Mueller EG et al.; All organisms modify the bases of their RNA after transcription . Relatively little is known about the functions that these chemical alterations serve and, with very few exceptions, even less has been established regarding the enzymology involved . One modified base of known function is 4-thiouridine at position 8 of certain bacterial tRNAs, which serves as a photosensor for near-UV light . A gene involved in the conversion of uridine at position 8 into 4-thiouridine has been identified by genetic screening and its role in 4-thiouridine generation has been confirmed biochemically . This same gene, thiI , has recently been shown to play a role in thiamin biosynthesis . The purification and characteristics of the purified protein are also reported.

Nucleic Acids Res, 1998 Jun 1, 26(11), 2593 - 7
Exonuclease IX of Escherichia coli; Shafritz KM et al.; The bacteria Escherichia coli contains several exonucleases acting on both double- and single-stranded DNA and in both a 5'-->3' and 3'-->5' direction . These enzymes are involved in replicative, repair and recombination functions . We have identified a new exonuclease found in E.coli, termed exonuclease IX, that acts preferentially on single-stranded DNA as a 3'-->5' exonuclease and also functions as a 3'-phosphodiesterase on DNA containing 3'-incised apurinic/apyrimidinic (AP) sites to remove the product trans -4-hydroxy-2-pentenal 5-phosphate . The enzyme showed essentially no activity as a deoxyribophosphodiesterase acting on 5'-incised AP sites . The activity was isolated as a glutathione S-transferase fusion protein from a sequence of the E.coli genome that was 60% identical to a 260 bp region of the small fragment of the DNA polymerase I gene . The protein has a molecular weight of 28 kDa and is free of AP endonuclease and phosphatase activities . Exonuclease IX is expressed in E.coli , as demonstrated by reverse transcription-PCR, and it may function in the DNA base excision repair and other pathways.

Nucleic Acids Res, 1998 Jun 1, 26(11), 2519 - 25
The environment of 5S rRNA in the ribosome: cross-links to the GTPase-associated area of 23S rRNA; Sergiev P et al.; Two photoreactive diazirine derivatives of uridine were used to study contacts between 5S rRNA and 23 rRNA in situ in Escherichia coli ribosomes . 2'-Amino-2'-deoxy-uridine or 5-methyleneaminouridine were introduced into 5S rRNA by T7 transcription . After incorporation of these uridine analogues into the transcript their amino groups were modified with 4-{3-(trifluoromethyl)-3 H -diazirin-3-yl}benzyl isothiocyanate or the N -hydroxysuccinimide ester of 4-{3-(trifluoromethyl)-3 H -diazirin-3-yl}benzoic acid respectively . 5S rRNA carrying the photoreactive diazirine groups (referred to as the 2'-aminoribose derivative and the 5-methyleneamino derivative respectively) was reconstituted into 50S subunits or 70S ribosomes . After mild UV irradiation cross-links formed to 23S rRNA were analysed by standard procedures . All of the observed cross-links involved residue U89 of the 5S rRNA . Three nucleotides of 23S rRNA were cross-linked to this residue with the 5-methyleneamino derivative, namely U958, G1022 and G1138 . With the 2'-aminoribose derivative a single cross-link was found, to U958 . The significance of these cross-links for our understanding of the structure and function of 5S rRNA and its environment in the ribosome are discussed.

Brain Res, 1998 Mar 30, 788(1-2), 49 - 59
Selective chemokine mRNA expression following brain injury; Hausmann EH et al.; Injury in non-neuronal tissues stimulates chemokine expression leading to recruitment of inflammatory cells responsible for orchestration of repair processes . The signals involved in directing repair of damage to the brain are less well understood . We hypothesized that following brain injury, chemokines are expressed and regulate the rate and pattern of inflammatory cell accumulation . The two chemokine subfamilies are alpha(alpha)-chemokines, which primarily function as neutrophil chemoattractants, and the beta(beta)-chemokines, which function primarily as monocyte chemoattractants . We assessed alpha and beta chemokine mRNA expression patterns and leukocyte accumulation following a cerebral cortical lesion . Cortical lesions were produced with and without addition of endotoxin, Escherichia coli lipopolysaccharide (LPS), which stimulates cytokine expression . We studied the expression of the beta-chemokines: monocyte chemoattractant protein (gene product JE; MCP-1/JE), macrophage inflammatory protein-1 alpha and beta (MIP-1alpha and MIP-1beta), and the regulated upon activation normal T expressed and secreted chemokine (RANTES) as well as the alpha-chemokines: interferon-gamma-inducible protein (IP-10) and N51/KC (KC; a murine homologue of MIP-2) . Changes in gene expression were analyzed by Northern analysis at different time points following injury . Leukocyte and macrophage densities were analyzed by immunohistochemistry at the same time intervals . All chemokines were elevated following cortical injury/endotoxin . MCP-1 and MIP-1alpha were elevated at 2 h and peaked 6 h, MIP-1beta peaked at 6 h, but declined more rapidly than MCP-1 or MIP-1alpha, and IP-10 peaked at 6 h and showed the most rapid decline . KC was elevated at 1 h, and peaked at 6 h following LPS . RANTES was elevated at 1 h and achieved a plateau level between 6 and 18 h, then declined . In contrast, sterile injuries produced in the absence of endotoxin only induced the mRNA of the beta-chemokine MCP-1, and its expression was delayed compared to the cortical injury/endotoxin group . The presence of chemokine message as early as 1 h indicates that expression of this class of molecules is an early response in the repair process following traumatic brain injury . Macrophage/microglia accumulation occurred more rapidly, activated microglia further from the lesion border, and more cells accumulated in cortical injury/endotoxin than in cortical lesions produced under sterile conditions . Thus, there was a positive correlation between beta-chemokine expression and the number of beta-chemokine responsive cells (i.e . microglia) accumulating in injury sites . This is the first comprehensive study using a panel of chemokine probes and specific marcophage/microglial markers to study in vivo activation of the brain following injury . Our data show that the brain is capable of expression of multiple chemokine genes upon appropriate stimulation (e.g . LPS-treatment) . The gradient of microglial activation is consistent with physical damage stimulating release of chemokines that diffuse from the injury site . These data strongly suggest that chemokines are instrumental in the initiation of repair processes following brain injury .

J Biochem (Tokyo), 1998 Apr, 123(4), 740 - 6
Molecular cloning, expression, and enzymatic characterization of rabbit hydroxysteroid sulfotransferase AST-RB2; Yoshinari K et al.; Cytosolic sulfotransferases, which consist of at least three gene families, play a major role in activation and detoxification of both endogenous and exogenous chemicals . We recently purified a rabbit sulfotransferase, AST-RB2, showing high activities to both hydroxysteroids and amines . To characterize this enzyme, a rabbit cDNA library was screened using anti-AST-RB2 antibodies . The isolated cDNA was judged to encode AST-RB2 (ST2A8) based on the amino acid sequences of peptide fragments obtained from purified AST-RB2 . The cDNA showed high similarity to other mammalian hydroxysteroid sulfotransferases (ST2) at the amino acid level (58-68%), but low similarity to aryl sulfotransferases (ST1) (less than 37%) . The protein expressed in Escherichia coli catalyzed sulfation of typical ST2 substrates . Therefore, ST2A8 was judged to belong to the ST2 family from both its primary structure and substrate specificity . The ST2A8 protein expressed in E . coli clearly differed from rat ST2A1 and ST2A2 on its localization (cytosol/insoluble fraction ratio) . ST2A8 had no activity to lithocholate, but showed the highest catalysis on dehydroepiandrosterone and testosterone among the four forms (ST2A1, ST2A2, ST2A3, and ST2A8), indicating a clear difference between ST2A forms in substrate specificity to endogenous chemicals.

J Biochem (Tokyo), 1998 Apr, 123(4), 693 - 700
Functional expression and enzymatic properties of two Sitophilus zeamais cysteine proteinases showing different autolytic processing profiles in vitro; Matsumoto I et al.; To characterize in more detail the cathepsin L-like cysteine proteinases from Sitophilus zeamais (SCPs) cloned in our previous study {Matsumoto et al . (1997) J . Biochem . 121, 464-476}, we established a system for their functional expression and purification using a glutathione S-transferase (GST) fusion gene vector from Escherichia coli . The proenzyme forms of two representative SCPs, proSCPc1 and proSCPg3, were expressed as GST-fusion proteins and purified on a glutathione Sepharose column . GST-proSCPc1 undergoes autoproteolytic cleavage into the mature form efficiently at acidic pH, and exhibits significant proteolytic activity toward various substrates including hemoglobin and Z-Phe-Arg-MCA . The enzymatic characteristics of the activated form of SCPc1 are similar to those of mammalian cathepsin L, but its pH optimum for the hydrolysis of hemoglobin is significantly lower . The other proSCP, GST-proSCPg3, which has a shorter COOH-terminal domain than SCPc1, undergoes almost no autolytic processing and shows only very slight proteolytic activity, although the other enzymatic characteristics of GST-proSCPg3 are similar to those of GST-proSCPc1.

J Biochem (Tokyo), 1998 Apr, 123(4), 684 - 92
Comparison of in vivo activities of 5'-connected and 3'-connected cis-acting ribozymes: selection of intracellularly active ribozymes using the gene for dihydrofolate reductase (DHFR) as a selective marker in Escherichia coli; Hamada M et al.; If ribozymes are to be exploited in vivo, it is necessary to select ribozymes that are functional in the intracellular environment . Ribozymes selected in the intracellular environment should retain their function in vivo as well as in vitro . We have devised a novel system for selection of active ribozymes from pools of active and inactive ribozymes using the gene for dihydrofolate reductase (DHFR) as a selective marker . In our first attempt, a sequence encoding either an active or an inactive ribozyme was connected upstream of the gene for DHFR . Each plasmid was designed such that, when the ribozyme was active, the ribozyme would cleave the target site and, as a result, the rate of production of DHFR would be high enough to endow resistance to trimethoprim (TMP) . However, a critical defect may be associated with introduction of a ribozyme upstream of the DHFR gene because, during actual screening for active ribozymes on the 5' side from a pool of random sequences, there is the danger of selecting sequences that are not related to the activity of ribozymes . Indeed, some upstream linker sequences affected the level of expression of the DHFR protein and, as a result, the resistance of Escherichia coli to TMP . Therefore, we newly constructed a 3'-connected ribozyme system, and activities in vivo of 5'-connected and 3'-connected ribozymes were compared . We found that the cleavage efficiencies in vivo were nearly identical for the two types of ribozyme, 24% for the 5'-side ribozyme and 23% for the 3'-side ribozyme, indicating that polysomes did not seem to inhibit the action of the 3'-connected ribozyme . In both cases, when cells were transformed with a 1 : 1 mixture of active and inactive ribozyme-coding plasmids, it was mainly the cells that harbored the active ribozyme that survived in the presence of TMP.

J Biochem (Tokyo), 1998 Apr, 123(4), 568 - 70
Crystallization and preliminary X-ray diffraction studies of a rice cysteine proteinase inhibitor, Oryzacystatin-I; Kudo N et al.; Oryzacystatin-I from rice seeds was overexpressed in Escherichia coli, purified, and crystallized by the sitting-drop vapor diffusion method . Crystals obtained with 2-methyl-2,4-pentanediol as a precipitant exhibited space group I4122, with unit cell parameters of a = b = 100.0 A, c = 54.2 A, and diffracted up to 2.8 A resolution at 100 K . The crystals have one molecule per asymmetric unit.

Nature, 1998 May 7, 393(6680), 91 - 4
Catalysis of homologous DNA pairing by yeast Rad51 and Rad54 proteins; Petukhova G et al.; The Saccharomyces cerevisiae RAD51 and RAD54 genes are both required for the occurrence of homologous recombination and for the repair of double-stranded DNA breaks . Previous studies have indicated that Rad51 protein, together with the single-stranded DNA-binding factor replication protein A (RPA), can promote the formation of heteroduplex DNA, which is a key intermediate in homologous recombination . Here we report the purification of the Rad54 protein to near homogeneity and the biochemical testing of its molecular function . We find that Rad54 protein possesses a double-stranded DNA-dependent ATPase activity, and that it interacts with the Rad51 protein . Addition of Rad54 protein to reactions containing Rad51 strongly stimulates the rate of pairing between homologous single-stranded and double-stranded DNA molecules . We conclude that Rad54 acts to overcome kinetic impediments that would limit homologous DNA pairing between recombining chromosomes in vivo.

Nature, 1998 May 7, 393(6680), 72 - 6
Hypothalamic CART is a new anorectic peptide regulated by leptin; Kristensen P et al.; The mammalian hypothalamus strongly influences ingestive behaviour through several different signalling molecules and receptor systems . Here we show that CART (cocaine- and amphetamine-regulated transcript), a brain-located peptide, is a satiety factor and is closely associated with the actions of two important regulators of food intake, leptin and neuropeptide Y . Food-deprived animals show a pronounced decrease in expression of CART messenger RNA in the arcuate nucleus . In animal models of obesity with disrupted leptin signalling, CART mRNA is almost absent from the arcuate nucleus . Peripheral administration of leptin to obese mice stimulates CART mRNA expression . When injected intracerebroventricularly into rats, recombinant CART peptide inhibits both normal and starvation-induced feeding, and completely blocks the feeding response induced by neuropeptide Y . An antiserum against CART increases feeding in normal rats, indicating that CART may be an endogenous inhibitor of food intake in normal animals.

Mol Reprod Dev, 1998 Jun, 50(2), 229 - 39
Molecular cloning and expression in Escherichia coli of cDNA encoding bonnet monkey (Macaca radiata) zona pellucida glycoprotein-ZP2; Jethanandani P et al.; Zona pellucida (ZP) glycoproteins have been proposed as candidate antigens for an immunocontraceptive vaccine . The efficacy of such a vaccine has to be evaluated in nonhuman primates, thus necessitating the characterization of their ZP glycoproteins . A bonnet monkey (Macaca radiata) ovarian cDNA lambda gt11 library was screened for ZP2 (bZP2) using full-length human ZP2 cDNA as a probe . Two identical full-length clones with an open reading frame of 2235 nt encoding a polypeptide of 745 aa residues were isolated . The deduced aa sequence of bZP2 revealed high sequence identity (94.2%) with human ZP2 . The bZP2 cDNA (115-1914 nt, 1.8 kb), excluding sequences coding for N-terminal signal sequence and C-terminal transmembranelike domain, was PCR amplified and Sac1-Sal1 restricted fragment cloned in frame downstream of the T5 promoter under the lac operator control in a pQE-30 vector . Recombinant bZP2 (r-bZP2) was expressed as a polyhistidine fusion protein in Escherichia coli strain M15 {pREP4} . Immunoblot with rabbit polyclonal antibodies against bZP2 synthetic peptide (corresponding to aa residues 429-444; K434 replaced by R and 1436 by V) revealed a major band of 68 kDa . Immunization of male rabbits with the r-bZP2 protein purified on Ni-NTA resin under denaturing conditions generated antibodies reactive with r-bZP2 in ELISA as well as with native protein as revealed by positive fluorescence of ZP of bonnet monkey ovary . The availability of r-bZP2 and its aa sequence will help in the development and evaluation of a contraceptive vaccine based on ZP2.

Int J Cancer, 1998 May 18, 76(4), 512 - 8
Sulfated glycoglycerolipid from archaebacterium inhibits eukaryotic DNA polymerase alpha, beta and retroviral reverse transcriptase and affects methyl methanesulfonate cytotoxicity; Ogawa A et al.; A sulfated glycoglycerolipid, 1-O-(6'-sulfo-alpha-D-glucopyranosyl)-2,3-di-O-phytanyl- sn-glycerol (KN-208), a derivative of the polar lipid isolated from an archaebacterium, strongly inhibited DNA polymerase (pol) alpha and pol beta in vitro among 5 eukaryotic DNA polymerases (alpha, beta, gamma, delta, and epsilon) . It also inhibited Escherichia coli DNA polymerase I Klenow fragment (E . coli pol I) and human immunodeficiency virus reverse transcriptase (HIV RT) . The mode of inhibition of these polymerases was competitive with the DNA template primer and was non-competitive with the substrate dTTP . KN-208 inhibited pol beta most strongly, with a Ki value of 0.05 microM, 10-fold lower than that for pol alpha (0.5 microM) and 60- or 140-fold lower than that for HIV RT (3 microM) or for E . coli pol I (7 microM), respectively . The loss of sulfate on the 6'-position of glucopyranoside of this compound completely abrogated inhibition . However, the hydrophilic part of KN-208, glucose 6-sulfate alone, showed no inhibition . Other sulfated compounds containing different hydrophobic structures, such as dodecyl sulfate and cholesterol sulfate, exhibited a much weaker inhibition . Our results suggest that the whole molecular structure of KN-208 is required for inhibition . KN-208 was shown to be modestly cytotoxic for the human leukemic cell line K562 . Interestingly, a subcytotoxic dose of KN-208 increased the sensitivity of the human leukemic cells to an alkylating agent, methyl methanesulfonate, while it did not potentiate the effects of ultraviolet light or of cisplatin.

Biochem Biophys Res Commun, 1998 Apr 28, 245(3), 670 - 8
Analysis in Escherichia coli of the effects of in vivo CpG methylation catalyzed by the cloned murine maintenance methyltransferase; Tollefsbol TO et al.; Due in part to the complexity of mammalian systems, some of the proposed biological influences of mammalian DNA methylation have not been fully established . Escherichia coli cells, which normally contain negligible CpG methylation, exhibited progressive slowing of replication and lengthened generation times when expressing the murine DNA maintenance methyltransferase . Genomic analysis indicated significant amounts of CpG methylation in expressing cells which was absent from control cells . Expressing cells exposed to the cytosine demethylating agent, 5-azacytidine, rapidly reverted to propagation levels of controls . Substitution of cysteine with alanine in the carboxyl-terminal region proline-cysteine dipeptide of the methyltransferase completely inactivated methylating activity and cells expressing the inactive enzyme replicated as well as controls . These findings strongly implicate a role of epigenetic de novo CpG methylation in modulating cellular propagation, demonstrate that the maintenance methyltransferase can de novo methylate in vivo, and show that the methyltransferase requires an active site cysteine for activity.

J Nutr Sci Vitaminol (Tokyo), 1998 Feb, 44(1), 137 - 49
Transcriptional regulation of rat calbindin expression during development determined by bacterially expressed protein; Fukushima A et al.; Calbindin-D9k expression in intestinal mucosal cells reveals a specific pattern during development in rats . It shows a low basal level in suckling and adult rats, but after weaning at 21 d of age, increases to three times that of the basal level for several days only, around 24 d . We attempted to clarify whether the regulation of developmental change was at the transcriptional or post-transcriptional level . The calbindin-D9k protein and mRNA concentrations during pre- and postweaned development were determined by Western blot and Northern blot analysis, respectively, and compared with calcium binding activity by 45Ca . For Western blot analysis, a corresponding antibody was raised in rabbit using a bacterially expressed fusion protein, glutathione S-transferase (GST, EC 2.5.1.18), and calbindin-D9k . Calbindin-D9k cDNA was linked to a GST gene within a molecule of vector plasmid and a fusion protein was expressed in Escherichia coli . There were significant (p < 0.001) correlations between calbindin-D9k protein, mRNA concentrations and calcium-binding activity: r = 0.90 for protein vs . mRNA, r = 0.93 for protein vs . binding activity and r = 0.95 for mRNA vs . binding activity . These results indicate that calbindin-D9k expression during postnatal development is regulated at the transcriptional level.

Biotechniques, 1998 May, 24(5), 796 - 800, 802
Microsatellite enrichment in organisms with large genomes (Allium cepa L.); Fischer D et al.; To exploit the polymorphism of repeat numbers in short tandem repeat (STR) sequences (microsatellites) as molecular markers, STRs must be isolated and PCR primers must be developed in flanking sequences . In species with large genomes such as Allium cepa L . (onion and shallot), an efficient selection procedure for genomic fragments containing STRs is a crucial step . Here we describe a nonradioactive method for microsatellite isolation based on affinity capture of single-stranded restriction fragments annealed to biotinylated microsatellite oligonucleotides (CA)10, (GAA)8 and (AAC)8 followed by adapter-mediated genomic PCR . Cloning of the products in E . coli and plasmid sequencing revealed more than 60% positive clones . Primers were designed in STR-flanking regions, and one or two bands were amplified in 13 diploid onion and five shallot accessions . Allelism of the bands was confirmed by product sequencing.

Biotechniques, 1998 May, 24(5), 789 - 94
UGA read-through artifacts--when popular gene expression systems need a pATCH; MacBeath G et al.; pET and similar vectors are widely used for efficient gene expression in Escherichia coli and subsequent protein purification, often by means of a C-terminal histidine (His) tag . We found that the TGA translation termination signal following the His-tag sequence in pET constructs gives rise to a significant fraction of read-through protein extended by 21 amino acids . Mass spectrometry indicated that tryptophan is inserted at the UGA (opal) stop codon in the examined non-opal suppressor strains; no evidence for translational frameshifting was detected . We have shown that the problem of obtaining heterogeneous protein preparations can easily be corrected . Plasmid pATCH1 provides a replacement sequence for the inefficient stop signal and can be used to repair both pET vectors and existing pET-based expression constructs . Our observation illustrates the largely ignored fact that a UGA codon is the worst choice for proper translation termination in efficient overexpression vectors.

Alcohol, 1998 May, 15(4), 277 - 80
Ethanol inhibition of phagocytosis and superoxide anion production by microglia; Aroor AR et al.; Ethanol consumption has been associated with aberrant immune responses resulting in increased susceptibility to infection including opportunistic infections of the central nervous system . We have investigated the effects of chronic ethanol treatment on phagocytosis and production of superoxide anion by microglia . Phagocytosis of radio-labeled opsonized E . coli was markedly suppressed by treating microglia with ethanol . The unstimulated synthesis of superoxide anion was not altered by ethanol treatment of microglia, but ethanol treatment effectively suppressed phorbol-12 myristate-13 acetate-stimulated microglia superoxide anion production . The results indicate that ethanol inhibition of microglia function may play a role in increased susceptibility for central nervous system infections, particularly in immunocompromised subjects.

Crit Care Med, 1998 May, 26(5), 912 - 6
A comparison among animal models of acute lung injury; Rosenthal C et al.; OBJECTIVES: To compare four widely used animal models of acute lung injury and to determine the changes in physiologic variables associated with each model . DESIGN: A prospective, controlled animal study . SETTING: An animal laboratory of a university-affiliated children's hospital . SUBJECTS: Four groups of anesthetized, paralyzed, and ventilated young Yorkshire pigs, weighing 35 to 45 kg . INTERVENTIONS: Acute lung injury was generated by four different methods: a) intrapulmonary arterial infusion of endotoxin of Escherichia colt; b) bronchoalveolar instillation of 0.05N of hydrochloric acid; c) repeated bronchoalveolar warm saline lavage; and d) intrapulmonary arterial infusion of oleic acid . After each acute lung injury procedure, the temporal changes in various physiologic variables were measured, starting at 60 mins and at 15-min intervals thereafter for a total of 165 mins . Systemic and mixed venous serum immunoreactive tumor necrosis factor (TNF)-alpha concentrations were also measured at the same time points . Analysis of variance for repeated measures was employed to determine the absolute and relative significance of the changes observed . MEASUREMENTS AND MAIN RESULTS: Systemic and mixed venous immunoreactive TNF-alpha did not change following any of the acute lung injury procedures . The animals' heart rates and systemic vascular resistances also did not change . Hydrochloric acid instillation as well as bronchoalveolar lavage resulted in significant hypoxemia with no other hemodynamic effects . Endotoxin infusion did not result in hypoxemia but caused significant increases in mean pulmonary arterial pressure and pulmonary vascular resistance and decreases in mean arterial pressure and cardiac output . Oleic acid infusion resulted in a marked hypoxemia with a pronounced increase in mean pulmonary arterial pressure and pulmonary vascular resistance . It also markedly reduced the mean arterial pressure, cardiac output, and the mixed venous PO2 . CONCLUSIONS: The surfactant depletion and hydrochloric acid instillation models produce acute hypoxemia in an otherwise hemodynamically stable animal . A brief endotoxin infusion provides a model for cardiovascular instability and pulmonary hypertension but fails to produce hypoxemia in the pig . The oleic acid infusion creates a model of marked cardiovascular instability, pulmonary hypertension, and profound hypoxemia . However, none of the acute lung injury models described was associated with the production of tumor necrosis factor.

Crit Care Med, 1998 May, 26(5), 905 - 11
Aminoguanidine attenuates endotoxin-induced acute lung injury in rabbits; Mikawa K et al.; OBJECTIVE: To assess the effect of aminoguanidine, a selective inducible nitric oxide synthase inhibitor, on endotoxin-induced acute lung injury in rabbits . DESIGN: Prospective, blinded, controlled laboratory study . SETTING: University research laboratory . SUBJECTS: Twenty-eight male rabbits . INTERVENTIONS: The animals were randomly assigned to receive one of four treatments (n = 7 for each group): infusion of saline only (S-S group), infusion of saline and aminoguanidine (S-AG group), infusion of Escherichia coli endotoxin (5 mg/kg over 60 mins) (E-S group), and infusion of endotoxin and aminoguanidine (E-AG group) . Fifteen minutes before infusion of endotoxin (E-S and E-AG groups) or saline (S-S and S-AG groups), the animals received an intravenous injection of 1 mg/kg of aminoguanidine (S-AG and E-AG groups) or saline (S-S and E-S groups) . The same dose of aminoguanidine or saline was given 1 hr after the end of endotoxin or saline infusion . The lungs of the rabbits were ventilated with 40% oxygen . MEASUREMENTS AND MAIN RESULTS: Hemodynamics, peripheral leukocyte counts, and PaO2 were recorded during the ventilation period (6 hrs) . After these observations were made, lung mechanics, cell fraction of bronchoalveolar lavage fluid, and concentrations of thromboxane A2 and prostacyclin metabolites in bronchoalveolar lavage fluid were determined . The wet weight/dry weight ratio of the lung and albumin concentrations in bronchoalveolar lavage fluid were analyzed as indices of pulmonary edema . Endotoxin decreased the lung compliance and PaO2 and increased the wet weight/dry weight ratio, neutrophil counts, and albumin concentrations in bronchoalveolar lavage fluid . The bronchoalveolar lavage fluid concentrations of thromboxane B2 in bronchoalveolar lavage fluid were increased by infusion of endotoxin . Aminoguanidine attenuated these changes . Endotoxin caused extensive morphologic lung damage, which was lessened by aminoguanidine . CONCLUSIONS: Aminoguanidine given intravenously before and after endotoxin attenuated endotoxin-induced lung injury in rabbits . These findings suggest that inducible nitric oxide synthase inhibition may be useful in the treatment of endotoxin-induced lung injury . However, further studies are required to determine the optimal dosage of aminoguanidine, when the inhibitor is given alone as therapy after lung injury.

J Immunol, 1998 May 15, 160(10), 5088 - 97
Escherichia coli bound to the primate erythrocyte complement receptor via bispecific monoclonal antibodies are transferred to and phagocytosed by human monocytes in an in vitro model; Kuhn SE et al.; We have prepared cross-linked, bispecific mAb complexes (heteropolymers) that facilitate rapid and quantitative binding of a prototype pathogen, Escherichia coli, to the complement receptor (CR1) on primate erythrocytes . Incubation of the erythrocyte-heteropolymer-E . coli complexes with freshly isolated human mononuclear cells leads to rapid removal of the E . coli from the erythrocytes, and phagocytosis and killing of the bacteria . The erythrocytes are not lysed or phagocytosed during this transfer reaction, but both heteropolymer and CR1 are removed from the erythrocytes along with the E . coli . These findings parallel observations made in previous in vivo experiments in which heteropolymers were used to facilitate clearance of innocuous prototype pathogens in a monkey model . It should now be possible to extend the heteropolymer paradigm to a live pathogen in a primate model.

J Protein Chem, 1998 Apr, 17(3), 245 - 54
Single-chain Fv of anti-idiotype 11-1G10 antibody interacts with antibody NC41 single-chain Fv with a higher affinity than the affinity for the interaction of the parent Fab fragments; Iliades P et al.; A single-chain Fv (scFv) fragment of anti-idiotype antibody 11-1G10, which recognizes an idiotope of anti-neuraminidase antibody NC41, was constructed by joining VH and VL domains with a (Gly4Ser)3 linker, with a pelB leader sequence, and two C-terminal FLAG tag sequences, and expressed in E . coli (10 mg/L) . The 11-1G10 scFv was isolated by affinity chromatography on an anti-FLAG M2 antibody column as a 2:1 mixture of monomer and dimer forms which were separated by Superdex 75 chromatography; monomer (at 100 microg/ml) was stable for 7 days at 21 degrees C and 30 days at 4 degrees C, whereas the dimer slowly dissociated to monomer to yield a 2:1 monomerdimer equilibrium mixture after 30 days at 4 degrees C . The dimer was bivalent, with each combining site binding an NC41 Fab to yield a stable complex of Mr approximately 156,000 . Binding affinities, determined in solution using a BIAcore biosensor, showed that the affinity for the interaction of 11-IG10 scFv monomer with NC41 scFv monomer was five- to six-fold higher than the interaction of the parent Fab pair . This is the first example of an scFv derived from a monoclonal antibody with a higher affinity than its parent Fab.

J Protein Chem, 1998 Apr, 17(3), 219 - 28
The effect of Ser 128 substitution on the structure and stability of cAMP receptor protein from Escherichia coli; Malecki J et al.; Kinetic measurements of denaturation and renaturation of two mutants of cAMP receptor protein (CRP) at position 128, namely Ser --> Ala and Ser --> Pro, were performed in order to assess changes introduced by the mutation in the quaternary structure and protein stability . No significant changes were found in the unfolding/refolding reactions . However, small perturbations in the dissociation of CRP dimer can be seen, which indicate that subunit interactions are influenced by the mutation . Studies of intrinsic fluorescence quenching of these two mutants are also reported, showing changes compared with wild-type protein . Near-UV circular dichroism measurements indicate, however, that Trp residues remain in the same environment as in the wild-type CRP . It is proposed that Ser at position 128 is involved in maintaining the proper domain alignment within CRP subunits.

Electrophoresis, 1998 Apr, 19(4), 536 - 44
Low molecular weight proteins: a challenge for post-genomic research; Rudd KE et al.; The EcoGene project involves the examination of Escherichia coli K-12 DNA sequences and accompanying annotation in the public databases in order to refine the representation and prediction of the entire set of E . coli K-12 chromosomally encoded protein sequences . The results of this ongoing effort have been deposited in the SWISSPROT protein sequence database as sequencing of the E . coli genome has progressed to completion in recent years . Through this continuing research, we have discovered that the prediction of low molecular weight (small) proteins, arbitrarily defined as protein sequences < or = 150 amino acids (aa) in length, is problematic and requires special attention . We describe the small protein subset of EcoGene and the approach used to derive this subset from the complete E . coli genome sequence and database annotations . These E . coli proteins have helped to identify new small genes in other organisms and to identify conserved residues (motifs) using database searches and multiple alignments . Two thirds of the E . coli small proteins have not been characterized experimentally . The careful application of computer and laboratory methods to the analysis of small proteins is needed for accurate prediction, verification and characterization . The problem of accurate protein sequence identification is not limited to small proteins or to E . coli; these problems are encountered to varying degrees throughout all sequence databases.

Biochem Biophys Res Commun, 1998 Apr 28, 245(3), 666 - 9
Role of carboxyl residues surrounding heme of human cytochrome b5 in the electrostatic interaction with NADH-cytochrome b5 reductase; Kawano M et al.; To identify the cytochrome b5 residues responsible for the electrostatic interaction with NADH-cytochrome b5 reductase (b5R), we prepared and characterized the cytochrome b5 mutants in which Glu41, Glu42, Glu63, Asp70, and Glu73 were replaced by Ala, utilizing site-directed mutagenesis and the expression system for cytochrome b5 in Escherichia coli . Apparent Km values of the wild type b5R for Glu42Ala cytochrome b5 and Asp70Ala cytochrome b5 were approximately three-fold and six-fold higher than that for the wild type cytochrome b5, respectively, while the kcat values for those mutants were not remarkably affected . In contrast, Glu41Ala, Glu63Ala, and Glu73Ala cytochrome b5 showed almost the same kinetic properties as the wild type cytochrome b5 . Furthermore, kinetic studies on combinations of the cytochrome b5 and b5R mutants suggested the interaction between Glu42 and Asp70 of cytochrome b5 and Lys125 and Lys41 of b5R, respectively, in the reaction.

Jpn J Ophthalmol, 1998 Mar-Apr, 42(2), 108 - 14
Acridine orange staining for rapid diagnosis of Acanthamoeba keratitis; Hahn TW et al.; Acanthamoeba keratitis is uncommon, but one of the most severe infectious diseases of the cornea . Delayed diagnosis or misdiagnosis as bacterial or herpes simplex keratitis leads to extensive corneal inflammation and profound visual loss . Therefore, accurate and rapid diagnosis of Acanthamoeba keratitis is essential for successful treatment and good prognosis . We evaluated the usefulness of acridine orange staining from corneal scrapings and contact lens solutions for the rapid diagnosis of four consecutive cases of Acanthamoeba keratitis . Gram stain and culture on nonnutrient agar plates with Escherichia coli overlay were also made . Corneal scrapings stained with acridine orange revealed yellow-to-orange polygonal, cystic structures consistent with the appearance of Acanthamoeba among inflammatory cells and the corneal epithelial cells . The contact lens case solutions of two patients also showed numerous cysts with double wall . Some organisms from the third patient were identified as Acanthamoeba castellani and others as Acanthamoeba lugdunensis . Based on the acridine orange staining results in four cases of Acanthamoeba keratitis, this stain is recommended as a simple and reliable method for the rapid diagnosis of this disease.

Arch Biochem Biophys, 1998 Apr 15, 352(2), 247 - 54
A kinetic study of site-directed mutants of Escherichia coli ADP-glucose pyrophosphorylase: the role of residue 295 in allosteric regulation; Meyer CR et al.; The effects of amino acid substitutions at residue 295 on the regulatory properties of Escherichia coli ADP-glucose pyrophosphorylase were studied . In previous studies, this residue, altered from proline to serine (P295S) in the gene of a mutant strain of E . coli, resulted in a high-activity form of enzyme {higher activity in absence of activator fructose 1,6-bisphosphate (FBP), higher apparent affinity for FBP and substrates, and lower apparent affinity for the inhibitor, AMP} . The effects of size and charge on this site were explored by replacing Pro with Gly, Asp, Asn, Gln, or Glu . All mutant enzymes were expressed and purified for kinetic analysis . All mutant enzymes, to varying extents, were in more active form than the wild-type enzyme . Enzymes with a substituted negative charge (P295D, P295E) had the highest activity in the absence of FBP, while the P295G enzyme was most similar to the wild type . The P295D and P295E enzymes had the lowest apparent affinities for AMP; this effect was partially abolished by the neutral substitutions P295N and P295Q . Another mutation, G336D, had previously been found to produce an even higher activity enzyme form . In order to examine interactions between substitutions at the 295 and 336 positions, the double mutant P295D-G336D was constructed and characterized . The double mutant enzyme was more active in the absence of FBP, with a higher affinity for FBP and a lower apparent affinity for AMP than either single mutated enzyme . The significance of residue 295 in regulation is discussed.

Arch Biochem Biophys, 1998 Apr 15, 352(2), 199 - 206
Production and characterization of two variants of human cystatin SA encoded by two alleles at the CST2 locus of the type 2 cystatin gene family; Saitoh E et al.; Two variants of cystatin SA encoded by two alleles at the CST2 locus of the type 2 human cystatin gene family were expressed in Escherichia coli . One, termed cystatin SA1, is identical to cystatin SA {S . Isemura, E . Saitoh, and K . Sanada J . Biochem . 102, 693-704, 1987} . Another, termed cystatin SA2, carries two amino acid substitutions (59Gly-->Asp; 120Glu-->Asp), one of which is in the so-called QXVXG region (the first hairpin loop) and another in the C-terminal portion of the molecule . Four recombinant cystatins {full-sized cystatin SA1, two N-terminally truncated cystatin SA1 lacking four residues (WSPQ) and six residues (WSPQEE), and full-sized cystatin SA2} were purified from the periplasmic fractions of E . coli cells . Two N-terminally truncated recombinant cystatin SA1 inhibited bovine cathepsin C with 2- to 20-fold lower Ki values than that of the full-sized one . In the inhibition of papain and ficin, however, both of the N-terminally truncated cystatin SA1 displayed a 10-fold higher Ki value than that of full-sized one . In the inhibition of papain, ficin, and recombinant human cathepsin K, recombinant cystatin SA2 showed, respectively, 3826-, 1090-, and 30-fold higher Ki values compared with those of SA1 . Recombinant cystatin SA2 inhibited bovine cathepsin C with a 50-fold lower Ki value compared with that of SA1 . Recombinant cystatin SA1 did not inhibit human cathepsin H but SA2 inhibited it slightly (Ki = 528 nM) . Neither of the recombinant variants inhibited bovine cathepsin B . Our data supply evidence indicating that the amino acid sequence of the first hairpin loop of the cystatin superfamily is important in the inhibition of papain, ficin, cathepsin C, cathepsin H, and cathepsin K.

J Surg Res, 1998 Feb 1, 74(2), 179 - 86
Gluconeogenesis and phosphoenergetics in rat liver during endotoxemia; Morikawa S et al.; BACKGROUND: During endotoxemia, glucose and energy metabolism varies depending on the stage, severity, and other conditions . In this study, gluconeogenesis from 13C-labeled alanine and phosphoenergetic state in rat liver during the acute phase of endotoxemia were concurrently observed by in vivo 13C and 31P NMR spectroscopy in a noninvasive manner . MATERIALS AND METHODS: Lipopolysaccharide from Escherichia coli (10 mg/kg) was injected intravenously followed by infusion of {3-13C}alanine . In vivo 13C and 31P NMR spectra were alternately collected for 90 min with a 2.0 Tesla CSI Omega System . RESULTS: In our experimental model, endotoxin increased the pulse rate without decreasing the blood pressure and elevated the blood sugar level, which suggests the so-called hyperdynamic state . Even under such conditions, a slight, but significant, impairment of the phosphoenergetic state in the liver (a decrease in ATP and an increase in Pi) was detected with 31P NMR spectroscopy . The 13C peaks of glucose C6 and Glu/Gln C2 of the liver in endotoxemia were significantly lower than those of the control, despite hyperglycemia in endotoxemia . CONCLUSIONS: NMR spectroscopic studies suggest that the endotoxin caused the inhibition of gluconeogenic activity from the infused {3-13C}alanine and the TCA cycle accompanied by a deterioration in the phosphoenergetic state even in the hyperglycemic phase . Since the blood sugar level might be influenced by the systemic utilization of glucose, such direct measurements should prove important in the in vivo evaluation of glucose and energy metabolism in the liver.

J Surg Res, 1998 Feb 1, 74(2), 119 - 24
Nitric oxide donor decreases neutrophil adhesion in both lung and peritoneum during peritonitis; Fukatsu K et al.; BACKGROUND: As nitric oxide (NO) is an antiadhesive molecule, exogenous NO may modulate neutrophil adhesion in organs . This study was designed to examine the effects of the NO donor SNAP (S-nitroso-acetyl penicillamine) on neutrophil adhesion at the inflammatory site and in remote organs, in peritonitis using a fluorescent microscopic method . MATERIALS AND METHODS: In experiment 1, rats (n = 12) were given saline or 10 micrograms/kg of SNAP intravenously followed by continuous infusion of saline, or of 2, 20, or 200 micrograms/kg/h SNAP until sacrifice . Ten minutes after injection of saline or SNAP, 10(7) Escherichia coli were injected into the peritoneal cavity . Five hours after challenge, 10(6) fluorescein-labeled neutrophils were infused . Peritoneal samples, lungs, liver, and kidney were harvested for counting of labeled neutrophils under epifluorescent microscopy . In experiment 2, rats (n = 25) were treated with saline or 10 micrograms/kg of SNAP intravenously and infused with saline or 20 micrograms/kg/h SNAP; E . coli was injected as in experiment 1 . Before or 5 h after challenge, hemodynamic data were obtained . Then, labeled neutrophils were infused for counting of neutrophil numbers in organs . Arterial blood gas data and the circulating neutrophil number were also determined . RESULTS: Experiment 1 . Twenty and 200 micrograms/kg/h SNAP infusions tended to reduce labeled neutrophil numbers in lungs, while all three SNAP doses decreased the peritoneal labeled neutrophil numbers . RESULTS: Experiment 2 . Five hours after bacterial injection, SNAP infusion simultaneously decreased both pulmonary and peritoneal labeled neutrophil numbers . SNAP had no effect on hemodynamic and blood gas data, or on circulating neutrophil numbers . CONCLUSION: NO donors may be useful for preventing neutrophil-associated lung injury, but should be used with caution in light of the possible adverse effects on host defense in the peritoneal cavity.

J Endod, 1997 Aug, 23(8), 517 - 21
Fibroblast growth in vitro suppressed by LPS-activated macrophages . Reversal of suppression by hydrocortisone; Metzger Z et al.; Activated macrophages are among the major constituents of the periapical granuloma . Their state of activation may persist for long periods after the local irritant is removed and may delay resolution and repair of the lesion . The effect of activated macrophages on fibroblast growth was studied in vitro . Circular fibroblast colonies were formed using a drop containing 7.5 x 10(5) murine dermal fibroblasts and allowed to grow for 7 days . When peritoneal exudate macrophages were added (0.5-3.0 x 10(6) cells/dish) and activated in vitro by LPS (1 microgram/ml), the fibroblast colony's growth was suppressed . LPS alone, at the concentration used, had no effect on the fibroblast growth . Hydrocortisone (> or = 10(-7) M) totally reversed the suppression, when added either simultaneously with or 6, 24, or 48 h after the LPS . The efficacy of late hydrocortisone treatment suggests that its effect was through prevention of the expression of the LPS activation of the macrophages . These findings may provide a possible clue to a pharmacological modulation of the healing processes that occur in the periapical lesion once its infective source had been eliminated.

Yonsei Med J, 1998 Apr, 39(2), 141 - 7
Factors affecting transformation efficiency of BCG with a Mycobacterium-Escherichia coli shuttle vector pYUB18 by electroporation; Cho SN et al.; BCG has been one of the vehicles for multi-recombinant vaccine . However, low transformation efficiency of BCG with plasmid DNA hampered studies involving expression of foreign antigens in BCG . In an effort to determine the optimal conditions, this study was initiated to investigate factors involved in the transformation of BCG with a Mycobacterium-Escherichia coli shuttle vector, pYUB18, by electroporation . Mycobacterium bovis BCG (strain 1173P2) was grown in Middlebrook (M) 7H9 broth containing albumin-dextrose-catalase and 0.05% tween 80, and transformed BCG was grown in M7H10 agar containing kanamycin for counting viable cells . Pretreatment of BCG with 10 mM CaCl2 improved the transformation efficiency, but overnight incubation of BCG with 1% glycine did not . The transformation efficiency in BCG also varied depending on voltage, resistance, and DNA concentration . The maximum transformation efficiency was obtained when the infinity resistance, 12.5 Kv/cm, and 100 ng of DNA were used, and reached 1.4 x 10(5) CFU/microgram of plasmid DNA, which is about 3-100 times greater than those from previous reports . The transformation conditions described in this study, therefore, will give us a better position for employing BCG as a vehicle for developing multi-recombinant vaccines.

Pharmacol Biochem Behav, 1998 Apr, 59(4), 835 - 41
Effects of prenatal morphine exposure on NK cytotoxicity and responsiveness to LPS in rats; Shavit Y et al.; Prenatal exposure to opiates can adversely affect fetal development, resulting in long-term growth retardation and impairments in physiological and behavioral functions . In the present study we studied long-term effects of prenatal morphine exposure on immune functions, including the activity of natural killer (NK) cells and the febrile and behavioral responses to lipopolysaccharide (LPS) . Pregnant Fischer 344 rats were given increasing doses of morphine in slow release emulsion during gestational days 12-18 . Control rats were injected with vehicle and were either pair fed to morphine rats or fed ad lib . Postnatal experiments were conducted when offspring were 10-12 weeks old . Compared to both control groups, rats prenatally exposed to morphine exhibited: 1) suppressed cytotoxic activity of NK cells; 2) reduced LPS-induced fever measured by a biotelemetric system; 3) reduced hyperalgesia measured by the hot-plate test at 30 min, and augmented hypoalgesia at 2-6 h post-LPS; 4) higher open-field activity in saline-treated animals, and more pronounced suppression of activity in LPS-injected animals; 5) LPS-induced reduction of food consumption, body weight, and social exploration, which did not differ from the reduction observed in control animals . These findings indicate that prenatal exposure to morphine induces long-term impairment of host-defense mechanisms, which may render the offspring more susceptible to infectious diseases.

JPEN J Parenter Enteral Nutr, 1998 May-Jun, 22(3), 127 - 35
Effects of insulin-like growth factor 1 on neutrophil and monocyte functions in normal and septic states; Balteskard L et al.; BACKGROUND: Insulin-like growth factor 1 (IGF-1) mediates anabolic actions in catabolic states and also influences the immune system . Endogenous IGF-1 production is suppressed in sepsis; replacement therapy is therefore a natural approach to obtain the protein anabolic and potentially immune-stimulating effects of IGF-1 . METHODS: Twenty-two piglets were randomized to three groups: an IGF-1 group (n = 8) receiving a continuous infusion of 1.3 mg/h of IGF-1, a nontreated septic control group (n = 8), and a nonseptic control group (n = 6) receiving saline . Phagocytosis and respiratory burst in porcine neutrophils were evaluated by flow cytometry (FCM); tumor necrosis factor (TNF) levels were measured in serum during the septic period . In addition, human neutrophils and monocytes were primed in vitro with IGF-1 and subsequently were stimulated with phorbol myristate acetate (PMA) or Escherichia coli; phagocytosis and respiratory burst were evaluated by FCM . RESULTS: Under nonseptic conditions, pretreatment with IGF-1 suppressed the ability of neutrophils to ingest bacteria (ie, the level of phagocytosis) 43.4% +/- 2.7% (IGF-1-treated) vs 55.8% +/- 3.4% (nontreated septic controls) and 57.3% +/- 3.34% (nonseptic controls) (p = .01) . When challenged by live E . coli infusion, phagocytosis increased in the IGF-1 group to the levels of the nontreated group . The respiratory burst showed a convincing priming effect of IGF-1 . After 4 hours of sepsis, the mean fluorescence intensity was 63.1 +/- 6.9 in the IGF-1 group and 40.7 +/- 3.0 in nontreated septic controls . The serum levels of TNF-alpha in the nontreated septic control group were twice those in the IGF-1-treated group, ie, 65.7 +/- 13.1 pg/mL in the nontreated septic controls and 31.5 +/- 7.5 pg/mL in the IGF-1 group (p = .03) . In vitro priming of human neutrophils and monocytes with IGF-1 and subsequent stimulation with PMA or E . coli demonstrated that IGF-1 enhanced both phagocytosis and respiratory burst . CONCLUSIONS: IGF-1 serves as a priming agent for biologic functions of leukocytes.

Invest Clin, 1998 Mar, 39(1), 19 - 28
{Development of an enzyme assay for detection of hepatitis B virus core IGM antibodies}; Uzcategui N et al.; Detection of IgM anti-core (anti-HBcAg) antibodies of Hepatitis B Virus (HBV) is an useful marker for hepatitis B virus (HBV) acute infection . The aim of this study was to perform an immunodiagnostic assay for the detection of IgM anti-HBcAg antibodies . Hepatitis B core antigen (HBcAg) was produced by a recombinant clone of Escherichia coli and used for the development of the immunoassay . An IgM capture enzyme immunoassay (EIA) was selected for the detection of IgM anti-HBcAg antibodies . A total of 110 human plasma or sera were tested by the capture EIA and a commercial assay . The capture EIA yielded 99% of sensitivity and 93% specificity, when compared with the commercial test . The capture EIA developed here is of interest for epidemiological studies, particularly for endemic regions in South America.

Nucleic Acids Symp Ser, 1997, (37), 321 - 2
Novel DNA detection system of flow injection analysis (2) . The distinctive properties of a novel system employing PNA (peptide nucleic acid) as a probe for specific DNA detection; Kai E et al.; In order to realize immediate detection of a double stranded DNA amplified by Polymerase Chain Reaction (PCR), we applied Peptide Nucleic Acid (PNA) to the probe of DNA detection system using Surface Plasmon Resonance (SPR) . We report our success in immediate detection of PCR products solution with high sequence-specificity.

Nucleic Acids Symp Ser, 1997, (37), 319 - 20
Removing tRNA from a cell-free protein synthesis system for use in protein production; Kanda T et al.; The cell-free system for biosynthesis of proteins is becoming an important tool for protein engineering . In particular, introduction of the unnatural amino acids is achieved though cell-free protein synthesis with the use of chemically acylated tRNA that recognizes a specific codon . In the original method, however, it was difficult to control the system through changing tRNA composition, as the endogenous tRNAs are involved in the reaction . Thus, in the present study, we digested the tRNA within Escherichia coli S30 extract with resin-bound RNase A, and estimated the protein synthesis activity . It was revealed that this digestion process does not damage the activity, if a protease inhibitor, phenylmethylsulfonyl fluoride (PMSF), is present in the digestion reaction.

Nucleic Acids Symp Ser, 1997, (37), 295 - 6
Recognition system of class II tRNA in Escherichia coli and yeast; Soma A et al.; The recognition system of class II tRNA, tRNA(Ser) and tRNA(Leu), in a yeast Saccharomyces cerevisiae was studied using T7 RNA polymerase transcription system . Yeast SerRS recognizes the long variable arm as in E . coli . However, the anticodon loop of tRNA(Leu), which has no effect on leucylation in E . coli, play a key role for recognition by LeuRS . Results suggest that the recognition style of yeast class II tRNA is substantially different from that of E . coli.

Nucleic Acids Symp Ser, 1997, (37), 287 - 8
Colicin E5 as a new type of cytotoxin, which cleaves a specific group of tRNAs; Masaki H et al.; The C-terminal active domain of colicin E5 has a novel ribonuclease activity which specifically cleaves queuine-containing tRNAs . Colicin E5 is a new type cytotoxin targeting a group of tRNAs.

Nucleic Acids Symp Ser, 1997, (37), 247 - 8
Novel detection system of flow injection analysis (1) . The existence of significant relation between secondary structure of DNA and sensitivity in signal detection; Sawata S et al.; Polymerase Chain Reaction (PCR) products was detected quantitatively using a flow injection type sensor, based on Surface Plasmon Resonance (SPR) . We used asymmetric PCR to amplify the two kinds of products; their DNA lengths are different . This novel design permitted us not only to detect PCR products with high-sensitivity, but also to develop a rapid DNA detection system for the sense of the genetic pathogen.

Nucleic Acids Symp Ser, 1997, (37), 191 - 2
Expression of Xenopus laevis translation initiation factor 4E (eIF-4E) by baculovirus-insect cell system; Miyoshi H et al.; A gene encoding Xenopus laevis eIF-4E was cloned into a transfer vector, and its gene expression was attempted in cells of E . coli, yeast and insect . Effective expression of the active eIF-4E was achieved in the soluble fraction of the insect cell Sf9, which was infected with the recombinant baculovirus . Overexpression of the eIF-4E protein caused remarkable change in the shape of the cells.

Nucleic Acids Symp Ser, 1997, (37), 185 - 6
Escherichia coli tmRNA (10Sa RNA) in trans-translation; Himeno H et al.; Here we show that Escherichia coli tmRNA (10Sa RNA) has a dual function both as an mRNA and as a tRNA in vitro . The function as a tRNA is prerequisite for the function as an mRNA . These observations strongly support the trans-translation hypothesis.

Nucleic Acids Symp Ser, 1997, (37), 165 - 6
Mechanisms of the genotoxic effects associated with 5-formyluracil: effect of exogenous 5'-formyl-2'-deoxyuridine; Ide H et al.; E . coli HB101 harboring plasmid pUC19 was grown in the presence of 5-formyl-2'-deoxyuridine (fdU) to evaluate the genotoxic and cytotoxic potentials associated with this DNA lesion . Cell growth was inhibited by fdU in a concentration-dependent manner, but increased mutation was not observed in the lacZ(alpha) gene of pUC19 . The lack of the mutagenic effect was attributed to poor utilization of fdU as a substrate by thymidine kinase, which converts exogenous thymidine analogs to the corresponding 5'-monophosphates in the salvage pathway.

Nucleic Acids Symp Ser, 1997, (37), 133 - 4
Role of the CCA end of tRNA and its vicinity in aminoacylation; Tamura K et al.; The CCA sequence is common to all the 3' ends of tRNAs and more than half of the tRNAs possess the G1C72 base pair at the end of acceptor stem . To study the role of these terminal trinucleotides and their vicinity in the aminoacylation process, not only the substitutions of these bases but also the nucleotide-additions at the 5' end of tRNA were introduced into many kinds of Escherichia coli tRNA transcripts and the effects on the aminoacylation activity with cognate aminoacyl-tRNA synthetase were investigated . Basically the requirement of the CCA sequence in aminoacylation differed among each amino acid specific tRNA . Some rough conclusions could be drawn from the present results . It was found that the G1C72 base pair functioned as the negative identity elements in many tRNAs . The nucleotide-addition experiments suggested that proper spatial arrangement and flexibility were important for aminoacylation reaction . Functional and evolutional implications of the invariant CCA sequence and its vicinity were discussed.

Nucleic Acids Symp Ser, 1997, (37), 131 - 2
Mechanism of substrate recognition by the ribotoxin, alpha-sarcin; Takeda E et al.; The substrate recognition and catalytic mechanisms of alpha-sarcin were explored with kinetic method by using synthetic 25-mer RNA mimicking the alpha-sarcin/ricin loop in 23S rRNA of E . coli ribosomes . The oligomer containing deoxy-G at the site of alpha-sarcin (G14) was a potent competitive inhibitor . The RNA having deoxy-G8 however, increases the Kcat value by about five times but without significant alteration on Km . Surprisingly, the deletion of G8 makes the oligomer become a strong noncompetitive inhibitor of the enzyme . These results suggested that there are at least two sites in the RNA substrate which are recognized by alpha-sarcin, one is the G8 bulge or at around its neighbor and the other is the GAGA in the sarcin/ricin loop of the rRNA.

Nucleic Acids Symp Ser, 1997, (37), 129 - 30
Quadruplex formation of oligonucleotides containing G clusters and their nuclease resistance; Shida T et al.; Oligoribonucleotides containing four G clusters, (UGnU)4 (n = 3-5), were synthesized and characterized by electrophoretic analysis and resistance to nucleases . Electrophoretic analyses indicate that the oligomers except for (UG3U)4 can form antiparallel-stranded monomeric quadruplexes in the presence of potassium cation . The oligomers used in this study are protected from endonuclease P1 and venom phosphodiesterase degradation . In addition, the oligomers indicate resistance to nucleases in human serum and E . coli S-100 fraction.

Nucleic Acids Symp Ser, 1997, (37), 125 - 6
Use of biotinylated-cysteinyl-tRNA as a non-RI probe in protein synthesis; Ohtsuka H et al.; A convenient method for the preparation of biotinylated aminoacyl-tRNA to use in the non-radioisotopic (non-RI) detection of cell-free translation products was developed . After aminoacylation of E . coli tRNA(Cys) with L-cystein, its sulfhydryl group was modified with N-(6-{Biotinamide}hexyl)-3'-(2'-pyridyl dithio) propionamide or 1-Biotin amido-4-(4'-{maleimidomethyl} cyclohexane-carboxamido) butane . These biotin-labelled cysteinyl-tRNA are expected to function as the non-RI probe for protein synthesis equally to or even better than the biotinylated lysyl-tRNA which is now commercially available.

Nucleic Acids Symp Ser, 1997, (37), 115 - 6
Codon reading properties of tRNA variants substituted within the anticodon loop; Ouchi R et al.; Effects of base substitution within tRNA anticodon loop on the codon reading activities were quantitatively analyzed with the use of a set of unmodified tRNA molecules with GGA anticodon . The first (position 32) and the last (position 38) nucleotides of the anticodon loop of the wild-type molecule was changed from C32A38 to U32A38, U32G38, and C32G38 . The codon reading activities of these variants relative to that of the wild type molecule were measured in a cell-free translation system . The reading of both the UCU and UCC codons were lower in all the three variants than in the wild-type molecule.

Proc Natl Acad Sci U S A, 1998 May 12, 95(10), 5511 - 5
Directed evolution of an aspartate aminotransferase with new substrate specificities; Yano T et al.; The substrate specificity of aspartate aminotransferase was successfully modified by directed molecular evolution using a combination of DNA shuffling and selection in an auxotrophic Escherichia coli strain . After five rounds of selection, one of the evolved mutants showed a 10(5)-fold increase in the catalytic efficiency (kcat/Km) for beta-branched amino and 2-oxo acids and a 30-fold decrease in that for the native substrates compared with the wild-type enzyme . The mutant had 13 amino acid substitutions, 6 of which contributed 80-90% to the total effect . Five of these six substitutions were conserved among the five mutants that showed the highest activity for beta-branched substrates . Interestingly, only one of the six functionally important residues is located within a distance of direct interaction with the substrate, supporting the idea that rational design of the substrate specificity of an enzyme is very difficult . The present results show that directed molecular evolution is a powerful technique for enzyme redesign if an adequate selection system is applied.

Proc Natl Acad Sci U S A, 1998 May 12, 95(10), 5495 - 500
Snapshot of a phosphorylated substrate intermediate by kinetic crystallography; Kack H et al.; The ATP-dependent enzyme dethiobiotin synthetase from Escherichia coli catalyses the formation of dethiobiotin from CO2 and 7, 8-diaminopelargonic acid . The reaction is initiated by the formation of a carbamate and proceeds through a phosphorylated intermediate, a mixed carbamic phosphoric anhydride . Here, we report the crystal structures at 1.9- and 1.6-A resolution, respectively, of the enzyme-MgATP-diaminopelargonic acid and enzyme-MgADP-carbamic-phosphoric acid anhydride complexes, observed by using kinetic crystallography . Reaction initiation by addition of either NaHCO3 or diaminopelargonic acid to crystals already containing cosubstrates resulted in the accumulation of the phosphorylated intermediate at the active site . The phosphoryl transfer step shows inversion of the configuration at the phosphorus atom, consistent with an in-line attack by the carbamate oxygen onto the phosphorus atom of ATP . A key feature in the structure of the complex of the enzyme with the reaction intermediate is two magnesium ions, bridging the phosphates at the cleavage site . These magnesium ions compensate the negative charges at both phosphate groups after phosphoryl transfer and contribute to the stabilization of the reaction intermediate.

Structure, 1998 Apr 15, 6(4), 439 - 49
How glutaminyl-tRNA synthetase selects glutamine; Rath VL et al.; BACKGROUND: Aminoacyl-tRNA synthetases covalently link a specific amino acid to the correct tRNA . The fidelity of this reaction is essential for accurate protein synthesis . Each synthetase has a specific molecular mechanism to distinguish the correct pair of substrates from the pool of amino acids and isologous tRNA molecules . In the case of glutaminyl-tRNA synthetase (GlnRS) the prior binding of tRNA is required for activation of glutamine by ATP . A complete understanding of amino acid specificity in GlnRS requires the determination of the structure of the synthetase with both tRNA and substrates bound . RESULTS: A stable glutaminly-adenylate analog, which inhibits GlnRS with a Ki of 1.32 microM, was synthesized and cocrystallized with GlnRS and tRNA2Gln . The crystal structure of this ternary complex has been refined at 2.4 A resolution and shows the interactions made between glutamine and its binding site . CONCLUSIONS: To select against glutamic acid or glutamate, both hydrogen atoms of the nitrogen of the glutamine sidechain are recognized . The hydroxyl group of Tyr211 and a water molecule are responsible for this recognition; both are obligate hydrogen-bond acceptors due to a network of interacting sidechains and water molecules . The prior binding of tRNAGln that is required for amino acid activation may result from the terminal nucleotide, A76, packing against and orienting Tyr211, which forms part of the amino acid binding site.

Structure, 1998 Apr 15, 6(4), 501 - 9
Three-dimensional reconstructions from cryoelectron microscopy images reveal an intimate complex between helicase DnaB and its loading partner DnaC; San Martin C et al.; BACKGROUND: DNA helicases play a fundamental role in all aspects of nucleic acid metabolism and defects in these enzymes have been implicated in a number of inherited human disorders . DnaB is the major replicative DNA helicase in Escherichia coli and has been used as a model system for studying the structure and function of hexameric helicases . The native protein is a hexamer of identical subunits, which in solution forms a complex with six molecules of the loading protein DnaC . DnaB is delivered from this complex onto the DNA template, with the subsequent release of DnaC . We report here the structures of the DnaB helicase hexamer and its complex with DnaC under a defined set of experimental conditions, as determined by three-dimensional cryoelectron microscopy . It was hoped that the structures would provide insight into the mechanisms of helicase activity . RESULTS: The DnaB structure reveals that six DnaB monomers assemble as three asymmetric dimers to form a polar, ring-like hexamer . The hexamer has two faces, one displaying threefold and the other sixfold symmetry . The six DnaC protomers bind tightly to the sixfold face of the DnaB hexamer . This is the first report of a three-dimensional structure of a helicase obtained using cryoelectron microscopy, and the first report of the structure of a helicase in complex with a loading protein . CONCLUSIONS: The structures of the DnaB helicase and its complex with DnaC reveal some interesting structural features relevant to helicase function and to the assembly of the two-protein complex . The results presented here provide a basis for a more complete understanding of the structure and function of these important proteins.

Structure, 1998 Apr 15, 6(4), 465 - 75
Structure and control of pyridoxal phosphate dependent allosteric threonine deaminase; Gallagher DT et al.; BACKGROUND: Feedback inhibition of biosynthetic threonine deaminase (TD) from Escherichia coli provided one of the earliest examples of protein-based metabolic regulation . Isoleucine, the pathway end-product, and valine, the product of a parallel pathway, serve as allosteric inhibitor and activator, respectively . This enzyme is thus a useful model system for studying the structural basis of allosteric control mechanisms . RESULTS: We report the crystal structure of TD at 2.8 A resolution . The tetramer has 222 symmetry, with C-terminal regulatory domains projecting out from a core of catalytic PLP-containing N-terminal domains . The subunits, and especially the regulatory domains, associate extensively to form dimers, which associate less extensively to form the tetramer . Within the dimer, each monomer twists approximately 150 degrees around a thin neck between the domains to place its catalytic domain adjacent to the regulatory domain of the other subunit . CONCLUSIONS: The structure of TD and its comparison with related structures and other data lead to the tentative identification of the regulatory binding site and revealed several implications for the allosteric mechanism . This work prepares the way for detailed structure/function studies of the complex allosteric behaviour of this enzyme.

J Mol Endocrinol, 1998 Apr, 20(2), 233 - 44
Binding characteristics of antibodies to the TSH receptor; Oda Y et al.; We have used fragments of the TSH receptor (TSHR) expressed in E . coli as glutathione S-transferase fusion proteins to produce rabbit polyclonal antibodies and a panel (n=5) of monoclonal antibodies to the extracellular fragment of the TSHR . The binding characteristics of the antibodies to linear, conformational, glycosylated and unglycosylated forms of the receptor in different assay systems have been investigated . The reactivity of these antibodies with the TSHR was assessed by Western blotting with both native and recombinant human TSHR expressed in CHO cells, immunoprecipitation of 35S-labelled full-length TSHR produced in an in vitro transcription/ translation system, immunoprecipitation of 125I-TSH/TSHR complexes, inhibition of 125I-TSH binding to the TSHR and fluorescence activated cell sorter (FACS) analysis of binding to CHO-K1 cells expressing the TSHR on their cell surface . Fab fragments of monoclonal antibodies were isolated, labelled with 125I and used to determine the affinity constants of these antibodies with receptor, bound and free Fab being separated by polyethylene glycol (PEG) precipitation . Rabbit polyclonal and mouse monoclonal antibodies reacted with the TSHR in Western blotting and one monoclonal antibody (3C7) was able to inhibit 125I-TSH binding to native human TSHR (74% inhibition), recombinant human TSHR (84% inhibition) and porcine TSHR (65% inhibition) . Affinity constant values for TSHR monoclonal antibody Fab fragments calculated using Scatchard analysis were about 10(7) M(-1) . Four out of five monoclonal antibodies reacted in FACS analysis with TSHR expressed on the surface of CHO-K1 cells . The FACS unreactive monoclonal (3C7) bound well to detergent solubilised TSH receptors and this emphasised the importance of using a combination of FACS analysis and radioactively-labelled probes in analysis of the TSH receptor . The monoclonal antibodies produced in this study were found to be of relatively low affinity but proved useful for detection of the receptor by Western blotting and by FACS analysis.

Trends Biochem Sci, 1998 Apr, 23(4), 138 - 43
The ins and outs of a molecular chaperone machine; Richardson A et al.; Genetic and biochemical work has highlighted the biological importance of the GroEL/GroES (Hsp60/Hsp10; cpn60/cpn10) chaperone machine in protein folding . GroEL's donut-shaped structure has attracted the attention of structural biologists because of its elegance as well as the secrets (substrates) it can hide . The recent determination of the GroES and GroEL/GroES structures provides a glimpse of their plasticity, revealing dramatic conformational changes that point to an elaborate mechanism, coupling ATP hydrolysis to substrate release by GroEL.

Plasmid, 1998, 39(3), 165 - 81
Nucleotide sequence and genetic characterization of the novel IncQ-like plasmid pIE1107; Tietze E; The analysis of the complete nucleotide sequence of the small resistance plasmid pIE1107 revealed a close similarity to the well-known IncQ plasmids . Highly conserved replication proteins and nearly identical origins of replication (oriV) suggest equivalent functions in the related replication systems . However, pIE1107 contains two copies of IncQ-oriV-like DNA which are slightly different regarding the iterons . Upon deletion of a silent copy of IncQ-oriV-like DNA the resulting plasmid is fully compatible with IncQ plasmids, indicating that there is no mutual communication between the replication control of the respective replicons . Experiments with cloned oriV DNA strongly suggest that the replication initiation protein of pIE1107 has specialized into the distinct target-iterons of its own oriV which differs only by a few nucleotides from the oriV of IncQ plasmids . Implications from the apparent highly specific protein-DNA recognition and from the incompatibility properties of pIE1107 for the evolution of a family of compatible, IncQ-like plasmids are discussed .

Eur J Immunol, 1998 Apr, 28(4), 1243 - 50
Mutational analysis of the role of ADP-ribosylation activity and GM1-binding activity in the adjuvant properties of the Escherichia coli heat-labile enterotoxin towards intranasally administered keyhole limpet hemocyanin; de Haan L et al.; The Escherichia coli heat-labile enterotoxin (LT) is known for its potent mucosal immunoadjuvant activity towards co-administered antigens . LT is composed of one A subunit, which has ADP-ribosylation activity, and a homopentameric B subunit, which has high affinity for the toxin receptor, ganglioside GM1 . In previous studies, we have investigated the role of the LTA and LTB subunits in the adjuvanticity of LT towards influenza virus hemagglutinin (HA), administered intranasally to mice . We now studied the adjuvant properties of LT and LT variants towards keyhole limpet hemocyanin (KLH), which, in contrast to HA, does not bind specifically to mucosal surfaces . It is demonstrated that LT mutants without ADP-ribosylation activity, as well as LTB, retain mucosal immunoadjuvant activity when administered intranasally to mice in conjunction with KLH . As with influenza HA, adjuvanticity of LTB required GM1-binding activity, whereas GM1-binding was not essential for adjuvant activity of LT . Furthermore, we found that also recombinant LTA alone acts as a potent mucosal adjuvant, and that this adjuvanticity is independent of ADP-ribosylation activity . It is concluded that binding of the antigen to mucosal surfaces does not play an essential role in the immunostimulation by LT and LT variants, and that both recombinant LTA and LTB represent powerful nontoxic mucosal adjuvants.

Eur J Immunol, 1998 Apr, 28(4), 1155 - 60
Differential IgE recognition of recombinant Aspergillus fumigatus allergens by cystic fibrosis patients with allergic bronchopulmonary aspergillosis or Aspergillus allergy; Hemmann S et al.; Allergic bronchopulmonary aspergillosis (ABPA), an intense inflammatory reaction to Aspergillus in the lung, is recognized as a severe complication in patients with cystic fibrosis (CF) . The diagnosis of ABPA in CF patients sensitized to Aspergillus fumigatus is complicated by interfering laboratory and clinical findings shared by the diseases . We have used cDNA encoding A . fumigatus allergens which were cloned from a cDNA library displayed on phage surface to produce recombinant proteins in Escherichia coli . Differential IgE responses to the allergens in A . fumigatus-sensitized CF patients with or without ABPA and CF controls without sensitization to A . fumigatus were demonstrated . A secreted ribotoxin (rAsp f 1) and a peroxisomal protein (rAsp f 3) were recognized by sera from A . fumigatus-sensitized CF-patients with or without ABPA . An intracellular manganese superoxide dismutase (rAsp f 6) and rAsp f 4, a protein with unknown function, were recognized exclusively by IgE from sera of CF patients with ABPA . Therefore, Asp f 4 and Asp f 6 represent specific markers for ABPA and allow a sensitive, fully specific diagnosis of the disease . The data suggest distinct IgE responses to colonization of the bronchial tree in CF patients with ABPA or A . fumigatus allergy and therefore a differential recognition of the pathogen in the two IgE-related inflammatory diseases.

Blood Cells Mol Dis, 1998 Mar, 24(1), 54 - 61
Expression and enzymatic characterization of human glucose phosphate isomerase (GPI) variants accounting for GPI deficiency; Kanno H et al.; To elucidate the structure-function relationships in glucose phosphate isomerase (GPI), we established an expression system for human GPI as a fusion protein with glutathione S-transferase (GST) in E . coli . The GST-GPI fusion protein showed affinities for the substrates glucose 6-phosphate (G6P) and fructose 6-phosphate (F6P) similar to those of the native enzyme purified from human red blood cells (RBC) . We expressed GPI cDNAs with four distinct disease-causing mutations and examined their enzymatic characteristics . Although each mutation caused reduced thermal stability, an amino acid substitution Thr-5-->Ile (T5I) exhibited marked thermal instability, suggesting that the amino-terminal of GPI is important for enzymatic stability . Thr-224 seemed not to be an essential residue, since the amino acid substitution Thr-224-->Met (T224M) showed normal substrate affinity in spite of a slight decrease in both specific activity and thermostability . Gln-343 and Asp-539 have been shown to be in close proximity to the putative catalytic sites, and the present study showed that both Gln-343-->Arg (Q343R) and Asp-539-->Asn (D539N) caused impaired substrate affinity; Q343R showed high Km for both G6P and F6P, whereas D539N showed significantly decreased affinity only for F6P . These results suggest that not only reduced enzymatic stability but also impaired kinetics may disturb RBC metabolism of the GPI variants associated with hereditary hemolytic anemia .

Gene, 1998 Mar 16, 209(1-2), 51 - 8
Identification of Btr-regulated genes using a titration assay . Search for a role for this transcriptional regulator in the growth and virulence of Bordetella pertussis; Wood GE et al.; Bordetella pertussis is the causative agent of the respiratory disease pertussis or whopoping cough . Btr, an oxygen-responsive transcriptional regulator of B . pertussis, is homologous to the FNR protein of E . coli . Using a murine respiratory model, we observed in the present study that Btr is important in growth and survival of B . pertussis in vivo . A titration assay was developed that identified genes containing Btr binding sites including B . pertussis sodB and btr, E . coli aspA and a new B . pertussis gene, brg1 . The brg1 gene encodes a protein similar to the LysR family of transcriptional regulators and its expression is activated threefold by Btr under anaerobic growth conditions but unaffected by Btr aerobically . The nucleotide sequence flanking brg1 encodes proteins with similarity to various metabolic enzymes . Putative overlapping promoters and a Btr binding site (FNR box) were identified in the DNA sequence between brg1 and the adjacent genes . These intervening sequences may represent sites for regulation by Btr and Brg1.

Gene, 1998 Mar 16, 209(1-2), 167 - 74
Gene structure, expression in Escherichia coli and biochemical properties of the NAD+ -dependent glyceraldehyde-3-phosphate dehydrogenase from Pinus sylvestris chloroplasts; Meyer-Gauen G et al.; Photosynthetic eukaryotes typically possess two distinct glyceraldehyde-3-phosphate dehydrogenases, an NAD+ -specific enzyme in the cytosol (GapC: EC 1.2.1.12) and an NADP+ -dependent enzyme in the chloroplast (GapAB: EC 1.2.1.13) . The gymnosperm Pinus sylvestris is an exception in that it is known to express a gene encoding a transit peptide-bearing GapC-like subunit that is imported into chloroplasts (GapCp), but the enzymatic properties of this novel GAPDH have not been described from any source . We have expressed the mature GapCp unit from Pinus in Escherichia coli and have characterized the active enzyme . GapCp has a specific activity of 89 units per milligram and is strictly NAD+ -dependent, showing no detectable activity with NADP+ . Values of the apparent Km for NAD+ and glyceraldehyde-3-phosphate were determined as 62 and 344 microM, respectively . The Pinus GapCpl gene possesses 12 introns, two in the region encoding the transit peptide and ten in the region encoding the mature subunit, all of which are found at positions strictly conserved across genes for higher plant GapC . A cDNA encoding a homologue of GapCp was isolated from the heterosporous fern Marsilea quadrifolia, indicating that NAD+ -dependent chloroplast GAPDH also occurs in other higher plants.

Gene, 1998 Mar 16, 209(1-2), 113 - 22
Molecular characterization of the Helicobacter pylori uvr B gene; Thompson SA et al.; Helicobacter pylori persists in the human stomach where it may encounter a variety of DNA-damaging conditions, including gastric acidity . To determine whether the nucleotide excision repair (NER) pathway contributes to the repair of acid-induced DNA damage, we have cloned the putative H . pylori NER gene, uvrB . Degenerate oligonucleotide primers based on conserved amino acid residues of bacterial UvrB proteins were used in PCR with genomic DNA from H . pylori strain 84-183, and the 1.3-kb PCR product from this reaction was used as a probe to clone uvrB from an H . pylori genomic library . This plasmid clone had a 5.5-kb insert containing a 2.0-kb ORF whose predicted product (658 amino acids; 75.9 kDa) exhibited 69.5% similarity to E . coli UvrB . We constructed an isogenic H . pylori uvrB mutant by inserting a kanamycin-resistance cassette into uvrB and verified its proper placement by Southern hybridization . As with uvrB mutants of other bacteria, the H . pylori uvrB mutant showed a greatly increased sensitivity to the DNA-damaging agents methylmethane sulfonate and ultraviolet radiation . The uvrB mutant also was significantly more sensitive than the wild-type strain to killing by low pH, suggesting that the H . pylori nucleotide excision repair (NER) pathway is involved in the repair of acid-induced DNA damage.

Oncogene, 1998 Apr 9, 16(14), 1879 - 84
Evaluation of an autoregulatory tetracycline regulated system; Gallia GL et al.; Tetracycline controlled gene expression systems have become powerful tools in the analysis of gene function in mammalian cell culture as well as transgenic animals and plants . The original description of a tetracycline-regulated gene expression system is based on two plasmids, one of which constitutively expresses a tetracycline-controlled transactivator protein (tTA), a fusion protein between the tetracycline repressor of E . coli and the transcriptional activation domain of the VP16 protein of herpes simplex virus . The second plasmid contains the gene to be regulated by tTA under the control of an inducible promoter which consists of seven copies of the tetracycline resistance operator (tetO) . Since this original description, many modifications have been described . In this report, we evaluate an autoregulatory tetracycline controlled system, in which the tTA is itself under the control of the tetO . We demonstrate that this autoregulatory tetracycline system produces adverse effects including cellular morphologic changes, growth rate attenuation and alterations in cell cycle distribution.

Gesundheitswesen, 1998 Mar, 60(3), 159 - 65
{Infections with enterohemorrhagic Escherichia coli (EHEC)--results of an epidemiologic survey in Bavaria for the April 1996 to May 1997 time frame}; Huber HC et al.; Using a report system of the Bavarian Public Health services 300 EHEC infections were registered within one year (Apr 1, 1996, to Mar 31, 1997) in Bavaria . These consisted of 22 cases of haemolytic uraemic syndrome (HUS) (mean age, 2.8 yr), 6 cases of incomplete HUS (1.3 yr), 188 cases of enteritis (3.3 yr) and 84 asymptomatic infections (12.9 yr), respectively . From this follows an incidence rate for all infections of approximately 2.5 per 100,000 inhabitants and for HUS in the age group of children and juveniles up to 18 years of approximately 0.8, respectively . Possible sources (paths) of infection registered in relevant frequencies were: raw milk consumption (18% of the infected), farm-animal associated contact (43%), and contact with patients suffering from diarrhoea (36%).

Endocrinology, 1998 May, 139(5), 2278 - 83
In vivo and in vitro evidence for the involvement of tumor necrosis factor-alpha in the induction of leptin by lipopolysaccharide; Finck BN et al.; To examine the role of tumor necrosis factor-alpha (TNF alpha) in mediating leptin secretion during an immunological challenge, we studied the effects of lipopolysaccharide (LPS) and TNF alpha on leptin secretion in endotoxin-sensitive C3H/HeOuJ (OuJ) mice, endotoxin-insensitive C3H/HeJ (HeJ) mice, and primary adipocytes cultured from both . Intraperitoneal injection of LPS increased plasma concentrations of TNF alpha and leptin in OuJ mice, but not in HeJ mice, suggesting a causal relationship between the induction of TNF alpha and leptin . Consistent with this idea, i.p . injection of recombinant murine TNF alpha increased plasma leptin in both OuJ and HeJ mice . To determine whether TNF alpha induces leptin secretion by acting directly on fat cells, primary adipocytes from OuJ and HeJ mice were cultured in the presence of TNF alpha or LPS . Whereas LPS was without effect on leptin secretion by adipocytes, TNF alpha induced a marked increase in the cell supernatant leptin concentration . These data demonstrate that TNF alpha plays a role in regulating the increase in leptin caused by LPS . Moreover, they show that TNF alpha can act directly on adipocytes to stimulate leptin secretion . Our results are consistent with the emerging view that leptin is a key hormone coupling immune system activity to energy balance.

Biochemistry, 1998 Apr 21, 37(16), 5746 - 54
Epimerization via carbon-carbon bond cleavage . L-ribulose-5-phosphate 4-epimerase as a masked class II aldolase; Johnson AE et al.; Studies indicating that the E . coli L-ribulose-5-phosphate 4-epimerase employs an "aldolase-like" mechanism are reported . This NAD+-independent enzyme epimerizes a stereocenter that does not bear an acidic proton and therefore it cannot utilize a simple deprotonation-reprotonation mechanism . Sequence similarities between the epimerase and the class II l-fuculose-1-phosphate aldolase suggest that the two may be evolutionarily related and that the epimerization may occur via carbon-carbon bond cleavage and re-formation . Conserved residues thought to provide the metal ion ligands of the epimerase have been modified using site-directed mutagenesis . The resulting mutants show low kcat values in addition to a reduced affinity for Zn2+ . These observations serve to establish that there is a structural link between between the active site geometry of the epimerase and the aldolase . In addition, the H97N mutant was found to catalyze the condensation of dihydroxyacetone and glycolaldehyde phosphate to produce a mixture of L-ribulose-5-phosphate and D-xylulose-5-phosphate . This observation of aldolase activity establishes that the epimerase active site is capable of promoting carbon-carbon bond cleavage . Furthermore, glycolaldehyde phosphate was shown to be a competitive inhibitor of the mutant enzyme (KI = 0.37 mM) but not of the wild-type enzyme . The mutation apparently causes the epimerase to become "leaky" and enables it to bind/generate the normal reaction intermediates from the unbound aldol cleavage products.

Biochemistry, 1998 Apr 21, 37(16), 5682 - 8
Intra- vs intersubunit communication in the homodimeric restriction enzyme EcoRV: Thr 37 and Lys 38 involved in indirect readout are only important for the catalytic activity of their own subunit; Stahl F et al.; EcoRV is a dimer of two identical subunits which together form one binding site for the double-stranded DNA substrate . Concerted cleavage of both strands of the duplex requires intersubunit communication to synchronize the two catalytic centers of EcoRV . Here we address the question of how contacts to the DNA backbone trigger conformational changes which lead to the activation of both catalytic centers . The structure of the specific EcoRV-DNA complex shows that a region including amino acids Thr 37 and Lys 38 is involved in interactions with the DNA backbone and is a candidate for intersubunit communication . Homodimeric EcoRV T37A and K38A variants have a 1000-fold reduced catalytic activity . To examine whether Thr 37 and Lys 38 of one subunit affect the catalytic center in the same subunit and/or in the other subunit, we have produced heterodimeric variants containing a Thr 37 --> Ala or Lys 38 --> Ala substitution in one subunit combined with a wild type (wt) subunit (wt/T37A and wt/K38A) or with a subunit which contains an amino acid substitution (Asp 90 --> Ala) in the active site (D90A/T37A and D90A/K38A) . Cleavage experiments with supercoiled pAT153 show that wt/T37A and wt/K38A preferentially nick the DNA . A steady-state kinetic analysis of the cleavage of an oligodeoxynucleotide substrate shows that the activity of wt/T37A and wt/K38A is half of that of wild type EcoRV, whereas D90A/T37A and D90A/K38A are almost inactive . These results demonstrate that Thr 37 and Lys 38 affect primarily the catalytic center in their own subunit and that both subunits of EcoRV can be activated independently of each other . We suggest that Thr 37 and Lys 38 control the catalytic activity of the active site in their own subunit by positioning alpha-helix B.

Biochemistry, 1998 Apr 21, 37(16), 5643 - 53
Conformational changes in the extrinsic manganese stabilizing protein can occur upon binding to the photosystem II reaction center: an isotope editing and FT-IR study; Hutchison RS et al.; Photosystem II catalyzes the light-driven oxidation of water and reduction of plastoquinone in oxygenic photosynthesis . The manganese stabilizing protein (MSP) of photosystem II is an extrinsic subunit that plays an important role in catalytic activity . This subunit can be extracted and re-bound to the photosystem II reaction center . Extraction is associated with decreased stability of manganese binding by the enzyme and by loss in high rates of oxygen evolution activity; reconstitution reverses these phenomena . Since little is known about the assembly of complex membrane proteins, we have employed isotope editing and vibrational spectroscopy to obtain information about any changes in secondary structure that occur in MSP upon functional reconstitution to photosystem II . The spectroscopic data obtained are consistent with substantial changes in conformation when MSP binds to photosystem II; approximately 30-40% of the peptide backbone undergoes a change in secondary structure . These conclusions were reached by comparing different aliquots, before and after binding, of the same 13{C}MSP sample . Analysis of amide I band line shapes through Fourier deconvolution and nonlinear regression suggests that binding of MSP to photosystem II is associated with a decrease in random structure and an increase in beta-sheet content . We conclude that binding of MSP to the reaction center can induce folding of MSP . Our results also indicate that, in solution, MSP can sample a variety of conformational states, which differ in hydrogen bonding of the peptide backbone.

Biochemistry, 1998 Apr 21, 37(16), 5372 - 82
Methionine synthase exists in two distinct conformations that differ in reactivity toward methyltetrahydrofolate, adenosylmethionine, and flavodoxin; Jarrett JT et al.; Methionine synthase (MetH) from Escherichia coli catalyzes the synthesis of methionine from homocysteine and methyltetrahydrofolate via two methyl transfer reactions that are mediated by the endogenous cobalamin cofactor . After binding both substrates in a ternary complex, the enzyme transfers a methyl group from the methylcobalamin cofactor to homocysteine, generating cob(I)alamin enzyme and methionine . The enzyme then catalyzes methyl transfer from methyltetrahydrofolate to the cob(I)alamin cofactor, forming methylcobalamin cofactor and tetrahydrofolate prior to the release of both products . The cob(I)alamin form of the enzyme occasionally undergoes oxidation to an inactive cob(II)alamin species; the enzyme also catalyzes its own reactivation . Electron transfer from reduced flavodoxin to the cob(II)alamin cofactor is thought to generate cob(I)alamin enzyme, which is then trapped by methyl transfer from adenosylmethionine to the cobalt, restoring the enzyme to the active methylcobalamin form . Thus the enzyme is potentially able to catalyze two methyl transfers to the cob(I)alamin cofactor: methyl transfer from methyltetrahydrofolate during primary turnover and methyl transfer from adenosylmethionine during activation . It has recently been shown that methionine synthase is constructed from at least four separable regions that are responsible for binding each of the three substrates and the cobalamin cofactor, and it has been proposed that changes in positioning of the substrate binding regions vis-a-vis the cobalamin binding region could allow the enzyme to control which substrate has access to the cofactor . In this paper, we offer evidence that methionine synthase exists in two different conformations that interconvert in the cob(II)alamin oxidation state . In the primary turnover conformation, the enzyme reacts with homocysteine and methyltetrahydrofolate but is unreactive toward adenosylmethionine and flavodoxin . In the reactivation conformation, the enzyme is active toward adenosylmethionine and flavodoxin but unreactive toward methyltetrahydrofolate . The two conformations differ in the susceptibility of the substrate-binding regions to tryptic proteolysis . We propose a model in which conformational changes control access to the cobalamin cofactor and are the primary means of controlling cobalamin reactivity in methionine synthase.

Biochemistry, 1998 Apr 21, 37(16), 5356 - 61
Characterization and functional role of the QH site of bo-type ubiquinol oxidase from Escherichia coli; Sato-Watanabe M et al.; Cytochrome bo is a four-subunit terminal ubiquinol oxidase in the aerobic respiratory chain of Escherichia coli that vectorially translocates protons not only via directed protolytic reactions but also via proton pumping . Previously, we postulated that a bound quinone in the high-affinity quinone binding site (QH) mediates electron transfer from the low-affinity quinol oxidation site (QL) in subunit II to low-spin heme b in subunit I as an electron gate and a transient electron reservoir {Sato-Watanabe, M., Mogi, T., Ogura, T., Kitagawa, T., Miyoshi, H., Iwamura, H., and Anraku, Y . (1994b) J . Biol . Chem . 269, 28908-28912} . In the present study, we carried out screening of ubiquinone analogues using a bound ubiquinone-free enzyme (DeltaUbiA1) that has been isolated from a ubiquinone biosynthesis mutant, and identified PC24 (2-chloro-4, 6-dinitrophenol), PC32 (2,6-dibromo-4-cyanophenol), and PC52 (2-isopropyl-5-methyl-4,6-dinitrophenol) as potent QH site inhibitors . PC15 (2,6-dichloro-4-nitrophenol) and PC16 (2, 6-dichloro-4-dicyanovinylphenol), potent QL site inhibitors, did not exhibit such a selective inhibition of the QH site . Binding studies using the air-oxidized DeltaUbiA enzyme showed that PC32 and PC52 have 4- to 7-fold higher affinity than ubiquinone-1 . Reconstitution of the QH site with PC32 and PC52 resulted in a decrease of the apparent Vmax value to 1/7 and 1/3, respectively, of the control activity . These findings suggest that structural features of the QL and QH sites are different, and provide further support for the involvement of the QH site in intramolecular electron transfer and facile oxidation of quinols at the QL site.

Biochemistry, 1998 Apr 21, 37(16), 5349 - 55
Mapping the active sites of 3-phosphoglycerate kinase and glycerol kinase with monoammine chromium(III) ATP; Lester LM et al.; The 12 isomers of monoammine chromium(III) ATP have been used to probe the ATP binding sites of yeast 3-phosphoglycerate kinase and glycerol kinase from Candida mycoderma . Inhibition studies of 3-phosphoglycerate kinase show a dramatic decrease in isomer binding only when the ammonia is in the Delta axial facial anti position . This suggests an open site architecture with only one strong contact point between the coordination sphere and the enzyme surface . These results agree well with the computer modeling studies of bidentate chromium ATP into the nucleotide site determined by X-ray crystallography {McPhillips, T., et al . (1996) Biochemistry 35, 4118-4127} . Both methods describe an open site strongly supporting the validity of the inhibition studies . Inhibition studies of glycerol kinase show significant decreases in binding for all the tested ammonia positions, suggesting a closed site architecture with many contacts between the coordination sphere and the surface of the enzyme . This is in good agreement with X-ray studies {Hurley, T., et al . (1993) Science 259, 673-677} on the Escherichia coli glycerol kinase . Inhibition studies of hexokinase previously reported {Rawlings, J., et al . (1993) Biochemistry 32, 11204-11210} more closely resemble those of 3-phosphoglycerate kinase, suggesting the surprising result that however closely hexokinase and glycerol kinase are related structurally the site around the coordination sphere in hexokinase is functionally open like that of 3-phosphoglycerate kinase.

Biochemistry, 1998 Apr 14, 37(15), 5249 - 57
Regulation of RhoA GTP hydrolysis by the GTPase-activating proteins p190, p50RhoGAP, Bcr, and 3BP-1; Zhang B et al.; The small GTP-binding protein RhoA becomes inactivated by hydrolyzing bound GTP to GDP through its intrinsic GTPase activity which is further stimulated by a family of Rho GTPase-activating proteins (GAPs) . Here we have compared the kinetics of interaction between recombinant RhoA and the RhoGAP domains of p190, p50RhoGAP, Bcr, and 3BP-1 . The intrinsic rate of GTP hydrolysis by RhoA is relatively slow when compared to other Rho-family GTPases such as Cdc42 or Rac1 with a rate constant of 0.022 min-1, which can be further stimulated at least 4000-fold by p190 or p50RhoGAP . The RhoGAP domains of Bcr and 3BP-1, which were thought to be inactive toward RhoA, are also found capable of stimulating the GTPase activity of RhoA in a dose-dependent manner . The supreme catalytic activities of p190 and p50RhoGAP toward RhoA reside mostly in their lower Km values (1.79 and 2.83 microM, respectively) which correlate well with their binding affinity for GMP-PNP-bound RhoA (2.18 and 2 . 47 microM, respectively), in contrast with Bcr and 3BP-1 which interact with the activated RhoA with much higher Km (89 microM) . However, the mechanisms of catalysis by p190 and p50RhoGAP are distinct in at least three aspects: (1) p50RhoGAP displays an effect of product inhibition by binding to the GDP-bound form of RhoA with a Kd of 6 microM in comparison with the Kd for p190 of 33 microM; (2) the Km of p190 increases drastically upon the increase of salt and Mg2+ concentrations, conditions under which only modest changes of Km for p50RhoGAP are observed; and (3) p50RhoGAP remains partially active toward the effector domain mutants of RhoA, Y34K, and T37A, whereas p190 is completely inactive toward Y34K and T37A . These results suggest that there exists a unique mechanism of functional interaction between RhoA and individual RhoGAP which involves distinct structural determinants of the small G-protein to cause the apparent differences in kinetic properties.

Biochemistry, 1998 Apr 14, 37(15), 5201 - 10
Escherichia coli cAMP receptor protein-DNA complexes . 2 . Structural asymmetry of DNA bending; Pyles EA et al.; The effect of DNA sequence variability and the degree of cyclic AMP receptor protein (CRP)-induced bending of the flanking ends of fluorescently labeled DNA were investigated by steady-state fluorescence and differential phase polarization studies in the presence and absence of CRP . Six sequences, including the primary CRP binding sites of lac P1 (class I) and gal P1 (class II), were studied . Excitation and emission spectra of CPM-DNA upon binding CRP were observed to be qualitatively similar to one another, regardless of the CRP binding site sequence examined or the location of the probe . This result implies that the probe is not interacting with the protein . However, the magnitude of the changes in the fluorescence intensities of sensitized emission spectra of CPM-DNA is apparently dependent on the DNA sequence, indicating that the environments of the flanking ends of DNA may be different from one another in the protein-DNA complex . Differential phase polarization results were qualitatively consistent with the fluorescence energy transfer measurements . The implication of this study supports the idea that the DNA is bent symmetrically in the lac-CRP complex but is asymmetrically bent in the gal-CRP complex . The sequence in the half-site in conjunction with the flanking sequence defines the geometry of the bent DNA . It appears that the CRP-induced bend in the DNA may also be class dependent . This may be an important feature used by the system to regulate transcription at different promoter sites.

Biochemistry, 1998 Apr 14, 37(15), 5194 - 200
Escherichia coli cAMP receptor protein-DNA complexes . 1 . Energetic contributions of half-sites and flanking sequences in DNA recognition; Pyles EA et al.; In Escherichia coli, the cyclic AMP receptor protein (CRP) serves as a sensor of the intracellular level of cyclic AMP . At increasing concentrations of cyclic AMP, CRP becomes activated upon binding a cyclic AMP molecule . The activated CRP is capable of regulating the expression of more than 20 genes by binding to specific DNA sites . The specific DNA sequences recognized by CRP consist of two-half-sites of the consensus sequence TGTGA......XCAXA . At present, the relative contributions of the two half-site and flanking sequences in the energetics of CRP recognition have not been quantitatively defined . A series of 20 DNA sequences was designed to dissect the contributions of individual half-sites and flanking sequences using the natural gal P1 and lac P1 sequences as the initial targets . The binding of CRP to these DNA sequences was monitored by fluorescence anisotropy . None of the individual half-sites or flanking sequences contributes more to the binding energetics than a random sequence . In the lac P1 sequence, the combination of both half-sites leads to a >100-fold increase in affinity compared to that of an individual half-site in CRP-DNA complex formation . The flanking sequence of lac P1 exhibits a 10- and 0-fold enhancement in affinity for CRP compared to that of a random sequence in the presence and absence of the two half-sites, respectively . The observations of the gal P1 sequence differ from those of the lac P1 sequence . The combination of both half-sites exhibits no significant increase in affinity, but the flanking sequence exhibits a 100-fold enhancement in the presence of the two half-sites . However, there is a disproportionate contribution from the flanking sequence proximal to the conserved TGTGA motif . The total energetics of the gal-CRP complex formation is essentially due to the presence of the conserved half-site and its adjacent flanking sequence . Thus, the relative contributions of the half-site and flanking sequences to the energetics of DNA recognition are operon specific.

Biochemistry, 1998 Apr 14, 37(15), 5173 - 83
Single amino acid substitutions at the N-terminus of a recombinant cytotoxic ribonuclease markedly influence biochemical and biological properties; Newton DL et al.; Onconase is a cytotoxic ribonuclease with antitumor properties . A semisynthetic gene encoding the entire protein sequence was constructed by fusing oligonucleotides coding for the first 15 and the last 6 of the 104 amino acids to a genomic clone that encoded the remaining amino acid residues {Newton, D . L., et al . (1997) Protein Eng . 10, 463-470} . The resulting protein product expressed in Escherichia coli exhibited little enzymatic or cytotoxic activity due to the unprocessed N-terminal Met amino acid residue . In this study, we demonstrate that modification of the 5'-region of the gene to encode {Met-(-1)}Ser or {Met-(-1)}Tyr instead of the native pyroglutamate results in recombinant onconase derivatives with restored activities . {Met-(-1)}rOnc(E1S) was more active than {Met-(-1)}rOnc(E1Y) in all assays tested . Consistent with the action of native onconase, {Met-(-1)}rOnc(E1S) was a potent inhibitor of protein synthesis in the cell-free rabbit reticulocyte lysate assay, degrading tRNA at concentrations that correlated with inhibition of protein synthesis . An interesting difference between the recombinant onconase derivatives and the native protein was their susceptibility to inhibition by the major intracellular RNase inhibitor, PRI (onconase is refractory to PRI inhibition) . {Met-(-1)}rOnc(E1S) and {Met-(-1)}rOnc(E1Y) inhibited protein synthesis in intact SF539 neuroblastoma cells with IC50's very similar to that of onconase (IC50 3.5, 10, and 10 microg/mL after 1 day and 0.16, 0.35, and 2.5 microg/mL after 5 days for onconase, {Met-(-1)}rOnc(E1S), and {Met-(-1)}rOnc(E1Y), respectively) . Similar to that of onconase, cytotoxic activity of the recombinant derivatives was potentiated by monensin, NH4Cl, and retinoic acid . Brefeldin A completely blocked the enhancement of cytotoxicity caused by retinoic acid with all three proteins . Thus, drug-induced alterations of the intracellular trafficking of the recombinant derivatives also resembles that of onconase . Stability studies as assessed in serum-containing medium in the presence or absence of cells at 37 degreesC showed that the recombinant proteins were as stable to temperature and cell culture conditions as the native protein . Therefore, exchanging the Glu amino acid residue at the amino terminus of onconase with an amino acid residue containing a hydroxyl group produces recombinant proteins with ribonuclease and cytotoxic properties similar to native onconase.

Biochemistry, 1998 Apr 14, 37(15), 5154 - 61
RNase H1 can catalyze RNA/DNA hybrid formation and cleavage with stable hairpin or duplex DNA oligomers; Li J et al.; Cleavage of a RNA target site by RNase H1 from Escherichia coli was examined in the presence of complementary DNA sequences in the form of single-stranded, duplex, and hairpin structures . The target site was a 15 nt sequence in the middle of a 79 nt RNA transcript . DNA molecules employed included seven single-stranded oligodeoxynucleotides 10 or 15 nt long, and five hairpin DNAs each with a 10 bp stem and 5 nt loop . The loop and 3' side of the stem of two of the hairpin DNAs were fully complementary to the target site, while the other hairpin DNAs had sequence changes . A 10 bp duplex DNA with one strand complementary to the target site was also employed . A gel electrophoresis mobility shift assay examined hybrid formation between the RNA and the single-stranded 15 nt DNA and two hairpin DNAs that contained 15 complementary bases . RNA titration of the 32P-labeled single-stranded DNA produced a shifted band indicative of RNA/DNA complex formation . No RNA/DNA complex was detected when the more stable (Tm = 71 degrees C) hairpin DNA was combined with excess RNA . The less stable hairpin DNA (Tm = 62 degrees C) showed a small amount ( approximately 8%) of hybrid formation . Thermodynamic analysis of RNA binding to the DNAs was in qualitative agreement with the results . Although no RNA/DNA hybrid was expected from thermodynamic calculations, a RNase H assay at 25 degrees C showed that hairpin or duplex DNAs with a 10 nt complementary sequence catalyzed RNA degradation . A complementary loop sequence in the hairpin DNA was not required . Cleavage of the RNA did not occur with hairpin DNAs containing three or four noncomplementary bases in the stem . The results show that RNase H can promote the formation and cleavage of a RNA/DNA hybrid between an RNA site and a base paired strand of a stable hairpin or duplex DNA at temperatures below their Tm.

Biochemistry, 1998 Apr 14, 37(15), 5096 - 106
Biochemical characterization and crystallographic structure of an Escherichia coli protein from the phosphotriesterase gene family; Buchbinder JL et al.; Phosphotriesterase homology protein (PHP) is a member of a recently discovered family of proteins related to phosphotriesterase, a hydrolytic, bacterial enzyme with an unusual substrate specificity for synthetic organophosphate triesters and phosphorofluoridates, which are common constituents of chemical warfare agents and agricultural pesticides . No natural substrate has been identified for phosphotriesterase, and it has been suggested that the enzyme may have evolved the ability to hydrolyze synthetic compounds in bacteria under selective pressure to meet nutritional needs . PHP, which has 28% sequence identity with phosphotriesterase, may belong to the family of proteins from which phosphotriesterase evolved . Here we report the cloning, expression, initial characterization, and high-resolution X-ray crystallographic structure of PHP . Biochemical analysis shows that PHP is monomeric and binds two zinc ions per monomer . Unlike phosphotriesterase, PHP does not catalyze the hydrolysis of nonspecific phosphotriesters . The structure, similar to that of phosphotriesterase, consists of a long, elliptical alpha/beta barrel and has a binuclear zinc center in a cleft at the carboxy end of the barrel at the location of the presumptive active site.

Biochim Biophys Acta, 1998 Mar 13, 1370(2), 337 - 46
Molybdate binding by ModA, the periplasmic component of the Escherichia coli mod molybdate transport system; Imperial J et al.; ModA, the periplasmic-binding protein of the Escherichia coli mod transport system was overexpressed and purified . Binding of molybdate and tungstate to ModA was found to modify the UV absorption and fluorescence emission spectra of the protein . Titration of these changes showed that ModA binds molybdate and tungstate in a 1:1 molar ratio . ModA showed an intrinsic fluorescence emission spectrum attributable to its three tryptophanyl residues . Molybdate binding caused a conformational change in the protein characterized by: (i) a shift of tryptophanyl groups to a more hydrophobic environment; (ii) a quenching (at pH 5.0) or enhancement (at pH 7.8) of fluorescence; and (iii) a higher availability of tryptophanyl groups to the polar quencher acrylamide . The tight binding of molybdate did not allow an accurate estimation of the binding constants by these indirect methods . An isotopic binding method with 99MoO42- was used for accurate determination of KD (20 nM) and stoichiometry (1:1 molar ratio) . ModA bound tungstate with approximately the same affinity, but did not bind sulfate or phosphate . These KDs are 150- to 250-fold lower than those previously reported, and compatible with the high molybdate transport affinity of the mod system . The affinity of ModA for molybdate was also determined in vivo and found to be similar to that determined in vitro .

J Comput Biol, 1998 Spring, 5(1), 57 - 72
Statistical modelling and phylogenetic analysis of a deaminase domain; Mian IS et al.; Deamination reactions are catalyzed by a variety of enzymes including those involved in nucleoside/nucleotide metabolism and cytosine to uracil (C-->U) and adenosine to inosine (A-->I) mRNA editing . The active site of the deaminase (DM) domain in these enzymes contains a conserved histidine (or rarely cysteine), two cysteines and a glutamate proposed to act as a proton shuttle during deamination . Here, a statistical model, a hidden Markov model (HMM), of the DM domain has been created which identifies currently known DM domains and suggests new DM domains in viral, bacterial and eucaryotic proteins . However, no DM domains were identified in the currently predicted proteins from the archaeon Methanococcus jannaschii and possible causes for, and a potential means to ameliorate this situation are discussed . In some of the newly identified DM domains, the glutamate is changed to a residue that could not function as a proton shuttle and in one instance (Mus musculus spermatid protein TENR) the cysteines are also changed to lysine and serine . These may be non-competent DM domains able to bind but not act upon their substrate . Phylogenetic analysis using an HMM-generated alignment of DM domains reveals three branches with clear substructure in each branch . The results suggest DM domains that are candidates for yeast, platyhelminth, plant and mammalian C-->U and A-->I mRNA editing enzymes . Some bacterial and eucaryotic DM domains form distinct branches in the phylogenetic tree suggesting the existence of common, novel substrates.

Proc Natl Acad Sci U S A, 1998 Apr 14, 95(8), 4386 - 91
Bcl-XL interacts with Apaf-1 and inhibits Apaf-1-dependent caspase-9 activation; Hu Y et al.; Recent studies indicate that Caenorhabditis elegans CED-4 interacts with and promotes the activation of the death protease CED-3, and that this activation is inhibited by CED-9 . Here we show that a mammalian homolog of CED-4, Apaf-1, can associate with several death proteases, including caspase-4, caspase-8, caspase-9, and nematode CED-3 in mammalian cells . The interaction with caspase-9 was mediated by the N-terminal CED-4-like domain of Apaf-1 . Expression of Apaf-1 enhanced the killing activity of caspase-9 that required the CED-4-like domain of Apaf-1 . Furthermore, Apaf-1 promoted the processing and activation of caspase-9 in vivo . Bcl-XL, an antiapoptotic member of the Bcl-2 family, was shown to physically interact with Apaf-1 and caspase-9 in mammalian cells . The association of Apaf-1 with Bcl-XL was mediated through both its CED-4-like domain and the C-terminal domain containing WD-40 repeats . Expression of Bcl-XL inhibited the association of Apaf-1 with caspase-9 in mammalian cells . Significantly, recombinant Bcl-XL purified from Escherichia coli or insect cells inhibited Apaf-1-dependent processing of caspase-9 . Furthermore, Bcl-XL failed to inhibit caspase-9 processing mediated by a constitutively active Apaf-1 mutant, suggesting that Bcl-XL regulates caspase-9 through Apaf-1 . These experiments demonstrate that Bcl-XL associates with caspase-9 and Apaf-1, and show that Bcl-XL inhibits the maturation of caspase-9 mediated by Apaf-1, a process that is evolutionarily conserved from nematodes to humans.

Proc Natl Acad Sci U S A, 1998 Apr 14, 95(8), 4327 - 32
Simultaneous determination of helical unwinding angles and intrinsic association constants in ligand-DNA complexes: the interaction between DNA and calichearubicin B; Zeman SM et al.; We present a helical unwinding assay for reversibly binding DNA ligands that uses closed circular DNA, topoisomerase I (Topo I), and two-dimensional agarose gel electrophoresis . Serially diluted Topo I relaxation reactions at constant DNA/ligand ratio are performed, and the resulting apparent unwinding of the closed circular DNA is used to calculate both ligand unwinding angle (phi) and intrinsic association constant (Ka) . Mathematical treatment of apparent unwinding is formally analogous to that of apparent extinction coefficient data for optical binding titrations . Extrapolation to infinite DNA concentration yields the true unwinding angle of a given ligand and its association constant under Topo I relaxation conditions . Thus this assay delivers simultaneous structural and thermodynamic information describing the ligand-DNA complex . The utility of this assay has been demonstrated by using calichearubicin B (CRB), a synthetic hybrid molecule containing the anthraquinone chromophore of (DA) and the carbohydrate domain of calicheamicin gamma1I . The unwinding angle for CRB calculated by this method is -5 . 3 +/- 0.5 degrees . Its Ka value is 0.20 x 10(6) M-1 . For comparison, the unwinding angles of ethidium bromide and DA have been independently calculated, and the results are in agreement with canonical values for these compounds . Although a stronger binder to selected sites, CRB is a less potent unwinder than its parent compound DA . The assay requires only small amounts of ligand and offers an attractive option for analysis of DNA binding by synthetic and natural compounds.

Proc Natl Acad Sci U S A, 1998 Apr 14, 95(8), 4293 - 8
Chromophore-assisted light inactivation and self-organization of microtubules and motors; Surrey T et al.; Chromophore-assisted light inactivation (CALI) offers the only method capable of modulating specific protein activities in localized regions and at particular times . Here, we generalize CALI so that it can be applied to a wider range of tasks . Specifically, we show that CALI can work with a genetically inserted epitope tag; we investigate the effectiveness of alternative dyes, especially fluorescein, comparing them with the standard CALI dye, malachite green; and we study the relative efficiencies of pulsed and continuous-wave illumination . We then use fluorescein-labeled hemagglutinin antibody fragments, together with relatively low-power continuous-wave illumination to examine the effectiveness of CALI targeted to kinesin . We show that CALI can destroy kinesin activity in at least two ways: it can either result in the apparent loss of motor activity, or it can cause irreversible attachment of the kinesin enzyme to its microtubule substrate . Finally, we apply this implementation of CALI to an in vitro system of motor proteins and microtubules that is capable of self-organized aster formation . In this system, CALI can effectively perturb local structure formation by blocking or reducing the degree of aster formation in chosen regions of the sample, without influencing structure formation elsewhere.

Proc Natl Acad Sci U S A, 1998 Apr 14, 95(8), 4215 - 8
A single-headed dimer of Escherichia coli ribosomal protein L7/L12 supports protein synthesis; Oleinikov AV et al.; During protein synthesis, the two elongation factors Tu and G alternately bind to the 50S ribosomal subunit at a site of which the protein L7/L12 is an essential component . L7/L12 is present in each 50S subunit in four copies organized as two dimers . Each dimer consists of distinct domains: a single N-terminal ("tail") domain that is responsible for both dimerization and binding to the ribosome via interaction with the protein L10 and two independent globular C-terminal domains ("heads") that are required for binding of elongation factors to ribosomes . The two heads are connected by flexible hinge sequences to the N-terminal domain . Important questions concerning the mechanism by which L7/L12 interacts with elongation factors are posed by us in response to the presence of two dimers, two heads per dimer, and their dynamic, mobile properties . In an attempt to answer these questions, we constructed a single-headed dimer of L7/L12 by using recombinant DNA techniques and chemical cross-linking . This chimeric molecule was added to inactive core particles lacking wild-type L7/L12 and shown to restore activity to a level approaching that of wild-type two-headed L7/L12.

Proc Natl Acad Sci U S A, 1998 Apr 14, 95(8), 4204 - 8
Brefeldin A-inhibited guanine nucleotide-exchange activity of Sec7 domain from yeast Sec7 with yeast and mammalian ADP ribosylation factors; Sata M et al.; The Saccharomyces cerevisiae Sec7 protein (ySec7p), which is an important component of the yeast secretory pathway, contains a sequence of approximately 200 amino acids referred to as a Sec7 domain . Similar Sec7 domain sequences have been recognized in several guanine nucleotide-exchange proteins (GEPs) for ADP ribosylation factors (ARFs) . ARFs are approximately 20-kDa GTPases that regulate intracellular vesicular membrane trafficking and activate phospholipase D . GEPs activate ARFs by catalyzing the replacement of bound GDP with GTP . We, therefore, undertook to determine whether a Sec7 domain itself could catalyze nucleotide exchange on ARF and found that it exhibited brefeldin A (BFA)-inhibitable ARF GEP activity . BFA is known to inhibit ARF GEP activity in Golgi membranes, thereby causing reversible apparent dissolution of the Golgi complex in many cells . The His6-tagged Sec7 domain from ySec7p (rySec7d) synthesized in Escherichia coli enhanced binding of guanosine 5'-{gamma-{35S}thio}triphosphate by recombinant yeast ARF1 (ryARF1) and ryARF2 but not by ryARF3 . The effects of rySec7d on ryARF2 were inhibited by BFA in a concentration-dependent manner but not by inactive analogues of BFA (B-17, B-27, and B-36) . rySec7d also promoted BFA-sensitive guanosine 5'-{gamma-thio}triphosphate binding by nonmyristoylated recombinant human ARF1 (rhARF1), rhARF5, and rhARF6, although the effect on rhARF6 was very small . These results are consistent with the conclusion that the yeast Sec7 domain itself contains the elements necessary for ARF GEP activity and its inhibition by BFA.

Biochemistry, 1998 Apr 7, 37(14), 4910 - 5
Proximity of helices VIII (Ala273) and IX (Met299) in the lactose permease of Escherichia coli; Wang Q et al.; Three double-Cys mutant pairs--Ala273-->Cys/Met299-->Cys, Thr266-->Cys/Ile303-->Cys, and Thr266-->Cys/Ser306-->Cys--were constructed in a functional lac permease construct devoid of Cys residues, and the excimer fluorescence or electron paramagnetic resonance (EPR) was studied with pyrene- or spin-labeled derivatives, respectively . After reconstitution into proteoliposomes, excimer fluorescence is observed with mutant Ala273-->Cys/Met299-->Cys, but not with the single-Cys mutants nor with mutants Thr266-->Cys/Ile303-->Cys or Thr266-->Cys/Ser306-->Cys . Furthermore, spin-spin interaction is also observed with mutant Ala273-->Cys/Met299-->Cys, but only after the permease is reconstituted into proteoliposomes . The results provide independent support for the conclusions that helix VIII is close to helix IX and that the transmembrane helices of the permease are more loosely packed in a detergent micelle as opposed to a phospholipid bilayer.

Biochemistry, 1998 Apr 7, 37(14), 4875 - 83
Cation-promoted association of Escherichia coli phosphocarrier protein IIAGlc with regulatory target protein glycerol kinase: substitutions of a Zinc(II) ligand and implications for inducer exclusion; Pettigrew DW et al.; In Escherichia coli, inducer exclusion is one mechanism by which glucose prevents unnecessary expression of genes needed for metabolism of other sugars . The basis for this mechanism is binding of the unphosphorylated form of the glucose-specific phosphocarrier protein of the phosphoenolpyruvate:glycose phosphotransferase system, IIAGlc (also known as IIIGlc), to a variety of target proteins to prevent uptake or synthesis of the inducer . One of these target proteins is glycerol kinase (EC 2.1.7.30, ATP:glycerol 3-phosphotransferase), which is inhibited by IIAGlc . Glycerol kinase is the only IIAGlc target protein for which the structure of the complex is known . Association of these two proteins forms an intermolecular binding site for Zn(II) with metal ligands contributed by each protein, and Zn(II) enhances IIAGlc inhibition {Feese, M., Pettigrew, D . W., Meadow, N . D., Roseman, S., and Remington, S . J . (1994) Proc . Natl . Acad . Sci . U.S.A . 91, 3544-3548} . Here, we show that the Zn(II) enhancement can be described quantitatively by a model with binding of Zn(II) to the complex with an apparent dissociation constant of less than 1 microM at pH 7.0 and 25 degreesC . Initial velocity studies show that IIAGlc is an uncompetitive inhibitor with respect to both substrates, and the mechanism of inhibition is not altered by Zn(II) . The Zn(II)-liganding residue contributed by glycerol kinase (Glu478) is substituted by using site-directed mutagenesis to construct the enzymes E478C, E478D, E478H, and E478Q . The substitutions have only small effects on the inhibition by IIAGlc in the absence of Zn(II), the catalytic properties, or other allosteric regulation . However, all of the substitutions abolish the Zn(II) enhancement of IIAGlc inhibition, and the X-ray crystallographic structures of the complexes of IIAGlc with the E478C and E478H mutants show these substitutions abolish binding of Zn(II) to the intermolecular site . These results support the hypothesis that Zn(II) enhances the affinity for complex formation by binding at the intermolecular site, i.e., cation promoted association . The high affinity for Zn(II) binding to the complex and the ability of the other four amino acid residues to efficiently substitute for Glu478 in all functions except binding of Zn(II) suggest that cation promoted association of these two proteins may have a role in inducer exclusion in vivo.

Biochemistry, 1998 Apr 7, 37(14), 4815 - 22
Sst2 is a GTPase-activating protein for Gpa1: purification and characterization of a cognate RGS-Galpha protein pair in yeast; Apanovitch DM et al.; Genetic studies in the yeast Saccharomyces cerevisiae have shown that SST2 promotes pheromone desensitization in vivo . Sst2 is the founding member of the RGS (regulators of G protein signaling) family of proteins, which in mammals act as GAPs (GTPase activating proteins) for several subfamilies of Galpha proteins in vitro . A similar activity for Sst2 has not been demonstrated, and it is not self-evident from sequence homology arguments alone . Here we describe the purification of Sst2 and its cognate Galpha protein (Gpa1) in yeast, and demonstrate Sst2-stimulated Gpa1 GTPase activity . His-tagged versions of Sst2 and Gpa1 were expressed in E . coli, and purified using Ni2+-agarose and ion exchange chromatography . Time-course binding experiments reveal that Sst2 does not affect the binding or release of guanine nucleotides . Similarly, steady-state GTPase assays reveal that Sst2 does not alter the overall rate of hydrolysis, including the rate-limiting nucleotide exchange step . Single-turnover GTPase assays reveal, however, that Sst2 is a potent stimulator of GTP hydrolysis . Sst2 also exhibits GAP activity for mammalian Goalpha, and the mammalian RGS protein GAIP exhibits GAP activity for Gpa1 . Finally, we show that Sst2 binds with highest affinity to the transition state of Gpa1 (GDP-AlF4--bound), and with much lower affinity to the inactive (GDP-bound) and active (GTPgammaS-bound) conformations . These experiments represent the first biochemical characterization of Gpa1 and Sst2, and provide a molecular basis for their well-established biological roles in signaling and desensitization.

J Biol Chem, 1998 Apr 10, 273(15), 9323 - 9
The mitogen-activated protein kinase phosphatase-3 N-terminal noncatalytic region is responsible for tight substrate binding and enzymatic specificity; Muda M et al.; We have reported recently that the dual specificity mitogen-activated protein kinase phosphatase-3 (MKP-3) elicits highly selective inactivation of the extracellular signal-regulated kinase (ERK) class of mitogen-activated protein (MAP) kinases (Muda, M., Theodosiou, A., Rodrigues, N., Boschert, U., Camps, M., Gillieron, C., Davies, K., Ashworth, A., and Arkinstall, S . (1996) J . Biol . Chem . 271, 27205-27208) . We now show that MKP-3 enzymatic specificity is paralleled by tight binding to both ERK1 and ERK2 while, in contrast, little or no interaction with either c-Jun N-terminal kinase/stress activated protein kinase (JNK/SAPK) or p38 MAP kinases was detected . Further study revealed that the N-terminal noncatalytic domain of MKP-3 (MKP-3DeltaC) binds both ERK1 and ERK2, while the C-terminal MKP-3 catalytic core (MKP-3DeltaN) fails to precipitate either of these MAP kinases . A chimera consisting of the N-terminal half of MKP-3 with the C-terminal catalytic core of M3-6 also bound tightly to ERK1 but not to JNK3/SAPKbeta . Consistent with a role for N-terminal binding in determining MKP-3 specificity, at least 10-fold higher concentrations of purified MKP-3DeltaN than full-length MKP-3 is required to inhibit ERK2 activity . In contrast, both MKP-3DeltaN and full-length MKP-3 inactivate JNK/SAPK and p38 MAP kinases at similarly high concentrations . Also, a chimera of the M3-6 N terminus with the MKP-3 catalytic core which fails to bind ERK elicits non selective inactivation of ERK1 and JNK3/SAPKbeta . Together, these observations suggest that the physiological specificity of MKP-3 for inactivation of ERK family MAP kinases reflects tight substrate binding by its N-terminal domain.

J Biol Chem, 1998 Apr 10, 273(15), 9312 - 22
Refolding of bacteriorhodopsin from expressed polypeptide fragments; Marti T; Bacteriorhodopsin is a heptahelical membrane protein that can be refolded to the native state following denaturation . To analyze the in vitro folding process with independent structural domains, eight fragments comprising two (AB, FG), three (AC, EG), four (AD, DG) or five (AE, CG) of the transmembrane segments were produced by expression in Escherichia coli . The polypeptides were purified to homogeneity by solvent extraction of E . coli membranes, repeated phase separation, and anion-exchange chromatography employing the C-terminal tail of bacteriorhodopsin for adsorption . Upon reconstitution into phospholipid/detergent micelles pairs of complementary fragments (AB.CG, AC.DG, AD.EG, and AE.FG) assembled in the presence of retinal to regenerate the characteristic bacteriorhodopsin chromophore with high efficiency . Together with previous studies, these results demonstrate that the covalent connections in each of the six interhelical loops are dispensable for a correct association of the helices . The different loops, however, contribute to the stability of the folded structure, as shown by increased susceptibilities toward denaturation in SDS and at acidic pH, and decreased Schiff base pKa values for the AB.CG, AC . DG, AD.EG, and AE.FG complexes, compared with the intact protein . Notably, the heptahelical bundle structure was also generated by all possible combinations of pairs of overlapping fragments, containing one (AC.CG, AD.DG, AE.EG), two (AD.CG, AE.DG), or three (AE.CG) redundant helices . The spectral properties of the chromophores indicate that the retinal-binding pocket of the AC.CG, AD.CG, and AE . CG complexes is formed by helices A and B of the respective N-terminal fragment and the C-terminal CG fragment, whereas the AD . DG, AE.DG, and AE.EG complexes are likely to adopt a heptahelical bundle structure analogous to AD.EG . The combined data show that the specificity of the helix assembly of bacteriorhodopsin is influenced by connectivities provided by the C-D and E-F surface loops.

J Biol Chem, 1998 Apr 10, 273(15), 9279 - 84
A keratinocyte-specific epoxygenase, CYP2B12, metabolizes arachidonic acid with unusual selectivity, producing a single major epoxyeicosatrienoic acid; Keeney DS et al.; The CYP monooxygenase, CYP2B12, is the first identified skin-specific cytochrome P450 enzyme . It is characterized by high, constitutive expression in an extrahepatic tissue, the sebaceous glands of cutaneous tissues . It is expressed exclusively in a subset of differentiated keratinocytes called sebocytes, as demonstrated by Northern blot analysis, in situ hybridization, and polymerase chain reaction . The onset of its expression coincides with the morphological appearance of sebaceous glands in the neonatal rat . Recombinant CYP2B12 produced in Escherichia coli epoxidizes arachidonic acid to 11,12- and 8,9-epoxyeicosatrienoic acids (80 and 20% of total metabolites, respectively) . The identification of arachidonic acid as a substrate for this skin-specific CYP monooxygenase suggests an endogenous function in keratinocytes in the generation of bioactive lipids and intracellular signaling.

J Biol Chem, 1998 Apr 10, 273(15), 9243 - 8
Nucleotide binding to the C-terminal nucleotide binding domain of ArsA . Studies with an ATP analogue, 5'-p-fluorosulfonylbenzoyladenosine; Ramaswamy S et al.; ArsA protein, the catalytic component of the plasmid-encoded anion-translocating ATPase in Escherichia coli, contains two consensus nucleotide binding domains, A1 and A2, that are connected by a flexible linker . ATP has previously been shown to cross-link to the A1 domain upon activation with UV light but not to the A2 domain . The ATP analogue, 5'-p-fluorosulfonylbenzoyladenosine (FSBA) was used to probe the nucleotide binding domains of ArsA . The covalently labeled protein was subjected to partial trypsin proteolysis, followed by Western blot analysis of the fragments with the anti-FSBA serum . The N-terminal amino acid sequence of the labeled fragment showed that FSBA binds preferentially to the C-terminal domain A2 both in the absence and the presence of antimonite . Occupancy of the two nucleotide binding sites was determined by protection from trypsin proteolysis . Trypsin cleaved the ArsA protein at Arg290 in the linker to generate a 32-kDa N-terminal and a 27-kDa C-terminal fragment . The 32-kDa fragment is compact and largely inaccessible to trypsin; however, the 27-kDa was cleaved further . Incubation with FSBA, which binds to the C-terminal domain, resulted in significant protection of the 27-kDa fragment . This fragment was not protected upon incubation with ATP alone, indicating that A2 might be unoccupied . However, upon incubation with ATP and antimonite, almost complete protection from trypsin was seen . ATP and FSBA together mimicked the effect of ATP and antimonite, implying that this fully protected conformation might be the result of both sites occupied with the nucleotide . It is proposed that the A1 site in ArsA is a high affinity ATP site, whereas the allosteric ligand antimonite is required to allow ATP binding to A2, resulting in catalytic cooperativity . Thus antimonite binding may act as a switch in regulating ATP binding to A2 and hence the ATPase activity of ArsA.

J Biol Chem, 1998 Apr 10, 273(15), 9202 - 7
Mismatch-, MutS-, MutL-, and helicase II-dependent unwinding from the single-strand break of an incised heteroduplex; Dao V et al.; Escherichia coli MutS, MutL, and DNA helicase II are sufficient to initiate mismatch-dependent unwinding of an incised heteroduplex (Yamaguchi, M., Dao, V., and Modrich, P . (1998) J . Biol . Chem., 273, 9197-9201) . We have studied unwinding of 6.4-kilobase circular G-T heteroduplexes that contain a single-strand incision, 808 base pairs 5' to the mismatch or 1023 base pairs 3' to the mispair as viewed along the shorter path between the two DNA sites . Unwinding of both substrates in the presence of MutS, MutL, DNA helicase II, and single-stranded DNA binding protein was mismatch-dependent and initiated at the single-strand break . Although unwinding occurred in both directions from the strand break, it was biased toward the shorter path linking the strand break and the mispair . MutS and MutL are thus sufficient to coordinate mismatch recognition to the orientation-dependent activation of helicase II unwinding at a single-strand break located a kilobase from the mispair.

J Biol Chem, 1998 Apr 10, 273(15), 9197 - 201
MutS and MutL activate DNA helicase II in a mismatch-dependent manner; Yamaguchi M et al.; MutS, MutL, and DNA helicase II are required for the mismatch-provoked excision step that occurs during Escherichia coli methyl-directed mismatch repair . In this study MutL is shown to enhance the unwinding activity of DNA helicase II more than 10-fold on a conventional helicase substrate in which a 35-residue oligonucleotide is annealed to a M13 circular single-stranded phage DNA under conditions where the two proteins are present at approximately molar stoichiometry with respect to the substrate . MutS- and MutL-dependent activation of DNA helicase II has also been demonstrated with a model substrate in which a 138-residue oligonucleotide was hybridized to a 138-nucleotide gap in an otherwise duplex 7,100-base pair circular DNA . Displacement of the oligonucleotide requires MutS, MutL, DNA helicase II, and ATP and is dependent on the presence of a mismatch within the hybrid region . Although DNA helicase II and Rep helicase share substantial sequence homology and features of mechanism, Rep helicase is inactive in this reaction.

J Biol Chem, 1998 Apr 10, 273(15), 9058 - 69
Functional and structural heterogeneity of the DNA binding site of the Escherichia coli primary replicative helicase DnaB protein; Jezewska MJ et al.; The structure-function relationship within the DNA binding site of the Escherichia coli replicative helicase DnaB protein was studied using nuclease digestion, quantitative fluorescence titration, centrifugation, and fluorescence energy transfer techniques . Nuclease digestion of the enzyme-single-stranded DNA (ssDNA) complexes reveals large structural heterogeneity within the binding site . The total site is built of two subsites differing in structure and affinity, although both occlude approximately 10 nucleotides . ssDNA affinity for the strong subsite is approximately 3 orders of magnitude higher than that for the weak subsite . Fluorescence energy transfer experiments provide direct proof that the DnaB hexamer binds ssDNA in a single orientation, with respect to the polarity of the sugar-phosphate backbone . This is the first evidence of directional binding to ssDNA of a hexameric helicase in solution . The strong binding subsite is close to the small 12-kDa domains of the DnaB hexamer and occludes the 5'-end of the ssDNA . The strict orientation of the helicase on ssDNA indicates that, when the enzyme approaches the replication fork, it faces double-stranded DNA with its weak subsite . The data indicate that the different binding subsites are located sequentially, with the weak binding subsite constituting the entry site for double-stranded DNA of the replication fork.

J Biol Chem, 1998 Apr 10, 273(15), 8958 - 64
Kinetic and thermodynamic studies of purine repressor binding to corepressor and operator DNA; Xu H et al.; The kinetic and thermodynamic parameters for purine repressor (PurR)-operator and PurR-guanine binding were determined using fluorescence spectroscopy and nitrocellulose filter binding . Operator binding affinity was increased by the presence of guanine as demonstrated previously (Choi, K . Y., Lu, F., and Zalkin, H . (1994) J . Biol . Chem . 269, 24066-24072; Rolfes, R . J., and Zalkin, H . (1990) J . Bacteriol . 172, 5637-5642), and conversely guanine binding affinity was increased by the presence of operator . Guanine enhanced operator affinity by increasing the association rate constant and decreasing the dissociation rate constant for binding . Operator had minimal effect on the association rate constant for guanine binding; however, this DNA decreased the dissociation rate constant for corepressor by approximately 10-fold . Despite significant sequence and structural similarity between PurR and LacI proteins, PurR binds to its corepressor ligand with a lower association rate constant than LacI binds to its inducer ligand . However, the rate constant for PurR-guanine binding to operator is approximately 3-fold higher than for LacI binding to its cognate operator under the same solution conditions . The distinct metabolic roles of the enzymes under regulation by these two repressor proteins provide a rationale for the observed functional differences.

J Biol Chem, 1998 Apr 10, 273(15), 8897 - 902
Degradation versus aggregation of misfolded maltose-binding protein in the periplasm of Escherichia coli; Betton JM et al.; The periplasmic fates of misfolded MalE31, a defective folding mutant of the maltose-binding protein, were determined by manipulating two cellular activities affecting the protein folding pathway in host cells: (i) the malEp promoter activity, which is controlled by the transcriptional activator MalT, and (ii) the DegP and Protease III periplasmic proteolytic activity . At a low level of expression, the degradation of misfolded MalE31 was partially impaired in cells lacking DegP or Protease III . At a high level of expression, misfolded MalE31 rapidly formed periplasmic inclusion bodies and thus escaped degradation . However, the manipulated host cell activities did not enhance the production of periplasmic, soluble MalE31 . A kinetic competition between folding, aggregation, and degradation is proposed as a general model for the biogenesis of periplasmic proteins.

J Biol Chem, 1998 Apr 10, 273(15), 8783 - 9
Site-directed mutagenesis of conserved aspartates, glutamates and arginines in the active site region of Escherichia coli DNA topoisomerase I; Zhu CX et al.; To catalyze relaxation of supercoiled DNA, DNA topoisomerases form a covalent enzyme-DNA intermediate via nucleophilic attack of a tyrosine hydroxyl group on the DNA phosphodiester backbone bond during the step of DNA cleavage . Strand passage then takes place to change the linking number . This is followed by DNA religation during which the displaced DNA hydroxyl group attacks the phosphotyrosine linkage to reform the DNA phosphodiester bond . Mg(II) is required for the relaxation activity of type IA and type II DNA topoisomerases . A number of conserved amino acids with acidic and basic side chains are present near Tyr-319 in the active site of the crystal structure of the 67-kDa N-terminal fragment of Escherichia coli DNA topoisomerase I . Their roles in enzyme catalysis were investigated by site-directed mutation to alanine . Mutation of Arg-136 abolished all the enzyme relaxation activity even though DNA cleavage activity was retained . The Glu-9, Asp-111, Asp-113, Glu-115, and Arg-321 mutants had partial loss of relaxation activity in vitro . All the mutants failed to complement chromosomal topA mutation in E . coli AS17 at 42 degreesC, possibly accounting for the conservation of these residues in evolution.

J Biol Chem, 1998 Apr 10, 273(15), 8646 - 51
Characterization of a b2delta complex from Escherichia coli ATP synthase; Dunn SD et al.; The delta subunit of Escherichia coli ATP synthase has been expressed and purified, both as the intact polypeptide and as delta', a proteolytic fragment composed of residues 1-134 . The solution structure of delta' as a five-helix bundle has been previously reported (Wilkens, S., Dunn, S . D., Chandler, J., Dahlquist, F . W., and Capaldi, R . A . (1997) Nat . Struct . Biol . 4, 198-201) . The delta subunit, in conjunction with delta-depleted F1-ATPase, was fully capable of reconstituting energy-dependent fluorescence quenching in membrane vesicles that had been depleted of F1 . A complex of delta with the cytoplasmic domain of the b subunit of F0 was demonstrated and characterized by analytical ultracentrifugation using bST34-156, a form of the b domain lacking aromatic residues . Molecular weight determination by sedimentation equilibrium supported a b2delta subunit stoichiometry . The sedimentation coefficient of the complex, 2.1 S, indicated a frictional ratio of approximately 2, suggesting that delta and the b dimer are arranged in an end-to-end rather than side-by-side manner . These results indicate the feasibility of the b2delta complex reaching from the membrane to the membrane-distal portion of the F1 sector, as required if it is to serve as a second stalk.

Protein Expr Purif, 1998 Apr, 12(3), 404 - 14
Recombinant fusion proteins for the industrial production of disulfide bridge containing peptides: purification, oxidation without concatamer formation, and selective cleavage; Dobeli H et al.; We report the biotechnical production of peptides of approximately 35-50 amino acids in length containing one intramolecular disulfide bridge, using a recombinant fusion tail approach . This method fills the technological gap when either (a) chemical synthesis fails due to known problematic peptide sequences or (b) if simple recombinant expression is unsuccessful due to degradation . The fusion tail described here serves several purposes: (i) it enables high expression levels in Escherichia coli to be achieved; (ii) it renders the fusion protein fairly soluble; (iii) it contains a histidine affinity tag for easy purification on Ni-chelate resins, which also serves as a catalyst for the oxygen-dependent formation of the disulfide bridge; and (iv) it suppresses the formation of concatamers during the oxidation process through steric hindrance . The purified fusion protein is then immobilized on a reversed phase column for two purposes: (i) chemical cleavage of the fusion tail by cyanogen bromide and (ii) subsequent purification of the peptide . A very hydrophilic fusion partner is required so that immobilization on the reversed phase column always occurs due to the peptide . Sensitive hydrophobic residues are thereby protected from the cleavage reagent while the cleaved hydrophilic fusion tail is easily separated from the hydrophobic peptide . The method is exemplified by eight peptides representing an immunodominant epitope of the human immunodeficiency virus, but may be useful for a significant variety of similar peptides .

Protein Expr Purif, 1998 Apr, 12(3), 381 - 9
Recombinant expression, purification, and characterization of three isoenzymes of aspartate aminotransferase from Arabidopsis thaliana; Wilkie SE et al.; Five different genes encoding isoenzymes of aspartate aminotransferase (AAT) have been identified in the plant Arabidopsis thaliana . cDNA sequences encoding three of these AAT isoenzymes, asp1 (mitochondrial), asp2 (cytosolic), and asp5 (plastid), were manipulated into bacterial expression vectors and the recombinant proteins expressed were purified from liquid culture using conventional methods . Yields of the purified isoenzymes varied from 11.5 mg/g wet wt cells (AAT5) to 0.95 mg/g wet wt cells (AAT2), an improvement of more than 1000-fold over typical yields of native isoenzymes obtained from plant tissues of other species . Analysis of the recombinant proteins on denaturing PAGE gels indicated subunit Mrs of between 44 and 45 K . Kinetic parameters (Km and kcat) obtained for all four substrates (aspartate, alpha-ketoglutarate, glutamate, and oxaloacetate) were consistent with values obtained for native AAT isoenzymes from other plant species . Further characterization of the purified recombinant enzymes alongside native enzymes from A . thaliana leaf tissue on AAT activity gels confirmed the identity of asp1 and asp2 as the mitochondrial and cytosolic AAT genes but indicated that asp5 may encode an amyloplastic rather than the chloroplastic enzyme .

Protein Expr Purif, 1998 Apr, 12(3), 305 - 14
Renaturation of 1-aminocyclopropane-1-carboxylate synthase expressed in Escherichia coli in the form of inclusion bodies into a dimeric and catalytically active enzyme; Huxtable S et al.; 1-Aminocyclopropane-1-carboxylate (ACC) synthase is a key enzyme regulating the biosynthesis of the plant hormone ethylene . A wound-inducible zucchini ACC synthase cDNA was isolated by reverse-transcription polymerase chain reaction (RT-PCR) and expressed in a heterologous Escherichia coli BL21(DE3)pLysS:pET30a protein expression system . A method was developed and optimized for the renaturation of the ACC synthase expressed in the form of inclusion bodies . The optimum conditions were found to be unfolding in a buffer containing 100 mM Mops, pH 9.5, 6 M urea, and 50 mM DTT, for 3 h at 4 degrees C and refolding by a combined process of dialysis and dilution in 100 mM Mops, pH 8, 30 mM Chaps, and 5 mM GSH at a protein concentration of 45 microg/ml . The purified enzyme has a specific activity of 90,000 U mg-1 and exhibits an apparent homogeneity on SDS-PAGE fractionation . Biochemical characterization of the refolded enzyme revealed a high degree of similarity to the enzyme purified from the soluble source . The refolded enzyme was found to be a dimer with a native size of 110 kDa, a Km of 23 microM, and a Vmax of 112,000 U mg-1 .

Blood, 1998 Apr 15, 91(8), 2722 - 30
A placebo-controlled study of recombinant human granulocyte-macrophage colony-stimulating factor administered during and after induction treatment for de novo acute myelogenous leukemia in elderly patients . Groupe Ouest Est Leucémies Aiguës Myéloblastiques (GOELAM); Witz F et al.; The complete remission (CR) rate after intensive chemotherapy for acute myelogenous leukemia (AML) remains low in elderly patients, mainly because of a higher infectious mortality rate related to neutropenia and an increased incidence of adverse prognostic factors . Granulocyte-macrophage colony-stimulating factor (GM-CSF) has been shown to potentially recruit leukemic blasts into cell cycle and improve cytotoxic effects when given during chemotherapy, and to shorten the duration of neutropenia when administered after chemotherapy . Two hundred forty patients aged 55 to 75 years who had newly diagnosed AML were randomly assigned to receive placebo or Escherichia coli-derived GM-CSF (5 micrograms/kg/d by 6-hour intravenous infusion) starting during induction chemotherapy on day 1 and continued through and after chemotherapy until recovery of neutrophils, or evidence of regrowth of leukemia, or up to day 28 . Induction chemotherapy consisted of idarubicin (8 mg/m2/d on days 1 to 5) and cytarabine (100 mg/m2/d on days 1 to 7) . The study drug was not administered subsequent to the induction course . Patients who achieved a CR received continuous maintenance therapy for 1 year with four quarterly reinduction courses; in the 55- to 64-year age subgroup, patients were randomly assigned to receive or not a consolidation course before maintenance therapy . The CR rate was similar in the GM-CSF and placebo groups (63% and 60.5%, respectively; P = .79) . The mortality, rate of resistant disease, and rate of regrowth of leukemia were also similar in both groups . The time to neutrophil recovery was shorter in patients who received GM-CSF (24 v 29 days; P = .0001), but the incidence and characteristics of infectious events were not different . The 2-year disease-free survival (DFS) rate was significantly improved in the GM-CSF group (48% v 21% in the placebo group; P = .003) . This effect was highly significant in the cohort of patients aged 55 to 64, but only marginal in patients >/=65 years of age . There was a trend toward a longer overall survival (OS) in the GM-CSF group (P = .082) . In summary, the administration of GM-CSF, concomitantly with chemotherapy and thereafter during induction course in AML, shortened the time to neutrophil recovery, but did not improve the CR rate in patients aged 55 to 75 . Nonetheless, DFS and OS were significantly prolonged in patients aged 55 to 64 treated with GM-CSF . These results are promising and further evaluation of myeloid growth factors in AML is warranted.

J Exp Med, 1998 Apr 6, 187(7), 1123 - 32
Mucosal adjuvanticity and immunogenicity of LTR72, a novel mutant of Escherichia coli heat-labile enterotoxin with partial knockout of ADP-ribosyltransferase activity; Giuliani MM et al.; Heat-labile Escherichia coli enterotoxin (LT) has the innate property of being a strong mucosal immunogen and adjuvant . In the attempt to reduce toxicity and maintain the useful immunological properties, several LT mutants have been produced . Some of these are promising mucosal adjuvants . However, so far, only those that were still toxic maintained full adjuvanticity . In this paper we describe a novel LT mutant with greatly reduced toxicity that maintains most of the adjuvanticity . The new mutant (LTR72), that contains a substitution Ala --> Arg in position 72 of the A subunit, showed only 0.6% of the LT enzymatic activity, was 100,000-fold less toxic than wild-type LT in Y1 cells in vitro, and was at least 20 times less effective than wild-type LT in the rabbit ileal loop assay in vivo . At a dose of 1 microg, LTR72 exhibited a mucosal adjuvanticity, similar to that observed with wild-type LT, better than that induced by the nontoxic, enzymatically inactive LTK63 mutant, and much greater than that of the recombinant B subunit . This trend was consistent for both the amounts and kinetics of the antibody induced, and priming of antigen-specific T lymphocytes . The data suggest that the innate high adjuvanticity of LT derives from the independent contribution of the nontoxic AB complex and the enzymatic activity . LTR72 optimizes the use of both properties: the enzymatic activity for which traces are enough, and the nontoxic AB complex, the effect of which is dose dependent . In fact, in dose-response experiments in mice, 20 microg of LTR72 were a stronger mucosal adjuvant than wild-type LT . This suggests that LTR72 may be an excellent candidate to be tested in clinical trials.

Gene, 1998 Mar 16, 209(1-2), 219 - 28
Cloning and heterologous expression of Entamoeba histolytica adenylate kinase and uridylate/cytidylate kinase; Sanchez LB et al.; We have isolated two cDNA clones encoding Entamoeba histolytica nucleotide kinases, EhAK and EhUK, expressed them in E . coli and performed functional studies of the recombinant enzymes . Nucleotide sequence analysis showed that EhAK and EhUK genes exhibited the features characteristic of E . histolytica genes, such as transcripts with relatively short 5' and 3' untranslated flanking regions containing the conserved E . histolytica transcription promoter elements located 5' to the initiation codon and a polyadenylation signal in the 3' UTR, a distinctive codon usage bias for A or T in the third position and an AT bias greater than 75% in the flanking regions of the transcripts . At the protein level, both enzymes belong to the short variant nucleoside monophosphate (NMP) kinases, which lack a 29amino acid LID region present in the long variant isoenzymes . EhAK was 30-38% identical to the members of the adenylate kinase (AK) family while EhUK was more similar (48-49% identity) to UMP/CMP kinases . Both enzymes used ATP as preferred phosphate-group donor but each one exhibited strict specificity for the acceptor NMP, EhAK for AMP and EhUK for the pyrimidine nucleoside monophosphates UMP and CMP . Biochemical characterization of the enzymes and phylogenetic reconstruction showed that EhUK is an authentic and well conserved member of the UMP/CMP kinase group while EhAK is the most divergent member known of the AK1 isoenzymes.

Gene, 1998 Mar 16, 209(1-2), 185 - 92
Cloning and analysis of the shiA gene, which encodes the shikimate transport system of escherichia coli K-12; Whipp MJ et al.; In Escherichia coli K-12, the shiA gene is involved in the uptake of shikimate . This gene has been cloned and its nucleotide sequence determined . The gene is predicted to encode a protein of 438 amino acids and lies adjacent to the amn gene . The hydropathy profile and the amino acid sequence indicate that the ShiA protein is a polytopic membrane protein that shows a homology with members of the major facilitator superfamily of transport proteins . Recombining an inactive form of the cloned gene into the chromosome creates mutants unable to transport shikimate . Introducing a wild-type gene on a multicopy plasmid into a shiA mutant restores the ability to transport shikimate . When this multicopy shiA plasmid is introduced into an aroE strain, this strain is now able to grow with shikimate as the aromatic supplement, consistent with the notion that dehydroshikimate (DHS) accumulated in an aroE strain prevents uptake of shikimate by competition . Expression of the shiA gene does not appear to be regulated by the TyrR protein, a repressor/activator that controls the expression of other genes involved with the biosynthesis or transport of the aromatic amino acids.

Gene, 1998 Mar 16, 209(1-2), 131 - 8
The largest subunit of mouse RNA polymerase II (RPB1) functionally substituted for its yeast counterpart in vivo; Singleton TL et al.; The full-length mouse RNA polymerase II (pol II) largest subunit (RPB1) gene was used to replace 5070 bp of the yeast Saccharomyces cerevisiae RPB1 gene via homologous recombination and gene replacement in vivo . Transcription of the mouse RPB1 gene using the yeast promoter in the haploid state was confirmed by Northern analysis . This strain of yeast is viable, indicating that mouse RPB1 is able to interact functionally with the other yeast RNA pol II subunits in vivo.

Gene, 1998 Mar 16, 209(1-2), 95 - 103
Spontaneous cAMP-dependent derepression of gene expression in stationary phase plays a role in recombinant expression instability; Grossman TH et al.; E . coli recombinant expression systems that utilize lac operon control elements to modulate gene expression are known to produce some amount of uninduced (leaky) gene expression . Previously, we showed that high levels of uninduced gene expression was a major cause of instability in the pET expression system . We show here that the pET system, in which the phage T7 RNA polymerase gene is expressed via lac operon control elements, exhibits leaky expression that increases markedly as cells grown in complex medium enter stationary phase . Moreover, we found that this phenomenon occurs with the chromosomal lac operon as well . Further investigation revealed that stationary phase leaky expression requires cyclic AMP, and that substantial leaky expression could be effected in log phase cells by adding cyclic AMP and acetate at pH6.0 . Finally, a comparison of otherwise isogenic cya and wild-type hosts showed that expression stability and plasmid maintenance in the cya host is greatly enhanced, even when cells are passaged repeatedly in non-selection medium . These findings both provide a method to enhance the stability of lac-based recombinant expression systems, and suggest that derepression of the lac operon in the absence of inducer may be part of a general cellular response to nutrient limitation.

Biochem J, 1998 Apr 1, 331 ( Pt 1), 309 - 16
A human homologue of Escherichia coli ClpP caseinolytic protease: recombinant expression, intracellular processing and subcellular localization; Corydon TJ et al.; We have recently cloned a human cDNA (hClpP) with significant sequence similarity to the ATP-dependent Escherichia coli ClpP protease {Bross, Andresen, Knudsen, Kruse and Gregersen (1995) FEBS Lett . 377, 249-252} . In the present study, synthesis, intracellular processing and subcellular localization of hClpP have been analysed in intact cells and in a cell-free system . Using pulse-labelling/immunoprecipitation of Chang cells transfected with the hClpP cDNA, we observed two major bands with apparent molecular masses of approx . 39 and 37 kDa . A pulse-chase experiment showed that these bands were converted into one mature-enzyme band with a molecular mass of approx . 32 kDa that was stable for at least 24 h . The 37 kDa band co-migrated with a band produced upon expression of full-length hClpP in E . coli, and the 32 kDa band co-migrated with the product of E . coli-expressed hClpP in which the 56 N-terminal residues had been deleted, indicating that the 37 kDa moiety represents the precursor and that approx . 56 residues are cleaved off during maturation . The processing of hClpP in intact cells was dependent on mitochondrial membrane potential . These results were confirmed in an import assay system using in vitro transcription and translation directed by the hClpP cDNA and isolated rat liver mitochondria . No protease activity towards a series of fluorogenic peptides could be observed in extracts of Chang cells overexpressing hClpP, indicating that the protease may not be active without co-factors . Immunofluorescence studies using confocal-laser-scanning microscopy showed co-localization of the hClpP and the mitochondrially located Hsp60 (heat-shock protein 60) . Taken together, the results reported here show that hClpP is localized inside mitochondria and that the trafficking and processing of hClpP resembles the typical biogenesis pathway for nuclear-encoded mitochondrial proteins.

Biochem J, 1998 Apr 1, 331 ( Pt 1), 79 - 87
DNA polymerase beta: effects of gapped DNA substrates on dNTP specificity, fidelity, processivity and conformational changes; Ahn J et al.; Pre-steady-state kinetic analysis was used to compare the catalytic properties of DNA polymerase beta (Pol beta) for single-base gap-filling and regular duplex DNA synthesis . The rate of polymerization (kpol) and the apparent equilibrium dissociation constant of dNTP (Kd) were determined with single-nucleotide gapped DNA substrates for all four possible correct base pairs and twelve possible incorrect base pairs, and the results were compared with those obtained previously with non-gapped primer/template duplex DNA substrates . For correct dNTP incorporation, the use of single-nucleotide gapped DNA led to significant decreases in the Kd of dNTP . Although kpol was little affected, the catalytic efficiency kpol/Kd increased significantly owing to the decreases in Kd . In contrast, for incorrect dNTP incorporation, the use of single-nucleotide gapped DNA substrates did not affect the Kd of dNTP appreciably but caused the kpol (and thus kpol/Kd) for incorrect dNTP incorporation to increase . As a consequence the fidelity of Pol beta was not significantly affected by the use of single-nucleotide gapped DNA substrates . In addition we show that under processive polymerization conditions the processivity of Pol beta increases in the gap-filling synthesis owing to a decreased rate of DNA dissociation . Finally, with a single-nucleotide gapped DNA substrate the rate-limiting conformational change step before chemistry was also observed . However, the preceding fast conformational change observed with duplex DNA substrates was not clearly detected . A possible cause is that in the complex with the gapped DNA, the 8 kDa N-terminal domain of Pol beta already exists in a closed conformation . This interpretation was supported by tryptic digestion experiments.

Glycobiology, 1998 Mar, 8(3), 245 - 57
Immunocytochemical localization of alpha2,3(N)-sialyltransferase (ST3Gal III) in cell lines and rat kidney tissue sections: evidence for golgi and post-golgi localization; Burger PC et al.; Sialylation is a biosynthetic process occurring in the trans compartments of the Golgi apparatus . Corresponding evidence is based on localization and biochemical studies of alpha2, 6(N)-sialyltransferase (ST6Gal I) as previously reported . Here we describe generation and characterization of polyclonal antibodies to recombinant rat alpha2,3(N)-sialyltransferase (ST3Gal III) expressed as a soluble enzyme in Sf9 cells or as a beta-galactosidase-human-ST3Gal III fusion-protein from E.coli , respectively . These antibodies were used to localize ST3Gal III by immunofluorescence in various cell lines and rat kidney tissue sections . In transiently transfected COS cells the antibodies directed to soluble sialyltransferase or the sialyltransferase portion of the fusion-protein only recognized the recombinant antigen retained in the endoplasmic reticulum . However, an antibody fraction crossreactive with beta-galactosidase recognized natively expressed ST3Gal III which was found to be colocalized with beta1, 4-galactosyltransferase in the Golgi apparatus of several cultured cell lines . Antibodies affinity purified on the beta-galactosidase-ST3Gal III fusion-protein column derived from both antisera have then been used to localize the enzyme in perfusion-fixed rat kidney sections . We found strong staining of the Golgi apparatus of tubular epithelia and a brush-border-associated staining which colocalized with cytochemical staining of the H+ATPase . This subcellular localization was not observed for ST6Gal I which localized to the Golgi apparatus . These data show colocalization in the Golgi apparatus and different post-Golgi distributions of the two sialyltransferases.

J Biochem Mol Toxicol, 1998, 12(4), 205 - 12
Molecular cloning, sequence, and expression of mouse flavin-containing monooxygenases 1 and 5 (FMO1 and FMO5); Cherrington NJ et al.; Full-length cDNA clones encoding FMO1 and FMO5 have been isolated from a library constructed with mRNA from the liver of a female CD-1 mouse . The derived sequence of FMO1 contains 2310 bases: 1596 in the coding region, 301 in the 5'-flanking region, and 413 in the 3'-flanking region . The sequence for FMO5 consists of 3168 bases; 1599 in the coding region, 812 in the 5'-flanking region, and 757 in the 3'-flanking region . The sequence of FMO1 encodes a protein of 532 amino acids with a predicted molecular weight of 59.9 kDa and shows 83.3% identity to human FMO1 and 83-94% identity to other FMO1 homologs . FMO5 encodes a protein of 533 amino acids with a predicted molecular weight of 60.0 kDa and 84.1% identity to human FMO5 and 83-84% identity to other FMO5 orthologs . Two GxGxxG putative pyrophosphate binding domains exist beginning at positions 9 and 191 for FMO1, and 10 and 192 for FMO5 . Mouse FMO1 and FMO5 were expressed in E . coli and show similar mobility to the native proteins as determined by SDS-PAGE . The expressed FMO1 protein showed activity toward methimazole, and FMO5 was active toward noctylamine . In addition, FMO1 was shown to metabolize radiolabeled phorate, whereas FMO5 showed no activity toward phorate.

RNA, 1998 May, 4(5), 542 - 50
Matching the crystallographic structure of ribosomal protein S7 to a three-dimensional model of the 16S ribosomal RNA; Tanaka I et al.; Two recently published but independently derived structures, namely the X-ray crystallographic structure of ribosomal protein S7 and the "binding pocket" for this protein in a three-dimensional model of the 16S rRNA, have been correlated with one another . The known rRNA-protein interactions for S7 include a minimum binding site, a number of footprint sites, and two RNA-protein crosslink sites on the 16S rRNA, all of which form a compact group in the published 16S rRNA model (despite the fact that these interactions were not used as primary modeling constraints in building that model) . The amino acids in protein S7 that are involved in the two crosslinks to 16S rRNA have also been determined in previous studies, and here we have used these sites to orient the crystallographic structure of S7 relative to its rRNA binding pocket . Some minor alterations were made to the rRNA model to improve the fit . In the resulting structure, the principal positively charged surface of the protein is in contact with the 16S rRNA, and all of the RNA-protein interaction data are satisfied . The quality of the fit gives added confidence as to the validity of the 16S rRNA model . Protein S7 is furthermore known to be crosslinked both to P site-bound tRNA and to mRNA at positions upstream of the P site codon; the matched S7-16S rRNA structure makes a prediction as to the location of this crosslink site within the protein molecule.

Cancer Res, 1998 May 1, 58(9), 1946 - 51
Adenovirus-mediated transduction of Escherichia coli uracil phosphoribosyltransferase gene sensitizes cancer cells to low concentrations of 5-fluorouracil; Kanai F et al.; 5-fluorouracil (5-FU), although a widely used chemotherapeutic agent, has a limited effect in the treatment of human solid tumors due to their resistance to the cytotoxic effects of 5-FU . Escherichia coli uracil phosphoribosyltransferase (UPRT) is a pyrimidine salvage enzyme that catalyzes the synthesis of UMP from uracil and 5-phosphoribosyl-alpha-1-diphosphate . The present study demonstrates that adenovirus-mediated transduction of E . coli UPRT gene results in marked sensitization of colon, gastric, liver, and pancreas cancer cell lines to low concentration of 5-FU in vitro . The in vitro bystander effect was observed when only 10% of the hepatoma Hep3B cells were infected with UPRT-expressing adenovirus . In addition, 5-FU treatment of human hepatoma or gastric cancer xenografts in nude mice transduced with UPRT was demonstrated to result in significant in vivo antitumor effects . The adenovirus vector transduction of the UPRT gene followed by 5-FU administration is representative of a new chemosensitization strategy for cancer gene therapy.

Cancer Res, 1998 May 1, 58(9), 1936 - 45
Isolation of human O6-alkylguanine-DNA alkyltransferase mutants highly resistant to inactivation by O6-benzylguanine; Xu-Welliver M et al.; The activity of O6-alkylguanine-DNA alkyltransferase (AGT) protects cells from killing by methylating or chloroethylating agents . AGT is strongly inhibited by O6-benzylguanine (ED50, 0.2 microM), and this drug is presently undergoing clinical trials to enhance chemotherapy by alkylating agents . Point mutations such as P140A (ED50, 5 microM) render AGT resistant to O6-benzylguanine (BG) . Selection for such mutants may prove to be a problem in the use of BG, and a better knowledge of the factors underlying resistance to BG will enable the rational design of improved inhibitors able to inactivate these mutants . BG-resistant AGT mutants may also be valuable for expression in bone marrow stem cells to reduce myelosuppression brought about by alkylating agents, to increase the therapeutic index of therapies including BG, and for use as a selectable marker to allow other genes to be expressed in such stem cells . We have therefore set up a general screen to obtain such mutants by using the ability of AGT to protect Escherichia coli GWR109 lacking endogenous AGT from killing by N-methyl-N'-nitro-N-nitrosoguanidine . When the cells were rendered permeable to BG by mutating the lipopolysaccharide membrane component forming strain TRG8, the protection by AGT expression was abolished by treating the cells with BG . The known P140A mutant was used to test the system and was highly selected for by treatment with 50 microM BG and 40 microg/ml N-methyl-N'-nitro-N-nitrosoguanidine . The sequence coding for PVP at positions 138-140 in AGT was replaced with a random nucleotide sequence, and this library was used to transform TRG8 . All of the 59 colonies analyzed having AGT activity that survived the selection from the pool of 36,000 transformants were resistant to BG . Many (69%) of these mutants contained lysine at position 140, and all of these showed the highest level of resistance with <10% loss of activity when crude cell extracts were incubated with 1.2 mM BG . This result was confirmed with three mutants (P138K/V139L/P140K, P138M/V139L/P140K, and P140K), which were purified to homogeneity . The next most common residues found at position 140 were arginine (7%) and asparagine (7%) . Studies carried out with purified preparations of mutants P140R and P140N revealed that these mutations also provided resistance to BG but to a lesser extent than P140K (ED50s of 190 and 7 microM, respectively) . These results indicate that: (a) this screening method can be used to evaluate BG resistance of single or multiple changes throughout the AGT sequence; and (b) replacement of proline-140 with lysine is the most effective point mutation at this site causing BG resistance and is more than 200 times more effective than replacement with alanine.

Cancer Res, 1998 May 1, 58(9), 1808 - 12
Expression of a novel antiapoptosis gene, survivin, correlated with tumor cell apoptosis and p53 accumulation in gastric carcinomas; Lu CD et al.; A novel inhibitor of apoptosis designated survivin has recently been found in many common human cancers but not in normal tissues . A potential distribution of survivin in gastric cancer and its implication for apoptosis inhibition have been investigated . Recombinant survivin expressed in Escherichia coli as a glutathione S-transferase fusion protein was used to raise a novel panel of mouse monoclonal antibodies . In an immunohistochemical analysis of 174 cases of gastric carcinomas (stages I-III), anti-survivin monoclonal antibody 8E2 (IgG1) reacted with 34.5% of cases (60 of 174 cases) with a variable number of tumor cells stained (20-100%) . In contrast, no expression of survivin in neighboring normal tissues was observed . When stratified for p53 and bcl-2 expression and apoptotic index, the expression of survivin significantly segregated with p53- and bcl-2-positive cases {56.1 versus 15.2% (P = 0.001) and 69.2 versus 31.6% (P = 0.006), respectively} and with a decreased apoptotic index as compared with that of survivin-negative tumors (0.97 +/- 0.64 versus 0.62 +/- 0.39%, P < 0.001) . These data identify a role for survivin in promoting aberrantly increased cell viability in gastric cancer and suggest a potential correlation between accumulated p53 and survivin expression in neoplasia.

Virology, 1998 Apr 25, 244(1), 230 - 42
Regeneration of the binding properties of adenovirus 12 early region 1A proteins after preparation under denaturing conditions; Grand RJ et al.; Adenovirus 12 early region 1A (Ad12 E1A) was expressed in Escherichia coli . Protein was purified in good yield in the presence of 8 M urea and then renatured by dialysis against dilute NH4HCO3 buffer . The affinity of this protein for pRb, C-terminal binding protein (CtBP), TATA binding protein (TBP), and SUG1 was similar to, or greater than, that of Ad12 E1A prepared by immunoaffinity chromatography under nondenaturing conditions . While the binding of the 266- and 235-amino-acid (aa) E1A components to TBP showed similar characteristics the larger E1A protein had a higher affinity for CtBP, pRb, and SUG1 . Using nuclear magnetic resonance (NMR) spectroscopy it was shown that structural perturbations occurred in the 266-aa protein in the presence of Zn2+ consistent with binding--no such changes were seen for the 235-aa protein . Limited proteolysis of the 266- and 235-aa E1A proteins gave rise to comparable polypeptide products, suggesting overall similarities in structure . However, the different affinities of the 266- and 235-aa proteins for the partner proteins and the differences seen in the NMR spectra from the two proteins suggested structural differences.

Biochem J, 1997 Nov 1, 327 ( Pt 3), 891 - 8
Purification of Escherichia coli acetohydroxyacid synthase isoenzyme II and reconstitution of active enzyme from its individual pure subunits; Hill CM et al.; The first step in the biosynthesis of branched-chain amino acids is catalysed by acetohydroxyacid synthase (EC 4.1.3.18) . The reaction involves the decarboxylation of pyruvate followed by condensation with either a second molecule of pyruvate or with 2-oxobutyrate . The enzyme requires as cofactors thiamine diphosphate, a divalent metal ion and, usually, FAD . In most bacteria the enzyme is a heterotetramer of two large and two small subunits . Escherichia coli contains three active isoenzymes and the present study concerns isoenzyme II, whose large and small subunits are encoded by the ilvG and ilvM genes respectively . Cloning these genes into a plasmid vector and overexpression in E . coli allowed a two-step purification procedure for the native enzyme to be developed . The level of expression is considerably higher from a vector that introduces a 50 residue N-terminal fusion containing an oligohistidine sequence on the large subunit . Purification to homogeneity was achieved in a single step by immobilized-metal-affinity chromatography . The kinetic properties of the native and fusion enzyme are indistinguishable with respect to the substrate pyruvate and the inhibitor chlorsulfuron . The individual subunits were expressed as oligohistidine-tagged fusion proteins and each was purified in a single step . Neither subunit alone has significant enzymic activity but, on mixing, the enzyme is reconstituted . The kinetic properties of the reconstituted enzyme are very similar to those of the fusion enzyme . It is proposed that the reconstitution pathway involves successive, and highly co-operative, binding of two small subunit monomers to a large subunit dimer . None of the cofactors is needed for subunit association although they are necessary for the restoration of enzymic activity.

Biochem J, 1997 Nov 1, 327 ( Pt 3), 847 - 51
RNA minihelices as model substrates for ATP/CTP:tRNA nucleotidyltransferase; Li Z et al.; Twenty-one RNA minihelices, resembling the coaxially stacked acceptor- /T-stems and T-loop found along the top of a tRNA's three-dimensional structure, were synthesized and used as substrates for ATP/CTP:tRNA nucleotidyltransferases from Escherichia coli and Saccharomyces cerevisiae . The sequence of nucleotides in the loop varied at positions corresponding to residues 56, 57 and 58 in the T-loop of a tRNA . All minihelices were substrates for both enzymes, and the identity of bases in the loop affected the interaction . In general, RNAs with purines in the loop were better substrates than those with pyrimidines, although no single base identity absolutely determined the effectiveness of the RNA as substrate . RNAs lacking bases near the 5'-end were good substrates for the E . coli enzyme, but were poor substrates for that from yeast . The apparent Km values for selected minihelices were 2-3 times that for natural tRNA, and values for apparent Vmax were lowered 5-10-fold.

Biochem J, 1997 Nov 1, 327 ( Pt 3), 675 - 84
A mutant phosphofructokinase produces a futile cycle during gluconeogenesis in Escherichia coli; Torres JC et al.; Strains of Escherichia coli bearing different forms of phosphofructokinase were used to assess the occurrence of futile cycling in cell resuspensions supplied with glycerol as gluconeogenic carbon source . A model was used to simulate results of different kinds of experiments for different levels of futile cycle . The main predictions of the model were experimentally confirmed in a strain with a mutant phosphofructokinase-2 (phosphofructokinase-2*) which is not inhibited by MgATP . The intracellular fructose 1, 6-bisphosphate concentration reaches significantly higher levels in the mutant-bearing strain than in strains with either phosphofructokinase-1 or -2 . Also, this strain showed a higher rate and level of in vivo radioactive labelling of fructose 1, 6-bisphosphate, from a trace of {U-14C}glucose supplied during gluconeogenesis, indicating higher kinase activity in these conditions . Cell resuspensions of the mutant-bearing strain produced higher levels of radioactively labelled CO2 when supplied with {U-14C}glycerol as the only carbon source . Simultaneously, fewer glycerol carbons were incorporated into HClO4-insoluble macromolecules . Finally, radioactive CO2 output was measured in resuspensions supplied with glycerol as the major carbon source with traces of either {1-14C}glucose or {6-14C}glucose . It was found that, whereas in the strains with either of the wild-type phosphofructokinase isoenzymes, radioactive CO2 output from {1-14C}glucose was higher than with {6-14C}glucose, the reverse is found for the strain with phosphofructokinase-2* . This result also agrees with the corresponding prediction of the model . Using the radioactivity flux rates predicted by the model, an explanation linking the futile cycle to the differential labelling of CO2 is advanced . Finally, on the basis of these results it is proposed that strains bearing phosphofructokinase-2* sustain higher rates of futile cycling during gluconeogenesis than strains bearing either of the wild-type isoforms of phosphofructokinase . The kinetic equations and parameter values used for the model simulations are given in Supplementary Publication SUP 50183 (8 pages), which has been deposited at the British Library Document Supply Centre, Boston Spa, Wetherby, West Yorkshire LS23 7BQ, U.K., from whom copies can be obtained on the terms indicated in Biochem . J . (1997) 321, 8.

Rocz Akad Med Bialymst, 1997, 42(1), 267 - 76
Influence of endotoxin on the level of tumor necrosis factor alfa and blood morphology in rats; Hryniewicz A et al.; The influence of endotoxin E.coli O127 B:8 given intravenously or intraperitoneally on the level of Tumor Necrosis Factor alfa (TNF-alpha), blood cells count and bone marrow morphology in rats was studied . The presented results show that endotoxin (3 mg/kg) given intravenously or intraperitoneally (1 or 3 times) provoked an appearance of TNF-alpha in the systemic circulation 24 hours after its injection . Endotoxin given intravenously evoked greater changes in blood morphology and bone marrow than that applicated intraperitoneally.

Rocz Akad Med Bialymst, 1997, 42(1), 205 - 12
Effect of endotoxin on the concentration of triacylglycerols in the liver of the rat; Hryniewicz A et al.; The aim of the present study was to examine an effect of endotoxin E.coli O127:B8 on the concentrations of triacylglycerol (TG) in the rat's liver . The rats were divided into the following groups: control, treated with one dose of endotoxin, treated with three doses of endotoxin . The rats had free access to food (pallet diet for rodents) and water . Endotoxin E.coli O127:B8 was injected interperitoneally in dose of 3 mg/kg b.w.--once or three times at intervals of 72 hrs . One dose of endotoxin decreased blood serum TG concentration, although the concentration of liver TG was increased after 24 hrs . Triple doses of endotoxin given at 72 hrs intervals increased the blood serum TG concentration after 24 hrs, but had no effect on the liver concentration.

Rocz Akad Med Bialymst, 1997, 42(1), 69 - 74
Nitroblue tetrazolium test in course of ulcerative colitis; Zagorski K et al.; Studies were carried out on the neutrophil activity in a course of a ulcerative colitis (UC) . Phagocytic ability of granulocytes was assessed in spontaneous and stimulated nitroblue tetrazolium (NBT) reduction test . The studies included 60 patients who had 82 relapses and 11 patients with remission . They were classified into mild, moderate and severe relapse according to severity of disease . Increased phagocytic ability of neutrophils in spontaneous and stimulated NBT reduction test was shown.

Rocz Akad Med Bialymst, 1997, 42(1), 5 - 12
Lithogenecity of bile in the pathogenesis of cholelithiasis; Dadan H et al.; The authors have made a review of the present literature relating to lithogenic bile as the main pathogenic factor in the creation of cholelithiasis . Lithogenic bile is formed in result of complex biochemical reactions taking place in bile under physiological conditions, as well as in pathology of the liver and the biliary tract . Significant clinical implications result from the analysis.

Mol Biol Evol, 1998 May, 15(5), 590 - 9
Detecting recombination from gene trees; Maynard Smith J et al.; In this article, a method is proposed for detecting recombination in the sequences of a gene from a set of closely related organisms . The method, the Homoplasy Test, is appropriate when the sequences are rather similar, differing by 1%-5% of nucleotides . It is effective in detecting relatively frequent recombination between a set of rather similar strains, in contrast to previous methods which detect rare or unique transfers between more distant strains . It is based on the fact that, if there is no recombination and if no repeated mutations have occurred (homoplasy), then the number of polymorphic sites, v, is equal to the number of steps, t, in a most-parsimonious tree . If the number of "apparent homoplasies" in the most-parsimonious tree, h = t-v, is greater than zero, then either homoplasies have occurred by mutation or there has been recombination . An estimate of the distribution of h expected on the null hypothesis of no recombination depends on Se, the "effective site number," defined as follows: if ps is the probability that two independent substitutions in the gene occur at the same site, then Se = 1/ps . Se can be estimated if a suitable outgroup is available . The Homoplasy Test is applied to three bacterial genes and to simulated gene trees with varying amounts of recombination . Methods of estimating the rate, as opposed to the occurrence, of recombination are discussed.

Spine, 1998 Apr 15, 23(8), 870 - 6
Effects of steroid and lipopolysaccharide on spontaneous resorption of herniated intervertebral discs . An experimental study in the rabbit; Minamide A et al.; STUDY DESIGN: Histologic examination was performed on autografted intervertebral disc materials of rabbit models, which were partially incised through a retroperitoneal approach at L1-L2 and grafted within the posterior epidural space at L4 . OBJECTIVE: To evaluate whether the resorption process of the herniated intervertebral disc is influenced and controlled by treatments with medications . SUMMARY OF BACKGROUND DATA: Regarding resorption of herniated intervertebral discs, recent studies of magnetic resonance images and histologic investigations of surgically resected specimens in lumber disc herniation patients have been reported . It has been shown that inflammatory factors may play an important role in the mechanism of resorption of the herniated intervertebral disc . However, little is known about the origin of newly formed vessels and inflammatory cells detected in herniated disc specimens from patients . In this study, The resorption process of disc material grafted into the epidural space was observed in a rabbit model . METHODS: Thirty-six adult rabbits were used . The L1-L2 intervertebral disc was partially incised through a retroperitoneal approach . The harvested disc material, which contains the nucleus pulposus and the anulus fibrosus were placed into the posterior epidural space at L4 of the same rabbit . The animals were divided into control, and steroid groups . The control group received no treatment after surgery . In the lipopolysaccharide group, rabbits were injected 1 mg/kg into the peritoneum immediately and at 7 days after surgery . In the steroid group, rabbits were injected with 1 mg/kg betamethasone into the epidural space daily from 1 to 7 days after surgery . Rabbits of each group were killed for histologic examination at 1, 2, 4, and 8 weeks after surgery . RESULTS: At 1 and 2 weeks after surgery, inflammatory cells and newly formed vessels were more frequently observed in the lipopolysaccharide group than in the control and steroid groups . At 4 weeks after surgery, derangement and loosening of collagen fibers were also observed in the lipopolysaccharide group . At 8 weeks after surgery, fragmentation and partial disappearance of matrix were observed in the control and lipopolysaccharide groups . Most of the intervertebral discs were replaced by fibrous tissues in the lipopolysaccharide group . However, the matrix of the intervertebral disc almost remained . CONCLUSIONS: Autologous intervertebral disc material grafted into the epidural space was penetrated by newly formed vessels produced from the epidural fat tissue and resolved as the result of inflammatory reaction . Lipopolysaccharide accelerated the replacement of grafted intervertebral disc by fibrous tissue, which suggests the resorption of the disc in the epidural space of the rabbit, whereas high-dose steroid suppressed the replacement.

J Virol, 1998 Apr, 72(4), 3185 - 95
Inducible gene expression from African swine fever virus recombinants: analysis of the major capsid protein p72; Garcia-Escudero R et al.; A method to study the function of individual African swine fever virus (ASFV) gene products utilizing the Escherichia coli lac repressor-operator system has been developed . Recombinant viruses containing both the lacI gene encoding the lac repressor and a strong virus late promoter modified by the insertion of one or two copies of the lac operator sequence at various positions were constructed . The ability of each modified promoter to regulate expression of the firefly luciferase gene was assayed in the presence and in the absence of the inducer isopropyl beta-D-thiogalactoside (IPTG) . Induction and repression of gene activity were dependent on the position(s) of the operator(s) with respect to the promoter and on the number of operators inserted . The ability of this system to regulate the expression of ASFV genes was analyzed by constructing a recombinant virus inducibly expressing the major capsid protein p72 . Electron microscopy analysis revealed that under nonpermissive conditions, electron-dense membrane-like structures accumulated in the viral factories and capsid formation was inhibited . Induction of p72 expression allowed the progressive building of the capsid on these structures, leading to assembly of ASFV particles . The results of this report demonstrate that the transferred inducible expression system is a powerful tool for analyzing the function of ASFV genes.

Mutat Res, 1997 Dec, 385(3), 251 - 8
Effects of 1,10-phenanthroline and hydrogen peroxide in Escherichia coli: lethal interaction; Furtado FA et al.; It has been observed that when Escherichia coli cells are treated simultaneously with phenanthroline and H2O2, there is a lethal interaction . In order to analyze the mechanism of this lethal interaction, wild-type and xthA mutant cells of E . coli were treated with 2.5 mM H2O2 and 1 mM phenanthroline . This treatment was preceded by treatments with different metal chelators (dipyridyl for Fe2+, desferal for Fe3+ and neocuproine for Cu2+) or conducted simultaneously to other treatments with chelators and radical scavengers (thiourea, ethanol and sodium benzoate) . The lethal interaction was observed in both the E . coli wild-type strain and xthA mutant strain, which is deficient in the exonuclease III repair enzyme . Nevertheless, the mutant strain was much more sensitive than the wild-type one . Dipyridyl pretreatment protected the cells against the lethal interaction, while desferal pretreament was unable to do so . This suggests that the lethal interaction requires Fe2+ and not Fe3+ ions . Ethanol and sodium benzoate were incapable of protecting bacterial cells against the lethal interaction . Even a 20-min pretreatment with benzoate did not confer protection . On the other hand, thiourea protected the cells completely . Based on our results, we propose that the lethal interaction may be caused not only by the reaction kinetics of phenanthroline and Fe, but also by the ability of phenanthroline to intercalate in DNA . After forming the mono and bis complexes, phenanthroline would serve as a shuttle and take the Fe2+ ions to the DNA . So, the Fenton reaction would take its course with the consequent generation of OH . radicals near DNA . This proximity to the DNA would protect the OH . radicals against the scavengers' action, thus optimizing the Fenton reaction.

Arch Esp Urol, 1997 Nov, 50(9), 972 - 5
{Ureteral hydronephrosis secondary to appendicular abscess}; Montero Sanchez M et al.; OBJECTIVE: To report a case of ureterohydronephrosis secondary to an undiagnosed appendiceal abscess . METHODS/RESULTS: Herein we describe a case of a 4-year-old girl with right ureterohydronephrosis arising from extrinsic compression of the right ureter due to an undiagnosed appendiceal abscess . The patient was treated with intravenous antibiotics and the abscess was drained . Regular isotope and US assessments showed both the residual retroperitoneal fibrosis and renoureteral dilation had decreased . CONCLUSIONS: Acute appendicitis is still the most common cause of emergency abdominal operations in children . Although the symptoms are easily recognizable and generally lead to the correct diagnosis in most cases, the peculiarities of childhood can lead to errors in the diagnosis resulting in the complications reported herein . We emphasize the usefulness of ultrasound in the diagnosis and conservative treatment is advocated.

Nippon Ishinkin Gakkai Zasshi, 1998, 39(2), 85 - 90
{Regulation of gene expression in Aspergilli}; Tsukagoshi N; The Aspergillus oryzae Taka-amylase A (TAA) gene has been used as a model gene to characterize the regulatory mechanisms of gene expression in Aspergilli . TAA gene contained a typical eukaryotic promoter with a TATA box and putative regulatory elements such as a CCAAT sequence in its 5'-noncoding region . A nuclear protein designated AnCP bound to the CCAAT sequence . Replacement of the CCAAT sequence with CGTAA was found to abolish the binding of AnCP and to have an inhibitory effect on taa promoter activity . Although AnCP was not purified to homogeneity, AnCP appeared to have an apparent molecular mass of approximately 120 kDa . In Saccharomyces cerevisiae, the CCAAT-binding HAP complex is a heteromeric protein comprising at least four subunits (yHAP2, yHAP3, yHAP4 and yHAP5) and is involved in the regulation of genes responsible for oxidative phosphorylation . A gene designated hapC with significant homology to yHAP3 has been isolated from A . nidulans by Hynes et al . We expressed hapC gene as a fusion protein with MalE in E . coli, purified HapC protein and prepared anti-HapC antiserum . The MalE-HapC fusion protein was able to substitute for the authentic HapC in AnCP . Furthermore, addition of the anti-HapC antiserum to the DNA binding reaction mixture retarded the mobility of the shifted band . These indicate clearly that HapC protein acts as a subunit of AnCP and that AnCP is a counterpart of the yeast Hap complex.

Epilepsia, 1997 Jul, 38(7), 759 - 66
Direct gene transfer into human epileptogenic hippocampal tissue with an adeno-associated virus vector: implications for a gene therapy approach to epilepsy; Freese A et al.; PURPOSE: Virus vectors capable of transferring genetic information into human cells provide hope for improved therapy in several neurological diseases, including epilepsy . We evaluated the ability of an adeno-associated virus (AAV) vector to transfer and cause expression of a lacZ marker gene in brain slices obtained from patients undergoing temporal lobectomy for control of medically intractable seizures . METHODS: Human brain slices were injected with an AAV vector (AAVlacZ) encoding Escherichia coli beta-galactosidase and incubated for as long as 24 h . The presence of lacZ mRNA . beta-galactosidase protein and enzymatic activity were assayed by reverse transcriptase polymerase chain reaction (rtPCR), immunocytochemistry, and the X-Gal technique, respectively . RESULTS: AAVlacZ directed the expression in human epileptogenic brain of E . coli beta-galactosidase that had functional activity . Expression was observed in < or =5 h and was sustained for as long as the slices were viable . Morphological analysis indicated that neurons were preferentially transfected, and there was no evidence of cytotoxicity . CONCLUSIONS: Our results confirm the feasibility of using AAV vectors to transfer genes into the human CNS and in particular, into neurons . Replacement of the lacZ gene with a functional gene modulating hippocampal neuronal physiology, might allow a localized genetic intervention for focal seizures based on the stereotaxic or endovascular delivery of such a vector system into the appropriate brain region.

Protein Eng, 1998 Jan, 11(1), 59 - 63
Identification of four amino acid residues essential for catalysis in human cytidine deaminase by site-directed mutagenesis and chemical modifications; Cambi A et al.; By site-directed mutagenesis on human cytidine deaminase (CDA), five mutant proteins were obtained: C65A, C99A, C102A, E67D and E67Q . The three cysteine mutants were completely inactive, whereas E67D and E67Q showed a specific activity about 200- and 200000-fold lower, respectively, than the wild-type CDA . Zinc analysis revealed that only E67D, E67Q and C65A contained 1 mol Zn2+/mol subunit as in the wild-type CDA . Kinetic measurements with the specific carboxylic group reagent N-ethoxy-carbonyl-2-ethoxy-1,2-dihydroquinoline performed on wild-type CDA suggest that Glu67 is essential for the catalytic process . Furthermore, when both native and denatured CDA was titrated with 5,5'-dithiobis(2-nitrobenzoic acid) six sulfhydryl groups were detected, whereas in the denatured and reduced enzyme nine such groups were found, according to the sequence data . When p-hydroxymercuriphenyl sulfonate was used, nine sulfhydryl groups were detectable and the release of 1 mol of zinc per mole of CDA subunit was revealed by the metal indicator dye 4-(2-pyridylazo)resorcinol . It seems plausible that the limiting step for the maintenance of zinc in the active site is the formation of coordination between Cys99 and Cys102, whereas Cys65 could lead the zinc to the correct position and orientation within the active site.

J Lab Clin Med, 1998 Apr, 131(4), 336 - 43
Endotoxin impairs agonist-induced calcium mobilization in bovine aortic myocytes by a nitric oxide-independent mechanism; Murray PT et al.; We hypothesized that endotoxin (LPS) would impair vasoconstrictor-agonist-induced calcium (Ca2+) mobilization by a nitric oxide (NO)-dependent mechanism . We incubated bovine aortic myocytes (passages 16 to 23) for 22 to 24 hours with 0 to 1.0 mg/ml Escherichia coli lipopolysaccharide (LPS) . Medium (Dulbecco's modified Eagle's medium (DMEM) + 10% fetal bovine serum (FBS)) was assayed for nitrite (chemiluminescence), and myocytes were loaded with fura-2 acetoxymethyl ester (fura-2AM), after which we assessed basal and thrombin (10 U/ml)-induced peak Ca2+ mobilization by microspectrofluorimetry . LPS (0.01 to 1.0 mg/ml) led to dose-dependent nitrite accumulation, which was blocked by coincubation with N(omega)-nitro-L-arginine methyl ester (L-NAME, 1 mmol/L) . LPS also impaired Ca2+ responses in a dose-dependent manner (from -13% at 0.1 mg/ml to -47% at 1.0 mg/ml, n = 8 to 43/dose) . However, coincubation with L-NAME did not ameliorate the Ca2+ mobilization defect (peak Ca2+ increments: control = 419 +/-30 nmol/L, vs LPS (1 mg/ml) = 206+/-18 nmol/L (mean+/-SE), n = 15; p < 0.001; control/L-NAME: 417+/-31 nmol/L vs LPS/L-NAME: 212+/-19 nmol/L; n = 17 p < 0.001), despite inhibition of associated nitrite accumulation in the medium (control vs LPS: p < 0.001; control/L-NAME vs LPS/L-NAME: p > 0.05; LPS vs LPS/L-NAME: p < 0.001) . Supplemental L-arginine augmented LPS-induced nitrite generation without affecting Ca2+ mobilization . Indomethacin failed to prevent the LPS-induced decrement in thrombin response, but did inhibit LPS-induced myocyte nitrite production, suggesting "crosstalk" between the NO-synthase and cyclo-oxygenase (COX) systems . These experiments suggest that LPS-induced vascular contractile impairment is at least partly mediated by an NO-independent impairment of agonist-induced myocyte Ca2+ mobilization . This further suggests that any important contribution of NO synthesis to LPS-induced contractile dysfunction must depend on impairment of the Ca2+ sensitivity of the contractile apparatus (i.e., pharmacomechanical coupling).

Ann R Coll Surg Engl, 1998 Jan, 80(1), 36 - 9
'Surgical' peritonitis in the CAPD patient; Miller GV et al.; Peritonitis is the most frequent cause for emergency hospital admission in continuous ambulatory peritoneal dialysis (CAPD) patients . Patients may present with 'surgical' peritonitis from other intra-abdominal pathology, but are treated initially as CAPD-related peritonitis . We present nine such cases, each failing to respond to standard conservative treatment, and ultimately coming to laparotomy . Of the nine patients, six survived, five transferring to long-term haemodialysis and one patient returning to CAPD . Failure to respond to standard measures should alert the physician to the possibility of an intra-abdominal emergency . The presence of enteric organisms, particularly E . coli, is an additional suspicious feature . The diagnosis may be difficult and we recommend early surgical referral and appropriate surgical measures (laparotomy rather than simple catheter removal) in order to decrease morbidity and mortality.

Microbiology, 1998 Apr, 144 ( Pt 4), 1077 - 84
Molecular analysis of Physarum haemagglutinin I: lack of a signal sequence, sulphur amino acids and post-translational modifications; Morita M; The cDNAs encoding haemagglutinin I from plasmodia of Physarum polycephalum have been cloned using PCR protocols . The composite haemagglutinin I cDNA sequence, derived from several overlapping clones from PCR fragments, spans 408 nt and the 315 bp ORF encodes a polypeptide of 104 aa without a typical signal sequence . The putative molecular mass deduced from the amino acid sequence (10,760.76 Da) corresponds exactly to that determined by electrospray ionization MS (10,759.86 +/- 0.15 Da), suggesting that haemagglutinin I is not subject to post-translational modification . Haemagglutinin I lacks sulphur amino acids and has a beta-sheet as the major secondary structure . Expression of the coding sequence in Escherichia coli yielded a product that exhibits the same sugar-binding specificity as natural haemagglutinin I . The deduced amino acid sequence shows little similarity to that of any known lectins and thus apparently represents a novel type of lectin.

Microbiology, 1998 Apr, 144 ( Pt 4), 937 - 45
Physiological function and regulation of flavocytochrome c3, the soluble fumarate reductase from Shewanella putrefaciens NCIMB 400; Gordon EH et al.; Shewanella putrefaciens produces a soluble flavocytochrome c under anaerobic growth conditions . This protein shares sequence similarity with the catalytic subunits of membrane-bound fumarate reductases from Escherichia coli and other bacteria and the purified protein has fumarate reductase activity . It is shown here that this enzyme, flavocytochrome c3, is essential for fumarate respiration in vivo since disruption of the chromosomal fccA gene, which encodes flavocytochrome c3, leads to a specific loss of the ability to grow with fumarate as terminal electron acceptor . Growth with nitrate, trimethylamine N-oxide (TMAO) and other acceptors was unaffected . The fccA gene is transcribed as a 2 kb monocistronic mRNA . An adjacent reading frame that bears limited sequence similarity to one of the membrane anchor subunits of E . coli fumarate reductase is not co-transcribed with fccA . Expression of the fccA gene is regulated by anaerobiosis and by the availability of alternative electron acceptors, particularly nitrate and TMAO . DNA sequences have been identified that are required for this regulation.

Chem Pharm Bull (Tokyo), 1998 Apr, 46(4), 697 - 703
Syntheses of HIV-protease inhibitors having a peptide moiety which binds to GP120; Asagarasu A et al.; Some HIV-protease inhibitor derivatives having an N-carbomethoxycarbonyl-prolyl-phenylalanine benzyl ester (CPF) moiety as a binding site to gp120 were designed and synthesized . Almost all the compounds bearing CPF on the phenoxyacetyl group showed protease-inhibitory activity . Compounds 25a and 25b, which have the CPF moiety at the ortho- and meta-positions of the phenoxyacetyl group, respectively, had anti-HIV activity, although the others showed only protease-inhibitory activity . These results suggest that 25b binds to gp120 and inhibits HIV protease.

Biopolymers, 1998 Jun, 45(7), 493 - 501
Resolution of Trp near UV CD spectra of calmodulin-domain peptide complexes into the 1La and 1Lb component spectra; Barth A et al.; Near uv CD spectra of Trp residues in proteins frequently show a complex line shape deriving from the overlap of 1La and 1Lb electronic transitions . This study presents an original empirical method of resolving these components, based on the near uv CD spectra of well-defined complexes of calmodulin domains with target peptides containing a single Trp residue and derived from the skeletal muscle myosin light chain kinase target sequence . Spectra of 4 complexes were used to obtain the 1La and 1Lb component spectra that were then used to analyze further complexes . The broad and featureless 1La spectrum is centered at 279 nm, the 1Lb spectrum shows vibrational fine structure with maxima at 274.9, 281.5, and 289.8 nm . The CD spectrum of most complexes could successfully be fitted with one 1La and one 1Lb spectrum, the 1Lb spectrum being negative for all complexes but the 1La spectrum showing either positive or negative sign . Spectra of some complexes, however, failed to be adequately represented by only one 1La and one 1Lb spectrum . Instead, they could be fitted with one 1Lb spectrum and two 1La spectra with different sign and position . The method is successful in identifying and quantitating the relative intensities of a two-component system, consistent with a single conformation for tryptophan in a protein, and provides a simple indication of cases where a more complicated explanation is required.

Eur J Biochem, 1998 Apr 1, 253(1), 292 - 9
Thiol:fumarate reductase (Tfr) from Methanobacterium thermoautotrophicum--identification of the catalytic sites for fumarate reduction and thiol oxidation; Heim S et al.; Most methanogenic Archaea contain an unusual cytoplasmic fumarate reductase which catalyzes the reduction of fumarate with coenzyme M (CoM-S-H) and coenzyme B (CoB-S-H) as electron donors forming succinate and CoM-S-S-CoB as products . We report here on the purification and characterization of this thiol:fumarate reductase (Tfr) from Methanobacterium thermoautotrophicum (strain Marburg) . The purified enzyme, which was composed of two different subunits with apparent molecular masses of 58 kDa (TfrA) and 50 kDa (TfrB), was found to catalyze the following reactions: (a) the reduction of fumarate with CoM-S-H and CoB-S-H (150 U/mg); (b) the reduction of fumarate with reduced benzyl viologen (620 U/mg); (c) the oxidation of CoM-S-H and CoB-S-H to CoM-S-S-CoB with methylene blue (95 U/mg); and (d) the reduction of CoM-S-S-CoB with reduced benzyl viologen (250 U/mg) . The flavoprotein contained 12 mol non-heme iron and approximately the same amount of acid-labile sulfur/mol heterodimer . The genes encoding TfrA and TfrB were cloned and sequenced . Sequence comparisons with fumarate reductases and succinate dehydrogenases from Bacteria and Eucarya and with heterodisulfide reductases from M . thermoautotrophicum and Methanosarcina barkeri revealed that TfrA harbors FAD-binding motifs and the catalytic site for fumarate reduction and that TfrB harbors one {2Fe-2S} cluster and two {4Fe-4S} clusters and the catalytic site for CoM-S-H and CoB-S-H oxidation.

Eur J Biochem, 1998 Apr 1, 253(1), 270 - 9
Isolation and characterization of an evolutionary precursor of human monoamine oxidases A and B; Sablin SO et al.; An interesting flavoprotein-type monoamine oxidase (MAO) was recently isolated from Aspergillus niger and cloned {Schilling, B . & Lerch, K . (1995a) Biochim . Biophys . Acta 1243, 529-537; Schilling, B . & Lerch, K . (1995b) Mol . Gen . Genet . 247, 430-438} . The properties of this MAO, as well as a substantial part of its amino acid sequence, resemble those of both MAO A and B from higher animals, raising the possibility that it may be an evolutionary precursor of these mitochondrial enzymes . It differs from MAO A and B in several respects, however, including the fact that it is soluble and of peroxisomal location and that the FAD is non-covalently attached . We have overexpressed the fungal enzyme (MAO-N) in Escherichia coli and isolated it in pure form . Since several of the observations of previous workers on MAO-N could not be reproduced, we have reexamined its substrate specificity, interaction with reversible and irreversible inhibitors and other catalytic and molecular properties . MAO-N has a considerably higher turnover number on many aliphatic and aromatic amines than either form of the mammalian enzyme . Some aspects of the substrate specificity resemble those of MAO B, while others are similar to MAO A, including biphasic kinetics in double reciprocal plots . Contrary to a previous report {Schilling, B . & Lerch, K . (1995a) Biochim . Biophys . Acta 1243, 529-537}, however, the fungal enzyme does not oxidize serotonin, norepinephrine, dopamine or other biogenic amines . MAO-N is irreversibly inhibited by stoichiometric amounts of both (-)deprenyl and clorgyline in a mechanism-based reaction, forming flavocyanine adducts with N5 of the FAD, like the mammalian enzymes, but inactivation is much faster with clorgyline than deprenyl, suggesting a closer resemblance to MAO A than B . The dissociation constants for a large number of reversible competitive inhibitors have been determined for MAO-N and comparison with similar values for MAO A and B again pointed to a greater similarity to the former than the latter.

Eur J Biochem, 1998 Apr 1, 253(1), 132 - 8
A fully active FAD-containing precursor remains folded up to its translocation across the chloroplast membranes; Ottado J et al.; The cytosolic and two recombinant precursors, containing 10 and 30 amino acid spacers between the transit peptide and the mature region of the chloroplast flavoprotein ferredoxin-NADP+ reductase (FNR), were expressed in Escherichia coli cells . These proteins were purified rendering fully active precursors that contained bound FAD . Neither the transit peptide nor the spacers affected the formation of the tightly folded enzyme structure . Protease treatment of the folded precursors resulted in a rapid removal of the transit sequence, rendering an enzymatically active resistant core, even at high protease concentration . All three preproteins could be efficiently imported by isolated pea chloroplasts . Addition of the enzyme substrate NADP+ to the import medium slightly decreased the polypeptide translocation . The precursor bound to isolated chloroplasts in the presence or absence of leaf extracts was as resistant to proteolysis as the folded precursor in solution . In contrast, the FNR precursor unfolded by urea was rapidly digested even at the lowest protease concentration . Together, our results indicate that precursor unfolding may take place during translocation but not during binding to chloroplast envelopes or by interaction with leaf extract soluble factors, and that this process is independent of the distance between the transit peptide and the folded mature region of the protein.

Eur J Biochem, 1998 Apr 1, 253(1), 57 - 66
Ca2+ and Zn2+ bind to different sites and induce different conformational changes in human calcyclin; Kordowska J et al.; Calcyclin (CaCY) is a member of the S100 subfamily of helix-loop-helix (EF-hand) calcium-binding proteins . Human CaCY was overexpressed in Escherichia coli and purified with an overall yield of 40 mg/l culture . Ca2+ and Zn2+ binding properties of CaCY were examined with respect to the oxidation state of the single Cys residue at position 3 . CaCY with the SH group either reduced, blocked or oxidized stays as a dimer as shown by analytical ultracentrifugation . Upon binding of Ca2+, CaCY exhibits 30% enhancement of the Tyr fluorescence, the apparent binding constant (Ka) being 2.8-5.8x10(4) M(-1) . Oxidized CaCY binds Ca2+ approximately twice as weakly than its reduced form . The affinity for Ca2+ is increased in the presence of caldesmon, which could be a potential target molecule . Fully reduced CaCY binds Zn2+ with an affinity of at least 1.0x10(7) M(-1) . As compared to Ca2+, Zn2+ binding results in a three times greater enhancement of the Tyr fluorescence . Saturation occurs at a Zn2+/CaCY ratio of 2:1 . The reactivity of Cys3 is reduced by Zn2+ binding, although oxidized CaCY still binds Zn2+ . On the basis of the effects of thiol-directed labels on the affinities for Ca2+ and Zn2+, the fluorescence changes accompanying the binding, and the CaCY reactivity with a hydrophobic probe, it was concluded that the two cations bind to CaCY at different sites: Ca2+ binds probably at the EF-hand type sites, whereas binding of at least one Zn2+ ion involves the Cys residue, and results in a different structural change.

Biochem J, 1998 May 15, 332 ( Pt 1), 75 - 80
An esterase from Escherichia coli with a sequence similarity to hormone-sensitive lipase; Kanaya S et al.; An esterase from Escherichia coli that is a member of the hormone-sensitive lipase (HSL) family was overproduced, purified and characterized . It is encoded by the ybaC gene and composed of 319 amino acid residues with an Mr of 36038 . The enzymic activity was determined by using various p-nitrophenyl esters of fatty acids as a substrate at 25 degreesC and pH 7.1 . The enzyme showed hydrolytic activity towards substrates with an acyl chain length of less than 8, whereas it showed little hydrolytic activity towards those with an acyl chain length of more than 10 . In addition, it showed little hydrolytic activity towards trioleoylglycerol and cholesterol oleate . Determination of the kinetic parameters for the hydrolyses of the substrates from C2 to C8 indicates that C4 and C5 substrates are the most preferred . Close agreement between the Mr determined by SDS/PAGE (37000) and column chromatography (38000) suggests that the enzyme exists in a monomeric form . It is an acidic protein with a pI value of 4.1 . The far-UV CD spectrum suggests that its helical content is 26.1% . Comparison of the amino acid sequence of this enzyme with those involved in the HSL family allows us to propose that Ser165, Asp262 and His292 constitute the catalytic triad of E . coli esterase.

Biochem J, 1998 May 15, 332 ( Pt 1), 35 - 41
Hydroxylated naphthoquinones as substrates for Escherichia coli anaerobic reductases; Rothery RA et al.; We have used two hydroxylated naphthoquinol menaquinol analogues, reduced plumbagin (PBH2, 5-hydroxy-2-methyl-1,4-naphthoquinol) and reduced lapachol {LPCH2, 2-hydroxy-3-(3-methyl-2-butenyl)-1, 4-naphthoquinol}, as substrates for Escherichia coli anaerobic reductases . These compounds have optical, solubility and redox properties that make them suitable for use in studies of the enzymology of menaquinol oxidation . Oxidized plumbagin and oxidized lapachol have well resolved absorbances at 419 nm (epsilon=3.95 mM-1 . cm-1) and 481 nm (epsilon=2.66 mM-1.cm-1) respectively (in Mops/KOH buffer, pH 7.0) . PBH2 is a good substrate for nitrate reductase A (Km=282+/-28 microM, kcat=120+/-6 s-1) and fumarate reductase (Km=155+/-24 microM, kcat=30+/-2 s-1), but not for DMSO reductase . LPCH2 is a good substrate for nitrate reductase A (Km=57+/-35 microM, kcat=68+/-13 s-1), fumarate reductase (Km=85+/-27 microM, kcat=74+/-6 s-1) and DMSO reductase (Km=238+/-30 microM, kcat=191+/-21 s-1) . The sensitivity of enzymic LPCH2 and PBH2 oxidation to 2-n-heptyl-4-hydroxyquinoline N-oxide inhibition is consistent with their oxidation occurring at sites of physiological quinol binding.

J Neurosurg, 1998 May, 88(5), 870 - 3
Selective intraarterial gene delivery into a canine meningioma; Chauvet AE et al.; OBJECT: The goal of this study was to evaluate gene delivery to a benign brain tumor . METHODS: A recombinant adenovirus vector bearing the Escherichia coli beta-galactosidase reporter gene was selectively injected into the vascular supply of a spontaneously occurring canine olfactory groove meningioma . The tumor and a small amount of peritumoral brain tissue were removed 5 days after viral injection and stained with X-Gal to assess gene delivery . The authors noted significant beta-galactosidase gene expression by the tumor, but not by surrounding brain tissue . No obvious viral-related cytotoxicity was noted . CONCLUSIONS: The authors found that meningiomas can be successfully transduced by adenovirus vectors by using endovascular techniques.

Am J Physiol, 1998 Apr, 274(4 Pt 1), G653 - 61
In vivo endotoxin enhances biliary ethanol-dependent free radical generation; Chamulitrat W et al.; Endotoxemia is associated with alcoholic liver diseases; however, the effect of endotoxin on the oxidation of ethanol is not known . We tested the hypothesis that endotoxin treatment enhances hepatic ethanol radical production . The generation of free radicals by the liver was studied with spin-trapping technique utilizing the primary trap ethanol (0.8 g/kg) and the secondary trap alpha-(4-pyridyl-1-oxide)-N-t-butylnitrone (4-POBN; 500 mg/kg) . Electron paramagnetic resonance (EPR) spectra of bile showed six-line signals, which were dependent on ethanol, indicating the trapping of ethanol-dependent radicals . Intravenous injections of Escherichia coli lipopolysaccharide (0.5 mg/kg) 0.5 h before 4-POBN plus ethanol treatment caused threefold increases of biliary radical adducts . EPR analyses of bile from {1-13C}ethanol-treated endotoxic rats showed the presence of species attributable to alpha-hydroxyethyl adduct, carbon-centered adducts, and ascorbate radical . The generation of endotoxin-induced increases of ethanol-dependent radicals was suppressed by 50% on GdCl3 (20 mg/kg i.v.) or desferrioxamine mesylate (1 g/kg i.p.) treatment . Our data show that in vivo endotoxin increases biliary ethanol-dependent free radical formation and that these processes are modulated by Kupffer cell activation and catalytic metals.

J Cell Sci, 1998 Mar, 111 ( Pt 6), 749 - 57
Dissection of the translocation and chaperoning functions of yeast BiP/Kar2p in vivo; Holkeri H et al.; We used the rat nerve growth factor receptor ectodomain (NGFRe) and Escherichia coli ss-lactamase to dissect the functions of Saccharomyces cerevisiae BiP/Kar2p in vivo . Both were fused to the Hsp150Delta-polypeptide, which promotes proper folding of heterologous proteins which otherwise are misfolded in the yeast ER . Hsp150Delta-NGFRe and Hsp150Delta-beta-lactamase acquired disulfides and were properly folded and ONcreted to the culture medium . When disulfide formation was prevented by incubating cells with dithiothreitol (DTT), Hsp150Delta-NGFRe remained in the endoplasmic reticulum (ER) . The occupancy of an otherwise partially used N-glycosylation site of reduced NGFRe was complete suggesting that, normally, folding and disulfide formation occurred as rapidly as N-glycosylation . Removal of DTT resulted in remarkably rapid disulfide formation and secretion, suggesting only mild conformational distortion of reduced NGFRe . In contrast, reduced Hsp150(Delta)-ss-lactamase was severely misfolded and attained a secretion competent conformation more slowly after reoxidation . When kar2-159 cells were incubated at permissive temperature 24 degrees C with DTT, the reporter proteins were retained in the ER . After shift of the cells to 34 degrees C to inactivate BiP/Kar2p irreversibly, and subsequent removal of DTT, most pre-accumulated Hsp150Delta-NGFRe was rapidly secreted, whereas Hsp150Delta-beta-lactamase was secretion incompetent . Thus, Hsp150Delta-NGFRe did not require BiP/Kar2p for conformational maturation, though translocation was dependent on BiP/Kar2p . Apparently proteins differ in their post-translocational requirements for BiP/Kar2p, indicating that translocation and chaperoning are distinct functions.

Med Microbiol Immunol (Berl), 1998 Mar, 186(4), 201 - 7
HIV-1 Nef binding protein expressed on the surface of murine blood cells; Okada H et al.; The Nef protein of HIV-1 binds to and induces apoptotic cytolysis of a broad spectrum of uninfected blood cells of humans and mice independently of CD95 (Fas) . A 24-kDa glycoprotein responsible for Nef binding and the Nef-induced apoptosis has been identified on the surface of human CD4+ T cells . Using mouse monoclonal antibodies (mAbs) against the human Nef-binding protein and flow cytometry, we analyzed the expression of a corresponding protein on murine cells . The mAbs were shown to bind to the surface of various murine cell lines including T and B lymphocytes and macrophages, in a fashion similar to the binding by soluble Nef protein . The mAbs competed with the Nef protein in binding to the cell surfaces . Immunoprecipitation of cell membranes revealed a 25-kDa protein recognized by the mAbs . Treatment of the soluble Nef protein with anti-Nef (C terminus) mAb, but not anti-Nef (N terminus) mAb, deprived the Nef of the cell binding activity, indicating that binding site is located in the C-terminal domain . Cross-linking of the cell-bound mAbs with secondary antibodies induced apoptotic cytolysis, which occurred independently of CD95 (Fas) . On the other hand, neither the mAbs nor the soluble Nef protein reacted with primary lymphocytes in a resting stage obtained from lymph nodes, thymus and spleen of 5-week-old mice . However, some of the cells, predominantly comprising CD4+ T cells, became positive for the both reactions after mitogenic stimulation with phytohemagglutinin, concanavalin A or lipopolysaccharide of Escherichia coli . These results suggest that the 25-kDa protein on murine cell surfaces corresponds to the human Nef binding protein and is responsible for the Nef-induced apoptosis, and that its expression on the cell surface depends on cellular activation.

J Struct Biol, 1998 Jan, 121(1), 76 - 80
Purification, crystallization, and preliminary X-ray crystallographic data analysis of small heat shock protein homolog from Methanococcus jannaschii, a hyperthermophile; Kim KK et al.; A gene coding for a small heat shock protein homolog from the hyperthermophilic methanogenic Archaeon Methanococcus jannaschii was cloned . This gene was overexpressed in Escherichia coli harboring rare codon tRNAs and its protein purified and crystallized . Crystals displayed the space group R3 with unit cell dimensions a = b = 171.46 A and c = 102.13 A in a hexagonal axis setting . These crystals grew in one week and diffracted to 3.2 A resolution . The presence of eight molecules in the asymmetric unit gives a Vm value of 2.2 A3/Da and a solvent content of 44% by volume . The 24-molecule complex is generated from a subunit by a combination of crystallographic threefold symmetry and three types of noncrystallographic symmetries (a two-, a three-, and a fourfold).

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