Escherichia coli

Infect Immun, 1996 Jun, 64(6), 2101 - 5
Hypothermic response of mice to ornithine-containing lipids and to endotoxin; Kawai Y et al.; The hypothermic response of mice to ornithine-containing lipids (Orn-Ls) of the form alpha-N-(3-acyloxyacyl)-ornithine and to endotoxin (Escherichia coli 0111:B4 lipopolysaccharide {LPS}) was studied . After the administration of Orn-L or LPS to C3H/HeSlc mice, body temperature decreases were determined at 30-min intervals by inserting a thermistor into the rectum of each mouse . When Orn-L (750 microg) or LPS (70 microg) was injected into the mice, body temperature decreases of 0.8 and 2.0 degrees C, respectively, occurred 1.8 to 2.0 h later . These body temperature decreases were completely suppressed by the preadministration of indomethacin . When anti-tumor necrosis factor alpha (TNF-alpha) antibody was administered before the administration of Orn-L or LPS, only the body temperature decrease by LPS was suppressed . The body temperature decrease by Orn-L was suppressed by anti-interleukin-1beta (IL-1beta) antibody preadministration . Next, in order to study IL-1beta and TNF-alpha mRNA expression in macrophages, peritoneal macrophages were collected 40 min after the administration of Orn-L or LPS to mice . The expression of IL-1beta mRNA by stimulation with Orn-L was as strong as that by stimulation with LPS, but the expression of TNF-alpha mRNA by stimulation with Orn-L was very weak . Our previous studies of in vitro macrophage activation by Orn-L proved that strong induction of IL-1 and prostaglandin E2 generation by Orn-L occurred (Y . Kawai and K . Akagawa, Infect . Immun . 57:2086-2091, 1989) . From these experiments, the weak body temperature decrease in mice caused by Orn-L was found to be mediated by cytokines different from those which mediate the strong body temperature decrease caused by LPS . Namely, it was caused by prostaglandin E2 being mediated by IL-1 but not by TNF-alpha.

Infect Immun, 1996 Jun, 64(6), 2047 - 55
Nucleotide sequence and expression of the gene encoding the major 25-kilodalton outer membrane protein of Brucella ovis: Evidence for antigenic shift, compared with other Brucella species, due to a deletion in the gene; Cloeckaert A et al.; The nucleotide sequences encoding the major 25-kDa outer membrane protein (OMP) (omp25 genes) of Brucella ovis 63/290, Brucella melitensis 16M, Brucella suis 1330, Brucella canis RM6/66, and Brucella neotomae 5K33 (all reference strains) were determined and compared with that of Brucella abortus 544 (P . de Wergifosse, P . Lintermans, J . N . Limet, and A . Cloeckaert, J . Bacteriol . 177:1911-1914, 1995) . The major difference found was between the omp25 gene of B . ovis and those of the other Brucella species; the B . ovis gene had a 36-bp deletion located at the 3' end of the gene . The corresponding regions of other Brucella species contain two 8-bp direct repeats and two 4-bp inverted repeats, which could have been involved in the genesis of the deletion . The mechanism responsible for the genesis of the deletion appears to be related to the "slipped mispairing" mechanism described in the literature . Expression of the 25-kDa outer membrane protein (Omp25) in Brucella spp . or expression from the cloned omp25 gene in Escherichia coli cells was studied with a panel of anti-Omp25 monoclonal antibodies (MAbs) . As shown by enzyme-linked immunosorbent assay (ELISA) and immunoelectron microscopy, Omp25 was exported to the outer membrane in E . coli expressing either the truncated omp25 gene of B . ovis or the entire omp25 genes of the other Brucella species . Size and antigenic shifts due to the 36-bp deletion were demonstrated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and immunoblotting and by the differences in binding patterns in ELISA of the anti-Omp25 MAbs at the cell surface of E . coli cells harboring the appropriate gene and of cells of B . ovis and other Brucella species . In particular, MAbs directed against discontinuous epitopes of the entire Omp25 showed the absence of, or a significant reduction in, antibody reactivity with the B . ovis truncated Omp25 . The results indicated that, as defined by the MAbs, exported Omp25 probably presents similar topologies in the outer membranes of E . coli and Brucella spp . and that the short deletion found in the omp25 gene of B . ovis has important consequences for the expression of surface B-cell epitopes which should be considered for the development of vaccines against B . ovis infection.

Infect Immun, 1996 Jun, 64(6), 1918 - 28
Pathogenicity of the diffusely adhering strain Escherichia coli C1845: F1845 adhesin-decay accelerating factor interaction, brush border microvillus injury, and actin disassembly in cultured human intestinal epithelial cells; Bernet-Camard MF et al.; The diffusely adhering Escherichia coli strain C1845 harboring the fimbrial F1845 adhesin can infect cultured human intestinal epithelial cells . The mechanism by which E . coli C1845 induces diarrheal illness remains unknown . This study investigated the injuries of cultured human intestinal cells promoted by E . coli C1845 . Membrane-associated decay accelerating factor was identified as the intestinal receptor for the F1845 fimbrial adhesin of the E . coli C1845 strain by using purified F1845 adhesin, antibody directed against the F1845 adhesin, and monoclonal antibodies directed against the decay accelerating factor . Using monolayers of Caco-2 cells apically infected with E . coli C1845 and examined by scanning and transmission electron microscopy, we observed that strain C1845 induced injury to microvilli (MV) characterized by elongation and nucleation of the MV . We observed that infection of T84 and Caco-2 cells by E . coli C1845 was followed by disassembly of the actin network in the apical and basal cell domains . MV injury was differentiation related: E . coli C1845 promoted MV injury only when the cells were fully differentiated . The disassembly of the actin network occurred in poorly differentiated and fully differentiated Caco-2 cells but not in undifferentiated cells . Moreover, apical actin disassembly was observed in fully differentiated Caco-2 cells infected with the laboratory strain E . coli HB101(pSSS1) expressing the F1845 adhesin . In conclusion, E . coli C1845 promotes MV lesion in human epithelial intestinal cells, resulting from disassembly of the actin network.

FEMS Microbiol Lett, 1996 Jun 1, 139(2-3), 189 - 93
RcsC-mediated induction of colanic acid by secretion of streptokinase in Escherichia coli K-12; Lee SH et al.; The introduction of a plasmid containing skc (streptokinase-coding gene) fused with ompA signal sequence into Escherichia coli K-12 strains, rendered the bacteria mucoid . Measurement of the synthesis of beta-galactosidase from a cps-lacZ fusion (lacZ fusion to a gene necessary for capsule synthesis) showed that the mucoid phenotype was due to induction of the capsular polysaccharide colanic acid synthesis . The introduction of a plasmid carrying skc fused with malE (gene encoding maltose-binding protein) also induced cps-lacZ expression, but intracellular expression of streptokinase in E . coli did not . The cps expression by secretion of streptokinase was diminished to the basal level in a cps-lacZ strain carrying a rcsC mutation . These results show that the secretion of streptokinase in E . coli induces colanic acid synthesis through the RcsC-dependent pathway.

FEMS Microbiol Lett, 1996 Jun 1, 139(2-3), 175 - 80
Suppressor mutations in alpha-subunit of RNA polymerase for a mutant of the positive regulator, OmpR, in Escherichia coli; Kato N et al.; The OmpR protein is a positive regulator specific for the Escherichia coli ompF and ompC genes . This protein functions in a phosphorylation-dependent manner through a presumed interaction with RNA polymerase . We previously isolated OmpR mutants which were suggested to be defective in transcription activation, but not in DNA binding (the so-called positive control (PC) mutant) . In this study we isolated mutants of the alpha-subunit of RNA polymerase which can suppress one of the putative PC mutants of OmpR . A crucial amino acid substitution was identified as {V264G} in the alpha-subunit, which is located in the helix H1 of the C-terminal domain, which has been claimed, based on mutational and structural analyses, to be involved in the interaction with other positive regulators including the well-characterized cAMP receptor protein.

FEMS Microbiol Lett, 1996 Jun 1, 139(2-3), 143 - 8
Isolation, characterisation and expression of the bacterioferritin gene of Rhodobacter capsulatus; Penfold CN et al.; The nucleotide sequence of the Rhodobacter capsulatus bacterioferritin gene (bfr) was determined and found to encode a protein of 161 amino acids with a predicted molecular mass of 18,174 Da . The molecular mass of the purified protein was estimated to be 18,176 . +/ 0.80 Da by electrospray mass spectrometry . The bfr was introduced into an expression vector, and bacterioferritin was produced to a high level in Escherichia coli . The amino acids which are involved in haem ligation, and those provide ligands in the binuclear metal centre in bacterioferritin from E . coli are conversed in the R . capsulatus protein . The sequences of bacterioferritins, ferritin-like proteins, and proteins similar to Dps of E . coli are compared, and membership of the bacterioferritin family re-evaluated.

Plant Cell, 1996 Jun, 8(6), 1027 - 39
The same Arabidopsis gene encodes both cytosolic and mitochondrial alanyl-tRNA synthetases; Mireau H et al.; In plants, all aminoacyl-tRNA synthetases are nuclearly encoded, despite the fact that their activities are required in the three protein-synthesizing cell compartments (cytosol, mitochondria, and chloroplasts) . To investigate targeting of these enzymes, we cloned cDNAs encoding alanyl-tRNA synthetase (AlaRS) and the corresponding nuclear gene, ALATS, from Arabidopsis by using degenerate polymerase chain reaction primers based on highly conserved regions shared between known AlaRSs from other organisms . Analysis of the transcription of the gene showed the presence of two potential translation initiation codons in some ALATS mRNAs . Translation from the upstream AUG would generate an N-terminal extension with features characteristic of mitochondrial targeting peptides . A polyclonal antibody raised against part of the Arabidopsis AlaRS revealed that the Arabidopsis cytosolic and mitochondrial AlaRSs are immunologically similar, suggesting that both isoforms are encoded by the ALATS gene . In vitro experiments confirmed that two polypeptides can be translated from AlATS transcripts, with most ribosomes initiating on the downstream AUG to give the shorter polypeptide corresponding in size to the cytosolic enzyme . The ability of the presequence encoded between the two initiation codons to direct polypeptides to mitochondria was demonstrated by expression of fusion proteins in tobacco protoplasts and in yeast . We conclude that the ALATS gene encodes both the cytosolic and the mitochondrial forms of AlaRS, depending on which of the two AUG codons is used to initiate translation.

Int Immunol, 1996 Jun, 8(6), 829 - 36
Quaternary structure of a carrier protein influences antigenicity and immunogenicity of an inserted T cell determinant; Janssen R et al.; To study the influence of the quaternary structure of the outer membrane protein PhoE of Escherichia coli on the presentation of an inserted T cell epitope, an epitope comprising amino acid residues 72-85 of myelin basic protein (MBP) was inserted at different sites in PhoE . This sequence is the critical T cell epitope in experimental autoimmune encephalomyelitis (EAE) in Lewis rats . The antigenicity and immunogenicity of two different conformational forms of the chimeric PhoE constructs, i.e . the denatured monomeric form and the native trimeric form, were studied . It appeared that the monomeric form, but not the native trimeric form of such PhoE constructs induced proliferation of the MBP72-85-specific T cell line Z1a . This conformational discrepancy was independent of the site in PhoE in which the epitope was inserted . Immunization with the monomeric form of PhoE constructs resulted in the priming of MBP72-85-specific T cells . In contrast, the trimeric form of these constructs was much less efficient in priming such cells . The differences between the monomeric and trimeric forms were most apparent when induction of EAE was studied . The monomeric form was encephalitogenic while the trimeric form was not . Furthermore, the antigen fine specificity, Vbeta usage and encephalitogenicity of T cells triggered by immunization with a monomeric PhoE construct appeared to be the same as those of T cell line Z1a, which was obtained after immunization with MBP, indicating that similar cells are triggered by immunization with the epitope either in PhoE or in its native context.

Nucleic Acids Res, 1996 Jun 1, 24(11), 2104 - 11
Asymmetric mutation around the recombination break point of immunoglobulin class switch sequences on extrachromosomal substrates; Li J et al.; Junctions at class switch recombination sites in the genome are characterized by a unique sequence feature . Nucleotide substitutions and small deletions are common on either of the two sides of the switch junction, but not on both together . We have previously reported an extrachromosomal substrate assay system for analyzing the recombination of class switch sequences . Here we have sequenced nine junctions on each side of the break point and compared these to 17 recombination junctions of control substrates from the same cells . Five of the nine switch recombination junctions have nucleotide substitutions and deletions, with multiple nucleotide changes being more common . Furthermore, mutations were found only on a single side of the junction, just as for the recombination of switch sequences in the genome . In contrast, only one of 17 control substrate junctions had a mutation, and this was a single nucleotide insertion . This difference is highly significant (P < 0.00007) and indicates that the fundamental recombination mechanism is likely to be similar for switch sequences in the chromosome and on minichromosome substrates.

Nucleic Acids Res, 1996 Jun 1, 24(11), 2067 - 72
Hdf1, a yeast Ku-protein homologue, is involved in illegitimate recombination, but not in homologous recombination; Tsukamoto Y et al.; Hdf1 is the yeast homologue of the mammalian 70 kDa subunit of Ku-protein, which has DNA end-binding activity and is involved in DNA double-strand break repair and V(D)J recombination . To examine whether Hdf1 is involved in illegitimate recombination, we have measured the rate of deletion mutation caused by illegitimate recombination on a plasmid in an hdf1 disruptant . The hdf1 mutation reduced the rate of deletion formation by 20-fold, while it did not affect mitotic and meiotic homologous recombinations between two heteroalleles or homologous recombination between direct repeats . Hence Hdf1 participates in illegitimate recombination, but not in homologous recombination, in contrast to Rad52, Rad50, Mre11 and Xrs2, which are involved in both homologous and illegitimate recombination . The illegitimate recombination in the hdf1 disruptant took place between recombination sites that shared short regions of homology (1-4 bp), as was observed in the wild-type . Based on the DNA end-binding activity of Hdf1, we discuss models in which Hdf1 plays an important role in the late step of illegitimate recombination.

Nucleic Acids Res, 1996 Jun 1, 24(11), 2044 - 52
Non-hydrogen bonding 'terminator' nucleosides increase the 3'-end homogeneity of enzymatic RNA and DNA synthesis; Moran S et al.; We report the use of novel non-polar nucleoside analogues as terminators of enzymatic RNA and DNA synthesis . Standard 'runoff' RNA synthesis by T7 RNA polymerase gives RNA products which have ragged ends as a result of transcription which often extends beyond the end of the template DNA strand . Similarly, the Klenow fragment of Escherichia coli DNA polymerase I tends to run past the end of the template strand during DNA synthesis . We report here that certain non-hydrogen-bonding nucleoside analogues, when placed at the downstream 5'-end of a template DNA strand, cause the polymerases to stop more abruptly at the last coding nucleotide . This results in a considerably more homogeneous oligonucleotide being produced . Three novel nucleosides are tested as potential terminators: 4-methylindole beta-deoxynucleoside (M), 1-naphthyl alpha-deoxynucleoside (N) and 1-pyrenyl alpha-deoxynucleoside (P) . Comparison is made to an abasic nucleoside (phi) and to unterminated synthesis . Of these, M is found to be the most efficient at terminating transcription, and both P and M are highly effective at terminating DNA synthesis . It is also found that the ability of a nucleoside to stall synthesis when it is internally placed in the template strand is not necessarily a good predictor of terminating ability at the end of a template . Such terminator nucleosides may be useful in the preparative enzymatic synthesis of RNA and DNA, rendering purification simpler and lowering the cost of synthesis by preventing the uptake of potentially costly nucleotides into unwanted products.

Nucleic Acids Res, 1996 Jun 1, 24(11), 2022 - 35
The NMR structure of 31mer RNA domain of Escherichia coli RNase P RNA using its non-uniformly deuterium labelled counterpart {the 'NMR-window' concept}; Glemarec C et al.; The NMR structure of a 31mer RNA constituting a functionally important domain of the catalytic RNase P RNA from Escherichia coli is reported . Severe spectral overlaps of the proton resonances in the natural 31mer RNA (1) were successfully tackled by unique spectral simplifications found in the partially-deuterated 31 mer RNA analogue (2) incorporating deuterated cytidines {C5 (>95 atom % 2H), C2' (>97 atom % 2H), C3' (>97 atom % 2H), C4' (>65 atom % 2H) and C5' (>97 atom % 2H)} {for the 'NMR-window' concept see: Foldesi,A . et al . (1992) Tetrahedron, 48, 9033; Foldesi,A . et al . (1993) J . Biochem . Biophys . Methods, 26, 1; Yamakage,S.-I . et al . (1993) Nucleic Acids Res., 21, 5005; Agback,P . et al . (1994) Nucleic Acids Res., 22, 1404; Foldesi,A . et al . (1995) Tetrahedron, 51, 10065; Foldesi,A . et al . (1996) Nucleic Acids Res., 24, 1187-1194} . 175 resonances have been assigned out of total of 235 non-exchangeable proton resonances in (1) in an unprecedented manner in the absence of 13C and 15N labelling . 41 out of 175 assigned resonances could be accomplished with the help of the deuterated analogue (2) . The two stems in 31mer RNA adopt an A-type RNA conformation and the base-stacking continues from stem I into the beginning of the loop I . Long distance cross-strand NOEs showed a structured conformation at the junction between stem I and loop I . The loop I-stem II junction is less ordered and shows structural perturbation at and around the G11 -C22 base pair.

Leukemia, 1996 Jun, 10(6), 978 - 83
Expression of receptor protein tyrosine kinase tif is regulated during leukemia cell differentiation; Dai W et al.; tif is a recently cloned and characterized cDNA predicting a transmembrane protein with a putative tyrosine kinase structure in its cytoplasmic domain . By analysis of the purified tif cytoplasmic domain expressed in Escherichia coli, we have demonstrated that tif is an active protein tyrosine kinase capable of autophosphorylation on tyrosine residues and this phosphorylation is inhibited by a tyrosine-specific inhibitor genistein . Northern blot analyses of various leukemia cell lines have revealed that tif mRNA expression is primarily confined to those bearing erythroid and megakaryocytic phenotypes . Megakaryocytic differentiation of K562 and HEL cells induced by phorbol 12-myristate 13-acetate is accompanied by down-regulation of tif mRNA expression . In addition, treatment of K562 and HEL with hexamethylene bis-acetamide, but not with hemin, decreases the steady-state level of tif mRNA . These combined results suggest that the receptor tyrosine kinase tif is involved in hematopoietic development.

Lab Invest, 1996 Jun, 74(6), 1051 - 9
A single-chain Fv reactive with the Goodpasture antigen; Ross CN et al.; Goodpasture's disease is defined by the presence of autoantibodies to the glomerular basement membrane and characterized clinically by rapidly progressive glomerulonephritis and pulmonary hemorrhage . P1, a murine monoclonal antibody to the Goodpasture antigen (the noncollagenous domain of the alpha 3 chain of type IV collagen, alpha 3(IV)NC1), has been a valuable reagent in investigating the pathogenesis of this disorder . The purpose of this study was to generate and characterize a recombinant form of P1 as a single-chain Fv (scFv) . First strand cDNA was made from RNA extracted from the P1 hybridoma cell line, and DNA encoding the antibody light and heavy chain variable domains was amplified by polymerase chain reaction, using universal oligonucleotides . The purified products were ligated sequentially into an expression plasmid separated by a sequence encoding a 15 amino acid flexible oligopeptide linker . The resulting scFv was expressed in E . coli . Functional scFv, designated HBR-3, was obtained by denaturing and refolding the expressed product . HBR-3 was shown by ELISA, immunoblotting, and immunohistologic techniques, to have the same specificity for alpha 3(IV)NC1 as P1 and autoantibodies from patients with Goodpasture's disease . HBR-3 and P1 were shown to have similar affinity for their mutual ligand . On sections of normal human kidney, the scFv bound only to glomerular basement membrane and distal tubular basement membrane . It did not bind to the glomerular basement membrane of patients with Alport's syndrome, in whom the Goodpasture antigen is often not expressed in an antigenic form . We have, therefore, generated a scFv which reproduces the specific binding properties of the parent monoclonal antibody, P1 . The potential of HBR-3 as a diagnostic reagent in Alport's syndrome has been demonstrated . The development of this recombinant molecule should permit new approaches to the investigation of Goodpasture's disease.

J Immunol, 1996 Jun 1, 156(11), 4274 - 9
Cloning of the cDNA for human IFN-gamma-inducing factor, expression in Escherichia coli, and studies on the biologic activities of the protein; Ushio S et al.; We have recently reported that a novel molecule, murine IFN-gamma-inducing factor (IGIF) produced by mouse liver cells, possesses potent biologic activities, including the induction of IFN-gamma production by spleen cells and the enhancement of NK cell cytotoxicity . In this paper, we report on the isolation of human IGIF cDNA clones from normal human liver cDNA libraries using murine IGIF cDNA as a probe . The amino acid sequence deduced from the human cDNA clones indicated a 193-amino acid precursor peptide and revealed 65% homology with that of murine IGIF . The amino acid sequence of IGIF also included an IL-1 signature-like sequence . Subsequently, the cloned cDNA was expressed in Escherichia coli, and preliminary studies on the biologic activities of the recombinant protein were performed . The recombinant human IGIF induced IFN-gamma production by mitogen-stimulated PBMC and enhanced NK cell cytotoxicity, in a manner similar to murine IGIF . In addition, recombinant human IGIF also augmented granulocyte-macrophage-CSF production and decreased IL-10 production, but had no effect on IL-4 production by Con A-stimulated PBMC . Based on these pleiotropic effects of IGIF, we propose that this novel cytokine be designated as IL-18.

Gene, 1996 Jun 1, 171(2), 209 - 13
A recombination-efficient baculovirus vector for simultaneous expression of multiple genes; Chatterji U et al.; The baculovirus system is an extremely powerful tool for expression of heterologous genes in eukaryotic environment . A multiple expression vector, pBacUCmP3, was constructed which harbored two copies of the Autographa californica nuclear polyhedrosis virus very late gene promoter and the Drosophila melanogaster 70-kDa heat-shock protein (hsp70) promoter with downstream unique restriction sites for cloning of three independent foreign genes . Co-transfection of pBacUCmP3 with Bsu36I-linearized viral DNA yields recombinant progeny viruses at very high frequencies . The utility of this multiple expression transfer vector was demonstrated using three heterologous reporter genes encoding the beta-subunit of the human chorionic gonadotropin hormone, firefly luciferase and the bacterial beta-galactosidase (beta Gal) enzyme . The expression of reporter genes, monitored at various times post-infection, confirmed that while beta-Gal synthesis was under the transcriptional control of the hsp70 promoter, the beta hCG and Luc proteins were synthesized as a function of polyhedrin promoter activation profile . This vector will be useful for multiple synthesis of proteins at different time points.

Am J Respir Crit Care Med, 1996 Jun, 153(6 Pt 1), 1831 - 7
The effects of recombinant human thrombomodulin on endotoxin-induced multiple-system organ failure in rats; Hasegawa N et al.; Activation of the coagulation system is postulated to play an important role in the pathogenesis of endotoxin-induced tissue injury . Thrombomodulin (TM) is an endothelial cell membrane glycoprotein receptor for thrombin . Once bound to TM, thrombin loses its procoagulant activity, which results in decreased clotting . In addition, the binding of thrombin to TM activates the endogenous anticoagulant pathway through protein C . We studied the effect of recombinant human TM (rh-TM) on endotoxin-induced multiple-system organ failure (MSOF) in Sprague-Dawley rats weighing 400 to 450 g: 2 mg/kg of rh-TM was injected (T1/2 = 4.5 h) 30 min prior to intravenous injection of 20 mg/kg of Escherichia coli endotoxin . The study presented here consisted of three separate experiments . Experiment 1: 24-h survival study . Experiment 2: multiple-system organ microthrombi study in which 125I-human fibrinogen was injected 30 min prior to an endotoxin or saline injection and tissue microthrombi formation was assessed by measuring the percentage of organ radioactivity (lung, heart, liver, and kidney) against total injected radioactivity (microthrombi index, MI) 2.25 h after an endotoxin or saline injection . Experiment 3: endotoxin-induced MSOF study in which 125I-rat albumin was injected 5 h after an endotoxin or saline injection, and endotoxin-induced organ injury was evaluated by measuring tissue wet-to-dry ratios (W/D) and tissue-to-plasma 125I-rat albumin concentration ratios (T/P) 8 h after the endotoxin or saline injection . Blood contamination in samples from Experiments 2 and 3 was corrected by using 131I-rat albumin measurements . Pretreatment with rh-TM improved the survival from 12 h through 23 h as compared with that of the endotoxin control group (p < 0.05) . However, at 24 h, after essentially all injected rh-TM had been eliminated, there was no difference in survival . Significant reductions in MI, W/D, and T/P in the organs sampled were observed in the rh-TM pretreated groups (p < 0.05) . In conclusion, rh-TM improved short-term but not overall survival and decreased MSOF in endotoxemic rats.

J Surg Res, 1996 Jun, 63(1), 287 - 92
Inhibition of phosphatidylinositol-3'-kinase prevents induction of endotoxin tolerance in vitro; Bowling WM et al.; Previous studies have shown an increase in the expression of phosphatidylinositol-3'-kinase (PI-3'-K) in macrophages from endotoxin tolerant (ET) rats . This implicates PI-3'-K cell signaling in attenuated macrophage responsiveness to lipopolysaccharide (LPS) . These experiments examined the effects of selective pharmacologic inhibition of PI-3'-K in an in vitro model of ET . To induce ET, RAW 264.7 macrophages cultured in RPMI 1640 with 10% fetal calf serum were initially exposed to 10 ng/ml LPS (E . coli 0111:B4) for 19 hr . Non-tolerant cells received an equal volume of phosphate buffered saline . Some cultures were also incubated with the specific PI-3'-K inhibitor wortmannin (10 nM) during this tolerizing period . Cells were then washed and re-challenged with 100 ng/ml LPS for 24 hr . Next, macrophage tumor necrosis factor-alpha (TNF-alpha) and nitrite production were measured as indicators of ET induction . Macrophage TNF-alpha production decreased significantly while nitrite production increased significantly following ET induction . Specific inhibition of PI-3'-K prevented this decrease in TNF-alpha and increase in nitrite production in ET macrophages . Production of each mediator returned to levels not different than in non-tolerant macrophages . In this in vitro model of macrophage ET, pharmacologic inhibition of the PI-3'-K signaling pathway prevented the induction of LPS tolerance as measured by the production of two inflammatory mediators.

J Surg Res, 1996 Jun, 63(1), 248 - 55
A novel tumor-derived mediator that sensitizes cytokine-resistant tumors to tumor necrosis factor; Marvin MR et al.; Therapeutic successes following treatment of murine tumors with tumor necrosis factor-alpha (TNF) have not been easily applied to clinical oncology because the concentrations of TNF required in humans induces systemic toxicity . This has led us to identify mediators which could sensitize tumors to the effects of TNF, permitting administration of lower doses and possible realization of the therapeutic potential of this cytokine . Our study reports the ability of a novel cytokine, endothelial-monocyte-activating polypeptide II (EMAP II), to sensitize initially resistant murine and human tumors to TNF-induced regression employing a murine model . Recombinant (r) EMAP II was purified from Escherichia coli transformed with a plasmid expressing mature EMAP II . The B16 melanoma, raised in C57BL/6 mice, or a human fibrosarcoma (HT-1080), grown in immunocompromised mice, was injected intratumorally with either vehicle or rEMAP II/heat-treated EMAP II (50-100 micrograms) followed by systemic TNF/heat-treated TNF (5 micrograms) and assessed for tumor volume, hemorrhage, and histologic appearance . Both the B16 melanoma and the HT-1080 human fibrosarcoma underwent thrombohemorrhagic and acute inflammatory changes concomitant with regression or significantly slowed growth after administration of intratumor EMAP II followed by systemic TNF . Omission or inactivation of either cytokine abrogated this effect . These results demonstrate that local treatment of certain tumors with EMAP II results in enhanced susceptibility to TNF-mediated induction of thrombohemorrhage and regression.

J Surg Res, 1996 Jun, 63(1), 209 - 14
Discordant reprogramming of LPS-stimulated cytokine gene transcription and secretion by macrophages after LPS pretreatment; West MA et al.; Dysregulated macrophage (Mphi) cytokine release occurs during systemic inflammation and may predispose to organ failure . We showed that Mphis pretreated (PreRx) in vitro with low-dose LPSp are "reprogrammed" to release less TNF and more IL-1 in response to subsequent LPS activation (LPSa) . The effects of this LPSp "reprogramming" on Mphi cytokine gene transcription were investigated in the present study . Murine peritoneal exudate Mphis were cultured in vitro 48 hr, then PreRx 24 hr +/- 100 ng/ml of LPSp . Cultures were stimulated with 0-1000 ng/ml LPSa and 6-hr supernatant TNF and IL-1 were measured using specific bioassays . Cytokine gene transcription was estimated 6 hr after LPSa using RT-PCR . PreRx with LPSp inhibited TNF and augmented IL-1 release by LPSa . PreRx with LPSp significantly inhibited cytokine gene transcription; however, messages for both TNF and IL-1 were detectable after high-dose LPSa . Despite LPSp inhibition of IL-1 transcription by most LPSa concentrations, IL-1 protein was augmented by PreRx . High-dose LPSa can override LPSp reprogrammed inhibition of cytokine gene transcription, but altered TNF and IL-1 protein release after LPSp may be regulated posttranscriptionally.

J Surg Res, 1996 Jun, 63(1), 185 - 92
Effects of lipopolysaccharide on intestinal injury; potential role of nitric oxide and lipid peroxidation; Mercer DW et al.; Nitric oxide can react with superoxide anion to form peroxynitrite . The resultant free radical can be rapidly protonated to yield even more toxic substances such as hydroxyl radical and nitric dioxide . The generation of either of these free radical species can promote lipid peroxidation and subsequent tissue injury if they are formed in excessive amounts . During sepsis, both nitric oxide synthesis and peroxynitrite production are substantially enhanced in a variety of tissues, effects which favor the development of lipid peroxidation . Consequently, this study was undertaken in conscious rats, to ascertain what effect lipopolysaccharide (LPS) has on inducible nitric oxide synthase expression in the small intestine and to determine whether this is associated with lipid peroxidation or morphologic injury . When examined by Western immunoblot analysis, significantly more inducible nitric oxide synthase immunoreactivity was detected in the ileum than in the jejunum 5 hr after treatment with intraperitoneal LPS (1 and 20 mg/kg) . Further, using the thiobarbituric acid assay as an index of lipid peroxidation, it was demonstrated that significantly more thiobarbituric acid reactive substances were present in the ileal mucosa than in the jejunal mucosa after LPS (20 mg/kg) administration . However, LPS (20 mg/kg) resulted in morphologic damage to both segments of the intestinal epithelium . These data indicate that the gut is a target during sepsis and that regional differences exist within the small bowel with respect to induction of nitric oxide synthase and lipid peroxidation following LPS treatment . Thus, while induction of nitric oxide synthase during endotoxic shock may still represent a mechanism of local intestinal damage, it is not necessarily associated with enhanced lipid peroxidation.

J Surg Res, 1996 Jun, 63(1), 143 - 6
Thalidomide inhibits TNF response and increases survival following endotoxin injection in rats; Schmidt H et al.; Sepsis is a leading cause of death following major trauma and complicated abdominal surgery . Tumor necrosis factor-alpha (TNF) is believed to be a central mediator in the inflammatory response syndrome . Numerous methods of blunting the TNF response in sepsis have been attempted with suggestion of increased survival and decreased organ injury . Thalidomide, shown in vitro to selectively inhibit TNF production, has been used clinically in states of chronic TNF elevation with encouraging results . In this study, we examined the effect of thalidomide administration in a rat model of acute septic shock . Femoral artery cannulation was performed and baseline TNF measured . Dose response was determined by giving varying doses of thalidomide by gavage . Rats were injected intraarterially with endotoxin and serial samples drawn . TNF was measured by ELISA . For survival, thalidomide was given by gavage and endotoxin injected intraperitoneally . Serum TNF elevation occurred after endotoxin injection with peak levels at 90 min . Thalidomide treated rats had lower TNF levels at all time points (P = <0.01 at 90 and 120 min), with the inhibition being dose dependent . Survival in treated rats exceeded that of untreated rats (53% vs 19%, P = <0.05) at 48 and 72 hr . In conclusion, we found that thalidomide administration leads to increased survival following acute endotoxemia, which may be due to the observed TNF inhibition.

Anal Biochem, 1996 Jun 1, 237(2), 174 - 81
Analysis of ligase chain reaction products via matrix-assisted laser desorption/ionization time-of-flight-mass spectrometry; Jurinke C et al.; A rapid and accurate detection of ligation products generated in ligase chain reactions (LCR) by using matrix-assisted laser desorption/ionization time-of-flight-mass spectrometry (MALDI-TOF-MS) is reported . LCR with Pfu DNA ligase was performed with a wild-type template and a template carrying a single point mutation within the Escherichia coli lacI gene as a model system . Starting from about 1 fmol of template DNA the ligation product generated in the positive reactions was analyzed with HPLC and MALDI-TOF-MS, whereby the need of proper sample purification prior to mass spectrometric analysis was demonstrated . A purification procedure with a high potential for automation using streptavidin-coated magnetic particles and ultrafiltration was introduced . Plasmid DNA and short single-stranded oligonucleotides have been used as template . A point mutation could be discriminated from the wild-type template due to the absence or presence of ligation product . This approach allows the rapid-specific detection of template DNA in femtomole amounts and moreover can distinguish between sequence variations in DNA molecules down to point mutations without the need for labeling, gel electrophoresis, membrane transfer, or hybridization procedures.

Scand J Immunol, 1996 Jun, 43(6), 626 - 32
Immunophenotyping of human bone marrow-derived macrophages; Koller M et al.; In vitro cultured cells derived from human bone marrow stimulated with macrophage-colony stimulating factor (M-CSF) or granulocyte-macrophage-colony stimulating factor (GM-CSF) were investigated for their immunophenotypic surface characteristics by flow cytometry . Approximately 80-90% of the cells showed morphological and histochemical features of macrophages and bore CD11a, CD11b, CD11c, CD14, CD29, CD32, CD33, CD44, CD45, CD54, CD64, CD71, HLA-DR antigen . The expression of CD4, CD25 and CD45RO were detected only in low density . Bone marrow-derived macrophages were negative for CD8, CD45RA, CD16 and CD23 . Functionally, macrophages were able to phagocytose opsonized Escherichia coli, but no oxidative bursts were induced either by fmlp or by opsonized bacteria . Cell surface phenotyping revealed a CD pattern very similar to monocyte-derived phagocytes, and was partially distinct from resident stromal macrophages.

J Cell Biol, 1996 Jun, 133(5), 985 - 96
Evidence using a green fluorescent protein-glucocorticoid receptor chimera that the Ran/TC4 GTPase mediates an essential function independent of nuclear protein import; Carey KL et al.; The Ran/TC4 GTPase is required for the nuclear accumulation of artificial karyophiles in permeabilized cell assays . To investigate Ran function in a physiologically intact setting using mammalian cells, we examined the effects of several Ran mutants on cell growth and on the nuclear translocation of a glucocorticoid receptor-green fluorescent protein fusion (GR-GFP) . Glucocorticoid receptor is cytosolic in the absence of ligand, but translocates to the nucleus on binding the agonist dexamethasone . After transfection into baby hamster kidney cells (BHK21), GR-GFP was detectable in living cells by direct fluorescence microscopy . Addition of dexamethasone caused a rapid translocation of the chimeric protein from the cytosol into the nucleus (t1/2 approximately 5 min) . Cotransfection with epitope-tagged, wild-type Ran led to expression of HA1-Ran that was approximately 1.6-fold higher than the level of the endogenous protein, but it had no deleterious effect on nuclear import of the GR-GFP . However, expression of the Ran mutants G19V, T24N, or a COOH-terminal deletion (delta C) mutant dramatically reduced the accumulation of GR-GFP in the nuclei . An L43E mutant of Ran was without significant effect on nuclear GR-GFP import . Identical results were obtained following micro-injection of recombinant Ran mutants into cells expressing GR-GFP . Significantly, all of the Ran mutants, including L43E, strongly inhibited cell growth . These results demonstrate the use of GR-GFP in real-time imaging of nuclear transport . They also show that multiple types of Ran mutant exert dominant effects on this process, and that normal Ran function requires cycling between the GTP- and GDP-bound states of the protein . Most importantly, the results with the L43E Ran mutant provide strong evidence that Ran mediates a function essential to cell viability that is independent of nuclear protein import.

J Bacteriol, 1996 Jun, 178(12), 3614 - 20
Polar allele duplication for transcriptional analysis of consecutive essential genes: application to a cluster of Escherichia coli fatty acid biosynthetic genes; Zhang Y et al.; The genes encoding acyl carrier protein and several key fatty acid biosynthetic enzymes are clustered at min 24 of the Escherichia coli chromosome . This cluster of genes is not transcribed as a classical operon, but rather multiple promoters are present and each gene is cotranscribed with at least one other gene . Transcripts specific for single genes ar also present . Transcription of acpP, the gene encoding acyl carrier protein, has been studied in detail . The acpP gene was shown to be transcribed from at least two different promoters by Northern (RNA) blot, primer extension, and deletion analyses, and the major promoter was mapped . We tested whether multiple promoters are necessary to produce acyl carrier protein by use of a new method whereby a transcriptional terminator was integrated into the chromosome upstream of the intact acpP gene . By use of this method (called polar allele duplication), we demonstrate that the promoter located immediately upstream of the coding sequence is sufficient for synthesis of this very abundant protein.

J Bacteriol, 1996 Jun, 178(12), 3550 - 6
In vivo stability of the Umu mutagenesis proteins: a major role for RecA; Frank EG et al.; The Escherichia coli Umu proteins play critical roles in damage-inducible SOS mutagenesis . To avoid any gratuitous mutagenesis, the activity of the Umu proteins is normally kept to a minimum by tight transcriptional and posttranslational regulation . We have, however, previously observed that compared with an isogenic recA+ strain, the steady-state levels of the Umu proteins are elevated in a recA730 background (R . Woodgate and D . G . Ennis, Mol . Gen . Genet . 229:10-16, 1991) . We have investigated this phenomenon further and find that another coprotease-constitutive (recA*) mutant, a recA432 strain, exhibits a similar phenotype . Analysis revealed that the increased steady-state levels of the Umu proteins in the recA* strains do indeed reflect an in vivo stabilization of the proteins . We have investigated the basis for the phenomenon and find that the mutant RecA* protein stabilizes the Umu proteins by not only converting the labile UmuD protein to the much more stable (and mutagenically active) UmuD' protein but by directly stabilizing UmuD' itself . In contrast, UmuC does not appear to be directly stabilized by RecA* but is instead dramatically stabilized in the presence of UmuD' . On the basis of these observations, we suggest that formation of a UmuD'C-RecA*-DNA quaternary complex protects the UmuD'C proteins from proteolytic degradation and as a consequence helps to promote the switch from error-free to error-prone mechanisms of DNA repair.

J Bacteriol, 1996 Jun, 178(12), 3457 - 61
The tolZ gene of Escherichia coli is identified as the ftsH gene; Qu JN et al.; Escherichia coli tolZ mutants are tolerant to colicins E2, E3, D, Ia, and Ib (Tol-), can grow on glucose but not on succinate or other nonfermentable carbon sources (Nfc-), and show temperature-sensitive growth (Ts) . A 1.8-kb DNA fragment that complemented the tolZ mutation was cloned . The DNA fragment was sequenced, and one open reading frame was found . This frame was identical to a part of the E . coli FtsH protein, an ATP-dependent metalloprotease that binds to the cytoplasmic membrane . The tolZ gene was located at 69 min on the E . coli genetic map, and the mutation was complemented by a plasmid carrying the ftsH gene, indicating that the tolZ gene is identical to the ftsH gene . The mutated tolZ21 gene was also cloned and sequenced and was found to have a single base change that caused an amino acid alteration of His-418 to Tyr in the FtsH protein . The tolZ21 mutant showed Hfl- (high frequency of lysogenization) and Std- (stop transfer-defective) pheno-types, both of which are due to a mutation in the ftsH (hflB) gene . However, the ftsH1, ftsH101, and hflB29 mutants did not show Tol- and Nfc phenotypes . The tolZ21 mutant was found to have a suppressor mutation, named sfhC, which allowed cells to survive . The sfhC mutation alone caused no Tol-, Nfc-, Ts, or Hfl- phenotypes in the tolZ21 mutant.

J Bacteriol, 1996 Jun, 178(12), 3426 - 33
Identification of major and minor chaperone proteins involved in the export of 987P fimbriae; Edwards RA et al.; The 987P fimbriae of Escherichia coli consist mainly of the major subunit, FasA, and two minor subunits, FasF and FasG . In addition to the previously characterized outer membrane or usher protein FasD, the FasB, FasC, and FasE proteins are required for fimbriation . To better understand the roles of these minor proteins, their genes were sequenced and the predicted polypeptides were shown to be most similar to periplasmic chaperone proteins of fimbrial systems . Western blot (immunoblot) analysis and immunoprecipitation of various fas mutants with specific antibody probes identified both the subcellular localizations and associations of these minor components . FasB was shown to be a periplasmic chaperone for the major fimbrial subunit, FasA . A novel periplasmic chaperone, FasC, which stabilizes and specifically interacts with the adhesin, FasG, was identified . FasE, a chaperone-like protein, is also located in the periplasm and is required for optimal export of FasG and possibly other subunits . The use of different chaperone proteins for various 987P subunits is a novel observation for fimbrial biogenesis in bacteria . Whether other fimbrial systems use a similar tactic remains to be discovered.

J Bacteriol, 1996 Jun, 178(12), 3418 - 25
The histone-like protein H-NS acts as a transcriptional repressor for expression of the anaerobic and growth phase activator AppY of Escherichia coli; Atlung T et al.; The transcriptional activator AppY is required for anaerobic and stationary-phase induction of the cyx-appA and hya operons of Escherichia coli, and expression of the appY gene itself is induced by these environmental conditions . The sequence of the appY gene and its promoter region is unusually AT rich . The nucleoid-associated protein H-NS has a DNA-binding specificity for intrinsically curved AT-rich DNA . Using a single-copy transcriptional appY-lacZ fusion, we have shown that appY gene expression is derepressed in hns mutants during aerobic exponential growth . In the hns mutant, growth phase and growth rate regulation under aerobic conditions was maintained, while ArcA-dependent anaerobic induction was greatly diminished . Judged by two-dimensional gel electrophoresis, the appY promoter fragment exhibits the features characteristic of curved DNA . Gel retardation assays showed that purified H-NS protein bound with high affinity to two different segments of the appY promoter region . The role of H-NS in the AppY regulatory cascade is discussed and compared with its function in the regulatory cascades of the AppY homologs CfaD and VirF.

J Bacteriol, 1996 Jun, 178(11), 3331 - 4
Cyclic AMP receptor protein positively controls gyrA transcription and alters DNA topology after nutritional upshift in Escherichia coli; Gomez-Gomez JM et al.; The expression of a transcriptional gyrA-lacZ gene fusion throughout the Escherichia coli growth cycle and the effect that mutation delta crp39 had on this expression were studied . The data obtained indicate that the expression of gyrA is growth phase dependent and under the positive control of the cyclic AMP receptor protein (CRP) . Complementation analysis of gyrA-lacZ expression with wild-type CRP or variant CRP pc (with a T-to-A mutation at position 158) in a CRP-deficient background suggests that this CRP action is mediated by a class I or class II CRP-dependent promoter(s) . Our results also indicate that CRP may be involved in the modulation of DNA topology in the transition from the lag period to the exponential phase of growth.

J Bacteriol, 1996 Jun, 178(11), 3252 - 9
Anaerobic biosynthesis of enterobactin Escherichia coli: regulation of entC gene expression and evidence against its involvement in menaquinone (vitamin K2) biosynthesis; Kwon O et al.; In Escherichia coli, isochorismate is a common precursor for the biosynthesis of the siderophore enterobactin and menaquinone (vitamin K2) . Isochorismate is formed by the shikimate pathway from chorismate by the enzyme isochorismate synthase encoded by the entC gene . Since enterobactin is involved in the aerobic assimilation of iron, and menaquinone is involved in anaerobic electron transport, we investigated the regulation of entC by iron and oxygen . An operon fusion between entC with its associated regulatory region and lacZ+ was constructed and introduced into the chromosome in a single copy . Expression of entC-lacZ was found to be regulated by the concentration of iron both aerobically and anaerobically . An established entC::kan mutant deficient in enterobactin biosynthesis was found to grow normally and synthesize wild-type levels of menaquinone under anaerobic conditions in iron-sufficient media . These results led to the demonstration of an alternate isochorismate synthase specifically involved in menaquinone synthesis encoded by the menF gene . Consistent with these findings, the entC+ strains were found to synthesize enterobactin anaerobically under iron-deficient conditions while the ent mutants failed to do so.

J Bacteriol, 1996 Jun, 178(11), 3201 - 6
mioC transcription, initiation of replication, and the eclipse in Escherichia coli; Bogan JA et al.; The potential role of mioC transcription as a negative regulator of initiation of chromosome replication in Escherichia coli was evaluated . When initiation was aligned by a shift of dnaC2(Ts) mutants to nonpermissive temperature (40 degrees C), mioC transcript levels measured at the 5' end or reading through oriC disappeared within one mass doubling . Upon return to permissive temperature (30 degrees C), the transcripts reappeared coordinately about 15 min after the first synchronized initiation and then declined sharply again 10 min later, just before the second initiation . Although these observations were consistent with the idea that mioC transcription might have to be terminated prior to initiation, it was found that the interval between initiations at permissive temperature, i.e., the eclipse period, was not influenced by the time required to shut down mioC transcription, since the eclipse was the same for chromosomes and minichromosomes which lacked mioC transcription . This finding did not, in itself, rule out the possibility that mioC transcription must be terminated prior to initiation of replication, since it might normally be shut off before initiation, and never be limiting, even during the eclipse . Therefore, experiments were performed to determine whether the continued presence of mioC transcription during the process of initiation altered the timing of initiation . It was found that minichromosomes possessing a deletion in the DnaA box upstream of the promoter transcribed mioC continuously and replicated with the same timing as those that either shut down expression prior to initiation or lacked expression entirely . It was further shown that mioC transcription was present throughout the induction of initiation by addition of chloramphenicol to a dnaA5(Ts) mutant growing at a semipermissive temperature . Thus, transcription through oriC emanating from the mioC gene promoter is normally inhibited prior to initiation of replication by the binding of DnaA protein, but replication can initiate with the proper timing even when transcription is not shut down; i.e., mioC does not serve as a negative regulator of initiation . It is proposed, however, that the reappearance and subsequent disappearance of mioC transcription during a 10-min interval at the end of the eclipse serves as an index of the minimum time required for the establishment of active protein-DNA complexes at the DnaA boxes in the fully methylated origin region of the chromosome . On this basis, the eclipse constitutes the time for methylation of the newly formed DNA strands (15 to 20 min at 30 degrees C) followed by the time for DnaA protein to bind and activate oriC for replication (10 min).

J Bacteriol, 1996 Jun, 178(11), 3194 - 200
Analysis of the traLEKBP sequence and the TraP protein from three F-like plasmids: F, R100-1 and ColB2; Anthony KG et al.; The sequence of a region of the F plasmid containing the traLEKBP genes involved in plasmid transfer was compared to the equivalent regions of two IncFII plasmids, R100-1 and ColB2 . The traLEK gene products of all three plasmids were virtually identical, with the most changes occurring in TraE . The TraB genes were also nearly identical except for an 11-codon extension at the 3' end of the R100-1 traB gene . The TraP protein of R100-l differed from those of F and ColB2 at its N terminus, while the ColB2 TraP protein contained a change of sequence in a predicted loop which was shown to be exposed in the periplasmic space by TnphoA mutagenesis . The effect of the altered TraP sequences was determined by complementing a traP mutant with clones expressing the traKBP genes of F, R100-1, and ColB2 . The traP mutation in pOX38 (pOX38-traP474), a derivative of F, was found to have little effect on pilus production, pilus retraction, and filamentous phage growth and only a moderate effect on transfer . The transfer ability of pOX38-traP474 was shown to be affected by mutations in the rfa (lipopolysaccharide) locus and in ompA in the recipient cell in a manner similar to that for the wild-type pOX38-Km plasmid itself and could be complemented with the traP analogs from R100-1 and ColB2 to give an F-like phenotype . Thus, the TraP protein appears to play a minor role in conjugation and may interact with TraB, which varies in sequence along with TraP, in order to stabilize the proposed transmembrane complex formed by the tra operon products.

J Bacteriol, 1996 Jun, 178(11), 3188 - 93
Isolation of cmr, a novel Escherichia coli chloramphenicol resistance gene encoding a putative efflux pump; Nilsen IW et al.; A novel gene designated cmr, which mapped to 18.8 min of the Escherichia coli K-12 genome, was shown to mediate resistance to chloramphenicol when it was expressed from a multicopy vector . The accumulation of chloramphenicol was significantly less in cells overexpressing cmr than in control cells harboring the vector without insert . After the addition of a proton motive force blocker, the level of accumulation of chloramphenicol in the resistant cells rapidly approached the levels found in sensitive cells carrying only the chromosomal cmr . Northern (RNA) blot analyses revealed that the cmr gene is expressed as a 1.3-kb transcript . This size corresponds very well with a predicted size of 1,293 nucleotides (nt) based on the mapping of the transcription initiation site to a G residue 24 nt upstream of the start codon and the presence of a putative rho-independent terminator sequence ending 36 nt downstream of the 1,233-nt open reading frame encoding the putative Cmr protein . The 411-residue-long derived amino acid sequence contains 12 putative transmembrane segments and displays significant sequence similarities to several known drug resistance protein sequences of the major facilitator family . We provide evidence strongly suggesting that the resistance mediated by Cmr involves active exclusion of chloramphenicol.

J Bacteriol, 1996 Jun, 178(11), 3091 - 8
Efficient homologous recombination in fast-growing and slow-growing mycobacteria; Baulard A et al.; Although homologous recombination is a major mechanism for DNA rearrangement in most living organisms, it has been difficult to detect in slowly growing mycobacteria by a classical suicide vector approach . Among the possible reasons for this are the low levels of transformation efficiency, the relatively high levels of illegitimate recombination, and the peculiar nature of the recA gene in slowly growing mycobacteria . In this report, we present an efficient homologous recombination system for these organisms based on the use of replicative plasmids which facilitates the detection of rare recombination events, because the proportions of recombined molecules increase over time . Intraplasmid homologous recombination in Mycobacterium smegmatis and Mycobacterium bovis BCG was easily selected by the reconstitution of an interrupted kanamycin resistance gene . Chromosomal integration via homologous recombination was selected by the expression of the kanamycin resistance gene under the control of a chromosomal promoter that was not present in the plasmid before recombination . This technique was termed STORE (for selection technique of recombination events) . All the clones selected by STORE had undergone homologous recombination, as evidenced by PCR analyses of the kanamycin-resistant clones . This technique should be applicable to all organisms for which homologous recombination has been difficult to achieve, provided the gene of interest is expressed.

J Bacteriol, 1996 Jun, 178(11), 3037 - 43
Redundant homosexual F transfer facilitates selection-induced reversion of plasmid mutations; Peters JE et al.; F plasmids use surface exclusion to prevent the redundant entry of additional F plasmids during active growth of the host cells . This mechanism is relaxed during stationary phase and nonlethal selections, allowing homosexual redundant plasmid transfer . Homosexual redundant transfer occurs in stationary-phase liquid cultures, within nongrowing populations on solid media, and on media lacking a carbon source . We examined the relationship between homosexual redundant transfer, which occurs between F+ hosts, and reversion of a plasmid-encoded lac mutant allele, lacI33omegalacZ . Sodium dodecyl sulfate (SDS) and mutations that prevent normal transfer to F- cells reduced redundant transfer and selection-induced reversion of the lacI33omegalacZ allele . A recA null mutation reduced redundant transfer and selection-induced reversion of the lacI33omegalacZ mutation . Conversely, a recD null mutation increased redundant transfer and selection-induced reversion of the lacI33omegalacZ allele . These results suggest an explanation for why SDS and these mutations affect reversion of the plasmid lacI33omegalacZ allele . However, a direct causal relationship between transfer and reversion remains to be established . These results suggest that Rec proteins play an active role in redundant transfer and/or that redundant transfer is regulated with the activation of recombination . Redundant homosexual plasmid transfer during a period of stress may represent a genetic response that facilitates evolution of plasmid-encoded functions through mutation, recombination, reassortment, and dissemination of genetic elements present in the populations.

Cancer Res, 1996 Jun 1, 56(11), 2535 - 8
Processing of a precursor of 72-kilodalton type IV collagenase/gelatinase A by a recombinant membrane-type 1 matrix metalloproteinase; Kinoshita T et al.; Membrane-type 1 matrix metalloproteinase that is associated with the proteolytic activation of progelatinase A was expressed as a recombinant fusion protein in Escherichia coli . The recombinant enzyme cleaved the propeptide sequence of gelatinase A in a sequence-specific manner . A mutant progelatinase A that has a substitution of Asn(66)-Leu to Ile-Val was not processed at all . The processing was blocked by tissue inhibitor of metalloproteinases-2 or BB-94 but not by tissue inhibitor of metalloproteinases-1 . Thus, membrane-type 1 matrix metalloproteinase is a direct activator of progelatinase A without requiring additional proteases.

Arch Biochem Biophys, 1996 Jun 1, 330(1), 199 - 208
The omega-hydroxlyation of lauric acid: oxidation of 12-hydroxlauric acid to dodecanedioic acid by a purified recombinant fusion protein containing P450 4A1 and NADPH-P450 reductase; Shet M et al.; The recombinant fusion protein rF450{mRat4Al/mRatOR}L1, containing the heme domain of P450 4A1 and the flavin domains of NADPH-P450 reductase, when incubated with dilaurylphosphatidylcholine (DLPC), Chaps, cytochrome b5, and a 20-fold excess of purified NADPH-P450 reductase, catalyzes the omega- oxidation of lauric acid at a rate of about 300 nmol/min/nmol P450 . This is the first report of a mammalian P450 enzyme with such a high turnover number . The resultant 12-hydroxydodecanoic acid {12-hydroxylauric acid (12-OH LA)} is further oxidized by the P450 oxygenase reaction to dodecanedioic acid (decane-1,10-dicarboxylic acid) via 12,12-dihydroxydodecanoic acid . Spectral binding studies show that 12-OH LA inhibits the binding of lauric acid to the active site of P450 with a Ki of about 1.9 microM . The construction and expression of recombinant P450 4A1 containing a six-member polyhistidine domain at the carboxy-terminus of the protein is described . Reconstitution experiments with this purified recombinant P450 4A1, DLPC, Chaps, b5, and purified NADPH-P450 reductase show results similar to those obtained with the purified fusion protein, albeit at lower turnover rates . The requirement for normal-phase HPLC in resolving the metabolites formed during lauric acid metabolism is demonstrated.

Arch Biochem Biophys, 1996 Jun 1, 330(1), 174 - 80
Reversible unfolding of Escherichia coli alkaline phosphatase: active site can be reconstituted by a number of pathways; Sarkar SN et al.; Acid-induced and guanidine hydrochloride (GdnCl)-induced reversible unfolding of Escherichia coli alkaline phosphatase (AP) was characterized under equilibrium conditions . The protein was exposed to extreme conditions of pH 2.0 or 6 M GdnCl and was subsequently returned to normal conditions . Associated changes in the protein structure was probed by various spectroscopic methods . The changes in the functional properties were monitored by measuring enzymatic activity, capacity to renature spontaneously upon removal of the denaturant, and renaturation in presence of various site-specific and nonspecific effector molecules, in the absence and presence of beta-mercaptoethanol . Analysis of the fluorescence and CD spectra showed that the unfolding of the organized structures was much more extensive in 6 M GdnCl than at pH 2.0 . Intrachain S-S bonds in each unfolded state were accessible to reduction by beta-mercaptoethanol . The effectors Zn2+ and ATP induced renaturation of active site only under reducing conditions, whereas Triton X-100 or alpha-crystallin needed the presence of some organized structure . The reconstituted protein from each denatured state without or with an effector showed different CD spectra . It is concluded that the active site domain of AP could be reconstituted independently of other structural domains in different pathways.

Proc Soc Exp Biol Med, 1996 Jun, 212(2), 174 - 84
The regulation of phospholipase-A2 (PLA-2) by cytokines expressing hematopoietic growth-stimulating properties; DellaPuca R et al.; Various growth factors released by macrophages and other cell types modulate normal hematopoiesis . The physiological mechanisms whereby these molecules interact with specific target cells are ill defined . Eicosanoids, the products of fatty acid metabolism, are known to regulate cell proliferation and differentiation . The release of membrane-bound phospholipid by phospholipase-A2 (PLA-2) is the first critical step in the initiation of membrane remodeling and eventually eicosanoid synthesis . We report here data that demonstrates how various cytokines exhibit a marked hydrolytic activity mediated through PLA-2 against both {1-14C} oleic acid- and {1-14C} arachidonic acid-labeled Escherichia coli (micelle) substrates . PLA-2 extracts were prepared from neutrophils elicited by injecting rats ip with 8% glycogen . The rate of hydrolysis of free fatty acids from the phospholipid substrate was found to be linear, rapid, and pH dependent and was calculated to be 30 nmoles of phospholipid/hr/mg protein lysate . Cytokines (i.e., interleukin-1 {IL-1, human and murine recombinant, alpha}, mouse lung cell-derived colony-stimulating factor {L-CSF}, granulocyte-macrophage colony-stimulating factor {murine recombinant GM-CSF}, tumor necrosis factor {murine recombinant TNF-alpha}, and granulocyte colony-stimulating factor {human recombinant, G-CSF} all induced PLA-2 activity with the release of free fatty acids above basal levels . In contrast, lipopolysaccharide (LPS), interleukin-2, (IL-2, human recombinant), and macrophage colony-stimulating factor (M-CSF) did not significantly activate PLA-2 hydrolysis . The activation of this membrane-bound enzyme-substrate complex by these growth factors may serve as a mechanism whereby the appropriate target cells expressing receptors respond through either direct or secondary signals leading to the formation of free fatty acids with the eventual synthesis of prostanoid or lipoxygenase products, resulting in cellular proliferation and differentiation.

J Virol, 1996 Jun, 70(6), 4136 - 41
Cloning, expression and characterization of the proteinase from human herpesvirus 6; Tigue NJ et al.; After the U53 gene encoding the proteinase from human herpesvirus 6 (HHV-6) was sequenced, it was expressed in Escherichia coli, and the activity of the purified, recombinant HHV-6 proteinase was characterized quantitatively by using synthetic peptide substrates mimicking the release and maturation cleavage sites in the polyprotein precursors of HHV-6, human cytomegalovirus (CMV), murine CMV, and Epstein-Barr virus . Despite sharing 40% identity with other betaherpesvirus proteinases such as human CMV proteinase, the one-chain HHV-6 enzyme was distinguished from these two-chain proteinases by the absence of an internal autocatalytic cleavage site.

J Virol, 1996 Jun, 70(6), 4110 - 5
The equine herpesvirus 1 glycoprotein gp21/22a, the herpes simplex virus type 1 gM homolog, is involved in virus penetration and cell-to-cell spread of virions; Osterrieder N et al.; Experiments to analyze the function of the equine herpesvirus 1 (EHV-1) glycoprotein gM homolog were conducted . To this end, an Rk13 cell line (TCgM) that stably expressed EHV-1 gM was constructed . Proteins with apparent M(r)s of 46,000 to 48,000 and 50,000 to 55,000 were detected in TCgM cells with specific anti-gM antibodies, and the gM protein pattern was indistinguishable from that in cells infected with EHV-1 strain RacL11 . A viral mutant (L11deltagM) bearing an Escherichia coli lacZ gene inserted into the EHV-1 strain RacL11 gM gene (open reading frame 52) was purified, and cells infected with L11deltagM did not contain detectable gM . L11deltagM exhibited approximately 100-fold lower titers and a more than 2-fold reduction in plaque size relative to wild-type EHV-1 when grown and titrated on noncomplementing cells . Viral titers were reduced only 10-fold when L11deltagM was grown on the complementing cell line TCgM and titrated on noncomplementing cells . L11deltagM also exhibited slower penetration kinetics compared with those of the parental EHV-1 RacL11 . It is concluded that EHV-1 gM plays important roles in the penetration of virus into the target cell and in spread of EHV-1 from cell to cell.

J Virol, 1996 Jun, 70(6), 4045 - 52
Chimeric hepatitis B virus core particles as probes for studying peptide-integrin interactions; Chambers MA et al.; An RGD-containing epitope from the foot-and-mouth disease virus (FMDV) VP1 protein was inserted into the e1 loop of the hepatitis B virus core (HBc) protein . This chimeric protein was expressed at high levels in Escherichia coli and spontaneously assembled into virus-like particles which could be readily purified . These fusion particles elicited high levels of both enzyme-linked immunosorbent assay- and FMDV-neutralizing antibodies in guinea pigs . The chimeric particles bound specifically to cultured eukaryotic cells . Mutant particles carrying the tripeptide sequence RGE in place of RGD and the use of a competitive peptide, GRGDS, confirmed the critical involvement of the RGD sequence in this binding . The chimeric particles also bound to purified integrins, and inhibition by chain-specific anti-integrin monoclonal antibodies implicated alpha 5 beta 1 as a candidate cell receptor for both the chimeric particle and FMDV . Some serotypes of FMDV bound to beta 1 integrins in solid- phase assays, and the chimeric particles competed with FMDV for binding to susceptible eukaryotic cells . Thus, HBc particles may provide a simple, general system for exploring the interactions of specific peptide sequences with cellular receptors.

J Virol, 1996 Jun, 70(6), 3688 - 97
Epitopes and hemagglutination binding domain on subgenus B:2 adenovirus fibers; Mei YF et al.; The adenovirus fiber serves as a ligand between the virus and the host cell receptor and manifests hemagglutination (HA) activity and antigenic domains . We have screened both the antigenic and immunogenic epitopes on the adenovirus fibers of subgenus B:2 by using recombinant fiber proteins (rfibers) expressed in Escherichia coli, synthesized peptides (P1 to P8), and the corresponding antisera . The results indicated that P4 (amino acids {aa} 201 to 220), P5 (aa 231 to 250), and P7 (aa 275 to 295) presented both antigenic and immunogenic epitopes in adenovirus type 11 prototype (Ad11p), Ad34a, and Ad11a fibers . P6 (aa 251 to 270) presented both epitopes in Ad11a fiber but only an antigenic epitope in other fibers . The C-terminal 20 amino acids of the fiber, corresponding to P8, manifested an epitope of low-level immunogenicity . P5, localized at the N-terminal aa 231 to 250, displayed an epitope that reacted with fibers of all the members of subgenus B analyzed . The rfibers of Ad11p and Ad34a displayed HA activity with monkey erythrocytes, though those of Ad11a did not . Mutagenesis of the rfibers revealed that neither the fragment replacements, 11p20211a, llp26011a,and 11a28011p, nor the Ad11p rfiber with the substitutions of Tyr-260-->H (Tyr260H)and Arg279Q displayed HA activity . The Ad11a fiber knob was sensitive to proteolytic digestion, whereas that of Ad11p was resistant . The results demonstrated that the decisive HA binding domain was presented at aa 260 to 280 and was conformation dependent . Nearby amino acids, aa 283 and 284, may also affect the HA function.

J Virol, 1996 Jun, 70(6), 3517 - 27
Identification and characterization of the pseudorabies virus UL3.5 protein, which is involved in virus egress; Fuchs W et al.; Alphaherpesvirus genomes exhibit a generally collinear gene arrangement, and most of their genes are conserved among the different members of the subfamily . Among the exceptions is the UL3.5 gene of pseudorabies virus (PrV) for which positional homologs have been detected in the genomes of varicella-zoster virus, equine herpesvirus 1, and bovine herpesvirus 1 but not in the genomes of herpes simplex virus types 1 and 2 . To identify and characterize the predicted 224 amino acid UL3.5 protein of PrV, a rabbit antiserum was prepared against a UL3.5 fusion protein expressed in Escherichia coli . In Western blot (immunoblot) analyses the antiserum detected a 30-kDa protein in the cytoplasm of PrV infected cells which was absent from purified virions . For functional analysis, UL3.5-expressing cell lines were established and virus mutants were isolated after the rescue of defective, glycoprotein B-negative PrV by insertion of the complementing glycoprotein B-encoding gene of bovine herpesvirus 1 at two sites within the UL3.5 locus . A PrV mutant carrying the insertion at codon 159 and expressing a truncated UL3.5 protein was still capable of efficient productive replication in noncomplementing cells . In contrast, a PrV mutant carrying the insertion at codon 10 of the UL3.5 gene did not express detectable UL3.5 protein and exhibited a dramatic growth deficiency on non-complementing cells with regard to plaque formation and one-step replication . Electron microscopical studies showed an accumulation of unenveloped capsids in the vicinity of the Golgi apparatus . This defect could be compensated by propagation on complementing UL3.5-expressing cell lines . Our results thus demonstrate that the PrV UL3.5 gene encodes a nonstructural protein which plays an important role in virus replication, presumably during virus egress . The functionally relevant domains appear to be located within the N-terminal part of the UL3.5 protein which also comprises the region exhibiting the highest level of homology between the predicted UL3.5 homologous proteins of other alphaherpesviruses.

J Virol, 1996 Jun, 70(6), 3423 - 31
Plasmid-like replicative intermediates of the Epstein-Barr virus lytic origin of DNA replication; Pfuller R et al.; During the lytic phase of herpesviruses, intermediates of viral DNA replication are found as large concatemeric molecules in the infected cells . It is not known, however, what the early events in viral DNA replication that yield these concatemers are . In an attempt to identify these early steps of DNA replication, replicative intermediates derived from the lytic origin of Epstein-Barr virus, oriLyt, were analyzed . As shown by density shift experiments with bromodeoxyuridine, oriLyt replicated semiconservatively soon after induction of the lytic cycle and oriLyt-containing DNA is amplified to yield monomeric plasmid progeny DNA (besides multimeric forms and high-molecular-weight DNA) . A new class of plasmid progeny DNA which have far fewer negative supercoils than do plasmids extracted from uninduced cells is present only in cells undergoing the lytic cycle of Epstein-Barr virus . This finding is consistent with plasmid DNAs having fewer nucleosomes before extraction . The newly replicated plasmid DNAs are dependent on a functional oriLyt in cis and support an efficient marker transfer into Escherichia coli as monomeric plasmids . Multimeric forms of presumably circular progeny DNA of oriLyt, as well as detected recombination events, indicate that oriLyt-mediated DNA replication is biphasic: an early theta-like mode is followed by a complex pattern which could result from rolling-circle DNA replication.

J Infect Dis, 1996 Jun, 173(6), 1428 - 36
Oral immunization with the B subunit of the heat-labile enterotoxin of Escherichia coli induces early Th1 and late Th2 cytokine expression in Peyer's patches; Nakagawa I et al.; The B subunit of heat-labile toxin (LT-B) from enterotoxigenic Escherichia coli has been shown to be a powerful mucosal immunogen . Oral immunization of mice with LT-B revealed that BALB/c (H-2d) and C57BL/6 (H-2b) mice gave high serum IgG and mucosal IgA responses specific for LT-B . However, ALY (H-2b) mice lacking intestinal Peyer's patches (PP) did not respond to oral LT-B with either serum or mucosal antibodies . These results indicate that PP lymphocytes supported both systemic and mucosal immune responses when the antigen was administered orally . Reverse transcription polymerase chain reaction analyses revealed that PP CD4 T cells expressed early Th1-type (interferon-gamma and interleukin {IL}-2) and late Th2-type (IL-4, -5, and -6) cytokine mRNA . These results suggest that the systemic and mucosal antibody responses induced by LT-B are regulated by a cooperation of PP Th1 and Th2 cells.

J Allergy Clin Immunol, 1996 Jun, 97(6), 1297 - 303
Complementary DNA cloning of the predominant allergen of bovine dander: a new member in the lipocalin family; Mantyjarvi R et al.; BACKGROUND: A number of allergenic proteins in animal danders have been characterized at the molecular level, but little is known of their biologic functions . We have found that the prevalence of IgE antibodies among patients with cattle-associated asthma is highest against a dander protein referred to as BDA20 . OBJECTIVE: The study was performed to characterize the molecular structure of BDA20,* the predominant allergen in bovine dander . METHODS: Clones encoding allergens were identified and isolated from a complementary DNA library by immunoblotting and DNA hybridization and sequenced . Recombinant proteins were produced in Escherichia coli . Immunoreactivity of the recombinant proteins and amino acid sequences of peptides obtained from native BDA20 after Lys-C cleavage were used to identify clones coding for BDA20 . RESULTS: In this article we report the cDNA and amino acid sequences of BDA20 . Homology comparisons showed that BDA20 belongs to the family of lipocalins . CONCLUSIONS: The results link a dander allergen to a group of functionally important proteins . Lipocalins are present in various body fluids and secretions of several animal species in which they function as carriers of small hydrophobic molecules, such as retinoids and pheromones . If allergenicity proves to be a property shared by lipocalins, our results will have considerable implications for allergen research.

J Clin Invest, 1996 Jun 1, 97(11), 2585 - 92
Endotoxin and cytokines decrease serum levels and extra hepatic protein and mRNA levels of cholesteryl ester transfer protein in syrian hamsters; Hardardottir I et al.; Endotoxin alters the metabolism of lipoproteins, including that of high density lipoprotein (HDL) . Cholesteryl ester transfer protein (CETP) facilitates exchange of HDL cholesterol for very low density lipoprotein (VLDL) triglyceride, leading to catabolism of HDL . We investigated the effects of endotoxin and cytokines on CETP in Syrian hamsters . Endotoxin induced a rapid and progressive decrease in serum CETP levels, by 48 h CETP had decreased to < 20% of control levels . Endotoxin also decreased CETP mRNA and protein levels in adipose tissue, heart, and muscle, the tissues with highest levels of CETP mRNA, providing a plausible mechanism for the endotoxin-induced decrease in circulating CETP . Dexamethasone did not mimic the effects of endotoxin on CETP, but the combination of tumor necrosis factor and interleukin-1 did, indicating that these cytokines may in part mediate the effects of endotoxin on CETP . The endotoxin-induced decrease in CETP may help maintain HDL cholesterol levels during infection and inflammation when increased triglyceride levels could drive the exchange of HDL cholesteryl ester for VLDL triglyceride . Maintaining circulating HDL may be important because HDL protects against the toxic effects of endotoxin and provides cholesterol for peripheral cells involved in the immune response and tissue repair.

J Clin Invest, 1996 Jun 1, 97(11), 2440 - 51
Fibrin deposition in tissues from endotoxin-treated mice correlates with decreases in the expression of urokinase-type but not tissue-type plasminogen activator; Yamamoto K et al.; The primary hypothesis of this report is that the formation and subsequent removal of fibrin in specific tissues during pathologic processes reflects temporal changes in the local expression of key procoagulant and fibrinolytic genes . To begin to test this hypothesis, we have used quantitative PCR assays and in situ hybridization analysis to examine the effects of endotoxin on the expression of specific genes in murine tissues, and to relate these changes to fibrin deposition/dissolution using immunohistochemical approaches . Endotoxin caused large increases in plasminogen activator inhibitor-1 mRNA and modest increases in tissue factor mRNA in most tissues examined . However, fibrin was only detected in the kidneys and adrenals of endotoxin-treated mice, and it was transient . Unexpectedly, changes in urokinase-type plasminogen activator mRNA but not tissue-type plasminogen activator mRNA correlated with fibrin deposition/dissolution in these tissues . Pretreatment of mice with the fibrinolytic inhibitor epsilon-aminocaproic acid before endotoxin increased both the number of fibrin-positive tissues and the duration of fibrin deposition in the kidneys and adrenals . These results suggest that the absence of fibrin in some tissues reflects ongoing local fibrinolysis, and that increases in plasminogen activator inhibitory and tissue fac- tor gene expression and decreases in urokinase-type plasminogen activator expression are necessary for tissue-specific fibrin deposition . Changes in tissue-type plasminogen activator gene expression do not appear to be essential for fibrin deposition/dissolution in this murine model of sepsis.

Nat Struct Biol, 1996 Jun, 3(6), 532 - 8
Crystal structure of the Escherichia coli dUTPase in complex with a substrate analogue (dUDP); Larsson G et al.; We have determined the structure of the homotrimeric dUTPase from Escherichia coli, completed with an inhibitor and substrate analogue, dUDP . Three molecules of dUDP are found symmetrically bound per trimer, each in a shallow cleft between adjacent subunits, interacting with evolutionary conserved residues . The interactions of the uracil ring and the deoxypentose with the protein are consistent with the high specificity of the enzyme with respect to these groups . The positions of the two phosphate groups and adjacent water molecules are discussed in relation to the mechanism and kinetics of catalysis . The role that dUTPase plays in DNA metabolism makes the enzyme a potential target for chemotherapeutic drugs: the results presented here will aid in the design and development of inhibitory compounds.

Nat Struct Biol, 1996 Jun, 3(6), 522 - 31
Hydrogen bonding and equilibrium isotope enrichment in histidine-containing proteins; Bowers PM et al.; We have measured deuterium/hydrogen fractionation in three histidine-containing proteins, ecHPr, ecHPr mutant S31A, and bsHPr, and in random coil peptides using NMR and mass spectrometry . The amide protons of unstructured peptides exhibit equilibrium enrichment for deuterium, in agreement with previous studies . Enrichment for both protium and deuterium was observed in both HPrs, with fractionation factors ranging from 0.63 to 1.41 . Enrichment for protium was seen in alpha-helical secondary structure . 'Strong' HBs previously identified by mutagenesis and thermodynamic measurements are significantly enriched for protium . Sites of protium enrichment are conserved in a structural context across species lines, though ecHPr and bsHPr share only 30% sequence identity, suggesting that strong HBs are conserved and may play an important role in stabilizing the folded state.

Invest Ophthalmol Vis Sci, 1996 Jun, 37(7), 1302 - 10
Expression and release of tumor necrosis factor-alpha by explants of mouse cornea; Sekine-Okano M et al.; PURPOSE . To elucidate a possible target of immunosuppressive agents widely used in the treatment of corneal disorders, the authors determined whether corneal cells are capable of expressing and releasing tumor necrosis factor-alpha (TNF alpha) on lipopolysaccharide (LPS) stimulation, and they investigated whether TNF alpha production can be modulated by pharmacologic agents . METHODS . Trephined central corneas from C57BL/6 mice were kept in culture for 3 days . Release of TNF alpha after a 24-hour stimulation with LPS (1 microgram/ml) into the culture medium was determined both by bioassay and by enzyme-linked immunosorbent assay . Expression of TNF alpha mRNA after 6-hour stimulation was examined by polymerase chain reaction . Immunofluorescent staining on cryostat sections of cultured corneas was performed to localize TNF alpha in the tissue . Corneal explants were pretreated with immunosuppressive agents (prednisolone, budesonide, cyclosporin A) for 48 hours, followed by 6-or 24-hour stimulation with LPS in the continuous presence of the agents . RESULTS . Lipopolysaccharide stimulated TNF alpha release into the culture medium . The addition of budesonide (10(-7) M) or prednisolone (10(-6) M) significantly inhibited LPS-induced TNF alpha release, whereas cyclosporin A (10(-7) - 10(-5) M) had no marked effect . Levels of TNF alpha mRNA in corneal explants increased fivefold after stimulation with LPS . Immunohistochemical staining revealed that TNF alpha was expressed in the epithelial cells . Budesonide markedly decreased mRNA expression and abolished immunostaining of TNF alpha stimulated by LPS . CONCLUSIONS . TNF alpha is produced and released by the epithelial cells of mouse central cornea in response to LPS . Contrary to cyclosporin A, corticosteroids such as prednisolone and budesonide potently inhibit TNF alpha production.

Invest Ophthalmol Vis Sci, 1996 Jun, 37(7), 1282 - 93
Ocular adenovirus gene transfer varies in efficiency and inflammatory response; Borras T et al.; PURPOSE . To study the effects of adenoviral gene transfer to the tissues of the anterior segment in vitro by rat and monkey lens organ cultures and in vivo by single injection into the anterior chamber of rabbits . METHODS . In vitro, intact lens cultures were exposed to 1 to 4 x 10(8) pfu Av1LacZ4 and Av1Luc1 in TC199 medium containing no serum or growth factors . Av1LacZ4 and Av1Luc1 are replication-deficient adenovirus vectors, carrying the reporter genes Escherichia coli LacZ and firefly luciferase, respectively . In vivo, the anterior chambers of eight rabbits were injected once with 20 mumol Av1LacZ4 (8 x 10(8) pfu) and evaluated 48 hours after injection . Enzyme activity of the reporter genes was measured biochemically and histochemically . RESULTS . In organ cultures, adenovirus delivers reporter genes efficiently to the ciliary processes but penetrates poorly into the capsulated lenses . Viral receptors, however, are present in rat lens epithelium, as in primary trabecular meshwork and other lens cell lines . In vivo, gene transfer was evident in corneal endothelium, iris anterior surface, and trabecular meshwork . Presence of the virus did not affect lens transparency or provoke external discomfort signs . Infected corneal endothelial cells were swollen and partly detached; 3 of 8 infected eyes showed a severe inflammatory response in chamber angle, anterior uvea, and limbal conjunctiva . CONCLUSIONS . These findings reveal the distinct gene transfer potential of each of the tissues of the anterior segment and emphasize the need to address the inflammatory response to these first-generation adenoviral vectors.

J Med Microbiol, 1996 Jun, 44(6), 453 - 63
Development and evaluation of an ELISA to detect Escherichia coli K88 (F4) fimbrial antibody levels; Vazquez F et al.; An enzyme-linked immunosorbent assay (ELISA) to determine IgG antibody levels against K88 (F4) fimbrial antigen from porcine enterotoxigenic Escherichia coli (ETEC) has been developed . The ELISA method was checked with serum samples obtained from rabbits and pigs, and the parameters affecting the method were also analysed . ELISA plates were optimally coated with K88 antigen 0.5 microgram/ml for testing rabbit antiserum or with 1.25 microgram/ml for testing pig serum . Optimal concentrations of H202 (0.5%) and orthophenylene-diamine (OPD) (0.125%) were chosen when a 10-min incubation period was used . The expression of antibody levels as enzyme-immunosorbent units (EIU) significantly decreased the variability of results between duplicate plates, when compared with the expression of results as direct OD values . ELISA-K88 applied to a field study with serum samples from 141 vaccinated and 52 unvaccinated sows was shown to be useful in differentiating between samples from vaccinated and unvaccinated animals.

J Med Microbiol, 1996 Jun, 44(6), 438 - 43
Use of gene probes and adhesion tests to characterise Escherichia coli belonging to enteropathogenic serogroups isolated in the United Kingdom; Scotland SM et al.; Nine hundred and twenty-five Escherichia coli isolates from cases of diarrhoea in the United Kingdom and belonging to enteropathogenic E . coli (EPEC) O serogroups were examined for virulence properties . The tests included adhesion to HEp-2 cells, the fluorescence actin staining (FAS) test (which correlates with the ability to cause attaching and effacing lesions) and DNA hybridisations with probes to detect sequences for eaeA (E . coli attaching and effacing factor), EAF (EPEC adherence factor), verocytotoxins VT1 and VT2, enteroaggregative E . coli and diffusely adherent E . coli . The O serogroups examined were 18, 26, 44, 55, 86, 111, 114, 119, 125, 126, 127, 128 and 142 . Six hundred and sixty strains (71.4%) hybridised with at least one of the DNA probes . Over 80% of strains in O serogroups 26, 55, 119, 125, 127 and 142 and 41% of strains of serogroups 86, 111, 114, 126 and 128 hybridised with the eae probe and most showed localised attachment and were FAS-positive . However, <10% of these eae probe-positive strains hybridised with the EAF probe . Eighty-four of 232 strains in O serogroups 44, 86, 111, and 126 were enteroaggregative . VT genes were detected in 57 of 402 strains in O serogroups 26, 55, 111 and 128 . Identification of EPEC by serogrouping was shown to be an effective method of identifying strains with pathogenic potential, although the organisms were diverse in their properties.

J Biol Chem, 1996 May 31, 271(22), 12833 - 9
Reconstitution of the steroid receptor.hsp90 heterocomplex assembly system of rabbit reticulocyte lysate; Dittmar KD et al.; Rabbit reticulocyte lysate contains a multiprotein system that assembles steroid receptors into a heterocomplex with hsp90 . In the case of the glucocorticoid receptor (GR), the receptor must be bound to hsp90 to bind steroid, and assembly of the GR.hsp90 complex restores the hormone binding domain of the receptor to the steroid binding conformation . Using both direct assay of heterocomplex assembly by Western blotting and indirect assay of assembly by steroid binding, it has previously been determined that the assembly system is both ATP/Mg2+-dependent and K+-dependent and that hsp70 and an acidic 23-kDa protein (p23) are required to form a functional GR.hsp90 complex . It is also thought that a 60-kDa protein (p60) may be required for progesterone receptor.hsp90 heterocomplex assembly, but a complete heterocomplex assembly system has never been reconstituted from individual components . In this work, we separate the proteins of rabbit reticulocyte lysate into three fractions by DEAE chromatography and then reconstitute the GR.hsp90 heterocomplex assembly system in a manner that requires the presence of each fraction . Fraction A contains most of the hsp70 and all of the p60 in lysate, and elimination of p60 by immunoadsorption inactivates this fraction, with bioactivity being restored by the addition of bacterially expressed human p60 . The activity of fraction A is replaced by a combination of highly purified rabbit hsp70 and lysate from p60-expressing bacteria . Fraction B contains hsp90, and its activity is replaced by purified rabbit hsp90 . Fraction C contains p23, and its activity is replaced in the recombined system by highly purified bacterially expressed human p23 . A minimal GR.hsp90 heterocomplex assembly system was reconstituted with purified rabbit hsp70 and hsp90 and bacterially expressed human p23 and p60 . This reports the first reconstitution of this apparently ubiquitous protein folding/heterocomplex assembly system.

J Biol Chem, 1996 May 31, 271(22), 13013 - 7
The mechanism of velocity modulated allosteric regulation in D-3-phosphoglycerate dehydrogenase . Cross-linking adjacent regulatory domains with engineered disulfides mimics effector binding; Al-Rabiee R et al.; D-3-Phosphoglycerate dehydrogenase (PGDH) (EC 1.1.1.95) from Escherichia coli is an allosterically regulated enzyme of the Vmax type . It is a tetramer of identical subunits and each subunit is made up of three identifiable domains, the cofactor binding domain, the substrate binding domain, and the regulatory domain . Each subunit contacts two other subunits through adjacent cofactor binding domains and through adjacent regulatory domains . L-Serine, the physiological effector, inhibits catalytic activity by apparently tethering regulatory domains from adjacent subunits together through the formation of hydrogen bonds to each subunit . This investigation demonstrates that cross-linking adjacent regulatory domains with engineered disulfides produces catalytic inhibition in the absence of inhibitor in a manner similar to that produced by the inhibitor . The inhibition due to cross-linking can be completely reversed in a concentration dependent manner by dithiothreitol . The active mutant enzyme, containing the engineered cysteines in the reduced state, retains its ability to be inhibited by L-serine, although at a 100-fold higher concentration . Hill plots of the serine inhibition of mutant and native enzyme indicate that the number of interacting sites remains at 2 in the mutant enzyme . The reversible inhibition of enzyme activity that results from tethering adjacent regulatory domains with engineered disulfides suggests that these domains move in some manner relative to one another during the active to inhibited state transition . These observations support the model which predicts that catalytic activity is regulated by the movement of rigid domains about flexible hinges and that effector binding prevents this by locking the regulatory domains in a state that produces an open active site cleft.

J Biol Chem, 1996 May 31, 271(22), 12885 - 90
In vitro insertion and assembly of outer membrane protein PhoE of Escherichia coli K-12 into the outer membrane . Role of Triton X-100; de Cock H et al.; The assembly of the in vitro synthesized outer membrane protein PhoE into purified outer membranes was investigated . The assembly appeared to be strongly stimulated by the presence of low amounts of Triton X-100 (optimal 0.08%, w/v) . The role of Triton X-100 in the in vitro system was further examined . Pretreating outer membranes with Triton X-100 did not make the membranes competent for correct assembly, indicating that the detergent did not act on the membrane but at the protein level . PhoE became assembly-incompetent with a half-life of approximately 12 min and 90 s at 37 degrees C in the absence and presence, respectively, of 0.08% Triton X-100 . Apparently, Triton X-100 induces an assembly-competent state in the PhoE protein with a very short half-life . Furthermore, the efficiency of correct assembly of PhoE was greatly reduced when outer membranes of deep rough lipopolysaccharide mutants were used, indicating an important role of lipopolysaccharides in the assembly of the porin.

J Biol Chem, 1996 May 31, 271(22), 13077 - 87
Purification and properties of human cytosolic folylpoly-gamma-glutamate synthetase and organization, localization, and differential splicing of its gene; Chen L et al.; Human cytosolic folylpolyglutamate synthetase (FPGS) was expressed in Escherichia coli and purified to homogeneity . Tetrahydrofolate and dihydrofolate were the most effective substrates, while 5-substituted folates were poor substrates . Most pteroyldiglutamates were better substrates than monoglutamates . The human FPGS gene spans 12 kilobases and contains 15 exons and 14 introns . A single FPGS gene was located to chromosome region 9q34.1 . Four exon 1 variants were identified, each of which was spliced to exon 2 . The exon 1 variant corresponding to the isolated cDNA contains two ATG codons and multiple transcription start sites in this region generates mitochondrial and cytosolic FPGS (Freemantle, S . J., Taylor, S . M., Krystal, G., and Moran, R . G . (1995) J . Biol . Chem . 270, 9579-9584) . Exons 1B and 1C, generated by alternate splicing in intron 1, and exon 1A, which is 5' to exon 1 and may encode an additional mitochondrial isoform, are preceded by a number of potential promoter sites . Chinese hamster ovary cell transfectants expressing FPGS activity in the mitochondria contained normal mitochondrial and low cytosolic folylpolyglutamate pools . Mitochondrial FPGS activity is required for mitochondrial folate accumulation, while cytosolic FPGS activity is needed for establishment of normal cytosolic folate pools . The reconstructed FPGS gene restored normal cytosolic and mitochondrial folate metabolism in hamster cells.

J Biol Chem, 1996 May 31, 271(22), 12767 - 74
Cloning and expression of human G/T mismatch-specific thymine-DNA glycosylase; Neddermann P et al.; Hydrolytic deamination of 5-methylcytosine leads to the formation of G/T mismatches . We have shown previously that these G/T mispairs are corrected to G/C pairs by a mismatch-specific thymine-DNA glycosylase, TDG, which we subsequently purified from human cells . Here we describe the cloning of the human cDNA encoding TDG . We have identified two distinct cDNA species that differ by 100 nucleotides at the 3'-untranslated region . These cDNAs contain a 410-amino acid open reading frame that encodes a 46-kDa polypeptide . The G/T glycosylase, expressed both in vitro and in Escherichia coli, migrated in denaturing polyacrylamide gels with an apparent size of 60 kDa . The substrate specificity of the recombinant protein corresponded to that of the cellular enzyme, and polyclonal antisera raised against the recombinant protein neutralized both activities . We therefore conclude that the cDNA described below encodes human TDG . Data base searches identified a serendipitously cloned mouse cDNA sequence that could be shown to encode the murine TDG homologue . No common amino acid sequence motifs between the G/T-specific enzyme and other DNA glycosylases could be found, suggesting that TDG belongs to a new class of base-excision repair enzymes.

J Biol Chem, 1996 May 31, 271(22), 12716 - 23
Structural and functional analysis of the plasminogen activator inhibitor-1 binding motif in the somatomedin B domain of vitronectin; Deng G et al.; Plasminogen activator inhibitor 1 (PAI-1) binds to the somatomedin B (SMB) domain of vitronectin (VN), a domain present in at least seven other proteins . In this study, we investigate the PAI-1 binding activity of these SMB homologs and attempt to more specifically localize the PAI-1 binding site within this domain . SMBVN and several of its homologs were expressed in Escherichia coli, purified, and tested for PAI-1 binding activity in a competitive ligand binding assay . Although recombinant SMBVN was fully active in this assay, none of the homologs bound to PAI-1 or competed with VN for PAI-1 binding . These inactive homologs are structurally related to SMBVN, having 33-45% sequence identity and containing all 8 cysteines at conserved positions . Thus, homolog-scanning experiments were conducted by exchanging progressively larger portions of the NH2- or COOH-terminal regions of active SMBVN with the corresponding regions of the inactive homologs . These experiments revealed that the minimum PAI-1-binding sequence was present in the central region (residues 12-30) of SMBVN . Alanine scanning mutagenesis further demonstrated that each of the 8 cysteines as well as Gly12, Asp22, Leu24, Try27, Tyr28, and Asp34 were critical for PAI-1 binding and were required to stabilize PAI-1 activity . These results indicate that the PAI-1 binding motif is localized to residues 12-30 of SMBVN and suggest that this motif is anchored in the active conformation by disulfide bonds.

J Biol Chem, 1996 May 31, 271(22), 12909 - 12
Identification of residues in alpha-macroglobulins important for binding to the alpha2-macroglobulin receptor/Low density lipoprotein receptor-related protein; Nielsen KL et al.; Variants of the receptor binding domain of both human alpha2-macroglobulin and the corresponding domain of hen egg white ovomacroglobulin have been expressed in Escherichia coli and refolded in vitro . Competition experiments with methylamine-treated alpha2-macroglobulin for binding to the multifunctional alpha2-macroglobulin receptor identify two Lys residues (residues 1370 and 1374 in human alpha2-macroglobulin) spaced by three amino acid residues as crucial for receptor binding . From this result and mutational evidence from other ligands for the alpha2-macroglobulin receptor, a tentative sequence motif for receptor binding is proposed.

J Biol Chem, 1996 May 31, 271(22), 13040 - 7
Isolation and characterization of the kininogen-binding protein p33 from endothelial cells . Identity with the gC1q receptor; Herwald H et al.; Kininogens, the precursor proteins of the vasoactive kinins, bind specifically, reversibly, and saturably to platelets, neutrophils, and endothelial cells . Two domains of the kininogens expose major cell binding sites: domain D3 that is shared by H- and L-kininogen and domain D5H that is exclusively present in H-kininogen . Previously we have mapped the kininogen cell binding sites to 27 residues of D3 ("LDC27") and 20 residues of D5H ("HKH20"", respectively (Herwald, H., Hasan, A . A . K., Godovac-Zimmermann, J., Schmaier, A . H., and Muller-Esterl, W . (1995) J . Biol . Chem . 270, 14634-14642; Hasan, A . A . K., Cines, D . B., Herwald, H., Schmaier, A . H., and Muller-Esterl, W . (1995) J . Biol . Chem . 270, 19256-19261) . The corresponding kininogen acceptor site(s) exposed by the cell surfaces are still poorly defined . Using a non-ionic detergent, Nonidet P-40, we have been able to solubilize kininogen binding sites from an endothelial cell line, EA.hy926, in their functionally active form . Affinity chromatography of the solubilized kininogen binding sites on HKH20, a synthetic peptide representing the D5H cell binding site, allowed us to isolate a 33-kDa protein ("p33") that binds specifically and reversibly to H-kininogen with a KD (apparent dissociation constant) of 9 +/- 2 nM . Preparative SDS electrophoresis followed by NH2-terminal amino acid sequence analysis identified the kininogen-binding protein p33 as the gC1q receptor ("gC1qR"), an extrinsic membrane protein that interacts with the globular domains of the complement component C1q . The purified p33 binds C1q with moderate affinity, KD = 240 +/- 10 nM . Recombinant expression of the corresponding cDNA in Escherichia coli demonstrated that p33 binds H-kininogen, but not L-kininogen . Peptide HKH20 but not peptide LDC27 inhibited binding of H-kininogen to the recombinant p33 in a concentration-dependent manner, indicating that H-kininogen binds to p33 via domain D5H . Recombinant p33 efficiently inhibited the binding of H-kininogen to EA.hy926 cells . Factor XII, but not prekallikrein, competed with H-kininogen binding to p33 . These findings suggest that an endothelial binding protein mediates the assembly of critical components of the kinin-generating pathway on the surface of endothelial cells, thereby linking the early events of kinin formation and complement activation.

J Mol Biol, 1996 May 31, 259(1), 15 - 26
DNA binding of PhoB and its interaction with RNA polymerase; Makino K et al.; We have identified the DNA-binding domain (DBD) of an Escherichia coli activator protein PhoB as its C-terminal 91 residues . Four amino acid positions in the PhoB DBD are found important for interaction with the RNA polymerase holoenzyme that contains the sigma 70 subunit . Assuming that the PhoB DBD is structurally similar to the histone H5 DBD, the four positions are placed around the turn region that connects two putative helices, 2 and 3 (helix 3 is likely to be the recognition helix) . The binding sites of PhoB, three with the sequence TGTCA and one of TTACA, are identified in the pstS promoter . The pstS promoter has intrinsic bending (or bendability), which is much enhanced upon binding PhoB . On the basis of the above, some aspects of the PhoB-DNA-RNA polymerase interaction are discussed.

Cell, 1996 May 31, 85(5), 773 - 9
Two-dimensional crystallography of TFIIB- and IIE-RNA polymerase II complexes: implications for start site selection and initiation complex formation; Leuther KK et al.; SUMMARY: Transcription factors IIB (TFIIB) and IIE (TFIIE) bound to RNA polymerase II have been revealed by electron crystallography in projection at 15.7 A resolution . The results lead to simple hypotheses for the roles of these factors in the initiation of transcription . TFIIB is suggested to define the distance from TATA box to transcription start site by bringing TATA DNA in contact with polymerase at that distance from the active center of the enzyme . TFIIE is suggested to participate in a key conformational switch occurring at the active center upon polymerase-DNA interaction.

Cell, 1996 May 31, 85(5), 761 - 71
Anatomy of a flexer-DNA complex inside a higher-order transposition intermediate; Lavoie BD et al.; SUMMARY: Escherichia coli HU, a nonsequence-specific histone- and HMG-like DNA-binding protein, was chemically converted into a series of HU-nucleases with an iron-EDTA-based cleavage moiety positioned at 16 rationally selected sites . Specific DNA cleavage patterns from each of these HU-nucleases allowed us to determine the precise localization, stoichiometry, and orientation of HU binding in the Mu transpososome, a multiprotein structure that mediates the chemical reactions in DNA transposition . Correlation of the DNA cleavage data with the position of the cleavage moiety in the HU three-dimensional structure indicates the presence of a dramatic DNA bend, for which the bend center, direction, and magnitude were assessed . The data, which directly localize selected HU amino acids with respect to DNA in the transpososome, were used as constraints for computer-based molecular modeling to derive the first snapshot of an HU-DNA interaction.

Biochim Biophys Acta, 1996 May 28, 1311(3), 181 - 8
Characterization of human platelet GTPase activating protein for the Ral GTP-binding protein; Bhullar RP et al.; RalA, a ras p21 related 27 kDa GTP-binding protein, was expressed as a fusion protein in Escherichia coli and purified to homogeneity using an immunoaffinity column . The purified protein was capable of binding and hydrolyzing GTP . Addition of platelet cytosolic or detergent solubilized particulate proteins stimulated the intrinsic GTPase activity of ralA by at least six-fold with maximal effect observed at pH 6.5 . Addition of platelet proteins denatured by boiling had no effect on ralA GTPase activity . Analysis of GTPase reaction products by thin layer chromatography demonstrated that in samples containing ralA, 78.5 +/- 6.3% of the radioactivity was recovered in the GTP form while samples containing ralA plus platelet cytosol or particulate proteins, only 7.5 +/- 0.2% and 9.0 +/- 1.4% of the radioactivity was in the GTP form respectively . The GTPase activating protein(s) in the cytosolic and particulate fraction was further characterized by measuring GAP activity in proteins eluted from gel slices after sodium dodecyl sulfate polyacrylamide gel electrophoresis . The ralA GTPase activating protein present in the cytosol and particulate fractions was recovered in a single gel slice of identical apparent molecular weight . The molecular mass of the ral specific GTPase activating protein was estimated to be 34 +/- 2 kDa . This protein did not stimulate the intrinsic GTPase activity of ras p21, G25K/CDC42Hs or rab3A GTP-binding proteins . Results demonstrate that in human platelets, the activity/function of ral-related GTP-binding protein(s) is under the regulation of a specific GTPase activating protein of molecular mass of 34 +/- 2 kDa that is distributed equally in the cytosol and particulate fraction.

Proc Natl Acad Sci U S A, 1996 May 28, 93(11), 5647 - 52
A truncated mutant (residues 58-140) of the hepatitis B virus X protein retains transactivation function; Kumar V et al.; The hepatitis B virus X protein (HBx) sequence (154 aa) has been divided into six regions (A-F) based on its sequence homology with X proteins of other mammalian hepadnaviruses . Regions A, C, and E are more conserved and include all the four conserved cysteines (C7, C61, C69, and C137) . To localize the regions of HBx important for transactivation, a panel of 10 deletion mutants (X5-X14) and 4 single point mutants (X1-X4), each corresponding to a conserved cysteine residue, was constructed by site-directed mutagenesis . A HBx-specific monoclonal antibody was developed and used to confirm the expression of mutants by Western blot . Transactivation property of the HBx mutants was studied on Rous sarcoma virus-long terminal repeat (RSV-LTR) in transient transfection assays . We observed that deletion of the most conserved region A or substitution of the N-terminal cysteine (C7) had no effect on transactivation . Deletion of the nonconserved regions B or F also had no deleterious effects . Deletions of regions C and D resulted in a significant loss of function . Substitution of both C61 and C69 present in region C, caused almost 90% loss of activity that could be partially overcome by transfecting more expression plasmid . The fully conserved 9 amino acid segment (residues 132 to 140) within region E including C137 appeared to be crucial for its activity . Finally, a truncated mutant X15 incorporating only regions C to E (amino acids 58-140) was able to stimulate the RSV-LTR quite efficiently, suggesting a crucial role played by this domain in transactivation function.

Proc Natl Acad Sci U S A, 1996 May 28, 93(11), 5624 - 8
A novel iron-regulated metal transporter from plants identified by functional expression in yeast; Eide D et al.; Iron is an essential nutrient for virtually all organisms . The IRT1 (iron-regulated transporter) gene of the plant Arabidopsis thaliana, encoding a probable Fe(II) transporter, was cloned by functional expression in a yeast strain defective for iron uptake . Yeast expressing IRT1 possess a novel Fe(II) uptake activity that is strongly inhibited by Cd . IRT1 is predicted to be an integral membrane protein with a metal-binding domain . Data base comparisons and Southern blot analysis indicated that IRT1 is a member of a gene family in Arabidopsis . Related sequences were also found in the genomes of rice, yeast, nematodes, and humans . In Arabidopsis, IRT1 is expressed in roots, is induced by iron deficiency, and has altered regulation in plant lines bearing mutations that affect the iron uptake system . These results provide the first molecular insight into iron transport by plants.

Proc Natl Acad Sci U S A, 1996 May 28, 93(11), 5590 - 4
Active barnase variants with completely random hydrophobic cores; Axe DD et al.; The central structural feature of natural proteins is a tightly packed and highly ordered hydrophobic core . If some measure of exquisite, native-like core packing is necessary for enzymatic function, this would constitute a significant obstacle to the development of novel enzymes, either by design or by natural or experimental evolution . To test the minimum requirements for a core to provide sufficient structural integrity for enzymatic activity, we have produced mutants of the ribonuclease barnase in which 12 of the 13 core residues have together been randomly replaced by hydrophobic alternatives . Using a sensitive biological screen, we find that a strikingly high proportion of these mutants (23%) retain enzymatic activity in vivo . Further substitution at the 13th core position shows that a similar proportion of completely random hydrophobic cores supports enzyme function . Of the active mutants produced, several have no wild-type core residues . These results imply that hydrophobicity is nearly a sufficient criterion for the construction of a functional core and, in conjunction with previous studies, that refinement of a crudely functional core entails more stringent sequence constraints than does the initial attainment of crude core function . Since attainment of crude function is the critical initial step in evolutionary innovation, the relatively scant requirements contributed by the hydrophobic core would greatly reduce the initial hurdle on the evolutionary pathway to novel enzymes . Similarly, experimental development of novel functional proteins might be simplified by limiting core design to mere specification of hydrophobicity and using iterative mutation-selection to optimize core structure.

Proc Natl Acad Sci U S A, 1996 May 28, 93(11), 5550 - 5
Molecular basis for dysfunction of some mutant forms of methylmalonyl-CoA mutase: deductions from the structure of methionine synthase; Drennan CL et al.; Inherited defects in the gene for methylmalonyl-CoA mutase (EC 5.4.99.2) result in the mut forms of methylmalonic aciduria . mut- mutations lead to the absence of detectable mutase activity and are not corrected by excess cobalamin, whereas mut- mutations exhibit residual activity when exposed to excess cobalamin . Many of the mutations that cause methylmalonic aciduria in humans affect residues in the C-terminal region of the methylmalonyl-CoA mutase . This portion of the methylmalonyl-CoA mutase sequence can be aligned with regions in other B12 (cobalamin)-dependent enzymes, including the C-terminal portion of the cobalamin-binding region of methionine synthase . The alignments allow the mutations of human methylmalonyl-CoA mutase to be mapped onto the structure of the cobalamin-binding fragment of methionine synthase from Escherichia coli (EC 2.1.1.13), which has recently been determined by x-ray crystallography . In this structure, the dimethylbenzimidazole ligand to the cobalt in free cobalamin has been displaced by a histidine ligand, and the dimethylbenzimidazole nucleotide "tail" is thrust into a deep hydrophobic pocket in the protein . Previously identified mut0 and mut- mutations (Gly-623 --> Arg, Gly-626 --> Cys, and Gly-648 --> Asp) of the mutase are predicted to interfere with the structure and/or stability of the loop that carries His-627, the presumed lower axial ligand to the cobalt of adenosylcobalamin . Two mutants that lead to severe impairment (mut0) are Gly-630 --> Glu and Gly-703 --> Arg, which map to the binding site for the dimethylbenzimidazole nucleotide substituent of adenosylcobalamin . The substitution of larger residues for glycine is predicted to block the binding of adenosylcobalamin.

Proc Natl Acad Sci U S A, 1996 May 28, 93(11), 5544 - 9
Interaction of cyclooxygenases with an apoptosis- and autoimmunity-associated protein; Ballif BA et al.; Cyclooxygenases (COXs) 1 and 2 are 72-kDa, intralumenal residents of the endoplasmic reticulum (ER) and nuclear envelope, where they catalyze the rate-limiting steps in the conversion of arachidonate to the physiologically dynamic prostanoids . Recent studies, including the generation of knockout mice, show COX-1 and COX-2 to have biologically distinct roles within cells and organisms . Also apparent is that arachidonate substrate is selectably metabolized by COX-2 after mitogen stimulation in many cells that contain both isoforms . Because COX-1 and COX-2 are highly conserved in all residues needed for catalysis and in their purified forms have almost identical kinetic properties, we have searched for COX-interacting ER proteins that might mediate these unique isoenzymic properties . Using COXs as bait in the yeast two-hybrid system, we identified autoimmunity- and apoptosis-associated nucleobindin (Nuc) as a protein that specifically interacts with both isoenzymes . COX-Nuc binding was substantiated by immunoprecipitation experiments, which showed that COX-1 and, to a lesser extent, COX-2 form complexes with Nuc in vitro . When overexpressed in COS-1 cells, Nuc was found to be extracellularly released . However, when Nuc was co-overexpressed with COX-1 or COX-2, its release was reduced by >80% . This finding suggests that COX isoenzymes participate in the retention of Nuc within the lumen of the ER, where COX may regulate the release of Nuc from the cell . It also identifies Nuc as a potential regulator of COXs through this interaction.

Proc Natl Acad Sci U S A, 1996 May 28, 93(11), 5522 - 6
The replication initiator protein pi of the plasmid R6K specifically interacts with the host-encoded helicase DnaB; Ratnakar PV et al.; The replication initiator protein pi of plasmid R6K is known to interact with the seven iterons of the gamma origin/enhancer and activate distant replication origins alpha and beta (ori alpha and ori beta) by pi-mediated DNA looping . Here we show that pi protein specifically interacts in vitro with the host-encoded helicase DnaB . The site of interaction of pi on DnaB has been localized to a 37-aa-long region located between amino acids 151 and 189 of DnaB . The surface of pi that interacts with DnaB has been mapped to the N-terminal region of the initiator protein between residues 1 and 116 . The results suggest that during initiation of replication, the replicative helicase DnaB is first recruited to the gamma enhancer by the pi protein . In a subsequent step, the helicase probably gets delivered from ori gamma to ori alpha and ori beta by pi-mediated DNA looping.

Proc Natl Acad Sci U S A, 1996 May 28, 93(11), 5460 - 5
Irreversible inactivation of interleukin 2 in a pump-based delivery environment; Tzannis ST et al.; The physical stability of pharmaceutical proteins in delivery environments is a critical determinant of biological potency and treatment efficacy, and yet it is often taken for granted . We studied both the bioactivity and physical stability of interleukin 2 upon delivery via continuous infusion . We found that the biological activity of the delivered protein was dramatically reduced by approximately 90% after a 24-hr infusion program . Only a portion of these losses could be attributed to direct protein deposition on the delivery surfaces . Analysis of delivered protein by size exclusion chromatography gave no indication of insulin-like, surface-induced aggregation phenomena . Examination of the secondary and tertiary structure of both adsorbed and delivered protein via Fourier-transform infrared spectroscopy, circular dichroism, and fluorescence spectroscopy indicated that transient surface association of interleukin 2 with the catheter tubing resulted in profound, irreversible structural changes that were responsible for the majority of the biological activity losses.

Proc Natl Acad Sci U S A, 1996 May 28, 93(11), 5191 - 6
Crystal structure at 2.6-A resolution of human macrophage migration inhibitory factor; Sun HW et al.; Macrophage migration inhibitory factor (MIF) was the first cytokine to be described, but for 30 years its role in the immune response remained enigmatic . In recent studies, MIF has been found to be a novel pituitary hormone and the first protein identified to be released from immune cells on glucocorticoid stimulation . Once secreted, MIF counterregulates the immunosuppressive effects of steroids and thus acts as a critical component of the immune system to control both local and systemic immune responses . We report herein the x-ray crystal structure of human MIF to 2.6 angstrom resolution . The protein is a trimer of identical subunits . Each monomer contains two antiparallel alpha-helices that pack against a four-stranded beta-sheet . The monomer has an additional two beta-strands that interact with the beta-sheets of adjacent subunits to form the interface between monomers . The three beta-sheets are arranged to form a barrel containing a solvent-accessible channel that runs through the center of the protein along a molecular 3-fold axis . Electrostatic potential maps reveal that the channel has a positive potential, suggesting that it binds negatively charged molecules . The elucidated structure for MIF is unique among cytokines or hormonal mediators, and suggests that this counterregulator of glucocorticoid action participates in novel ligand-receptor interactions.

Biochemistry, 1996 May 28, 35(21), 6900 - 10
Vaccinia virus mRNA (guanine-7-)methyltransferase: mutational effects on cap methylation and AdoHcy-dependent photo-cross-linking of the cap to the methyl acceptor site; Mao X et al.; The (guanine-7-)methyltransferase domain of the vaccinia virus mRNA capping enzyme is composed of the C-terminal portion of the D1 subunit, D1(498-844), heterodimerized with the D12 protein . In order to identify protein structural elements involved in cap methylation, we introduced eight alanine substitution mutations within two sequence motifs of D1(498-844)-(594)VLAIDFGNG(602) and (681)IHYSF(685)--that are conserved in the cap methyltransferase from yeast . The D1(498-844)-Ala proteins were coexpressed in bacteria with the D12 subunit, and the recombinant D1(498-844)/D12 heterodimers were purified . Alanine substitutions at five positions--Asp-598, Gly-602, Ile-681, Ser-684, and Phe-685--had little or no effect on methyltransferase activity . Mutations at three conserved residues were deleterious . Alanine substitution at Gly-600 reduced the specific activity to 4% of that of the wild-type protein . Substitutions at His-682 and Tyr-683 reduced activity to 4% and 0.05%, respectively . By further mutating Tyr-683 to Phe and Ser, we established that the aromatic group was essential for cap methylation, whereas the hydroxyl moiety was dispensable . Specific binding of the methyltransferase to the RNA cap was demonstrated by UV cross-linking to {32P}GMP-labeled capped poly(A) . Label transfer occurred exclusively to the D1(498-844) subunit and was competed by the cap analogs GpppA and m7GpppA . Cap-specific cross-linking to m7GpppA(pA)n was stimulated by AdoHcy, whereas cross-linking to GpppA(pA)n was unaffected by AdoHcy, but stimulated by AdoMet . We suggest that occupancy of the methyl donor site either enhances the affinity for the cap guanosine or alters the protein interface so that a photoreactive moiety is brought closer to the cap structure . The catalytically defective H682A, Y683A, and Y683S mutant methyltransferases were unable to cross-link to the cap in the presence of AdoHcy . The catalytically defective G600A mutant did cross-link to the cap in the presence of AdoHcy, suggesting that this mutation affects the chemical step of transmethylation.

Biochemistry, 1996 May 28, 35(21), 6795 - 805
New approach to the study of transient protein conformations: the formation of a semiburied salt link in the folding pathway of barnase; Oliveberg M et al.; We use in this study a novel kinetic approach to determine the H+ titration properties of a semiburied salt link in the transition state for unfolding of barnase . The approach is based on changes in the pH dependence of the kinetics upon mutation of a target residue . This makes it relatively insensitive to the absolute value of the stability and, thereby, to artifacts caused by structural rearrangements around the site of mutation . The semiburied salt bridge studied here is between Asp93 and Arg69 . Mutation of either residue significantly destabilized the protein, and the pKa value of Asp93 is severely lowered in the native state to below 1 because of the ionic interaction with Arg69 . The Asp93-Arg69 salt link appears to be formed early in the folding process; the pKa value of Asp93 in the transition state (approximately 1) is similar to that in the native state, and deletion of the ionic interaction with Arg69 substantially destabilizes the folding intermediate and changes the kinetic behavior from multistate to two-state or close to two-state, depending on the conditions . The results suggest that the formation of ionic interactions within clusters of hydrophobic residues can be important for early folding events and can control kinetically the folding pathway . This is not because of the inherent stability of the salt link but because the presence of two unpaired charges is very unfavorable . The data reveal also that fractional phi values are consistent with a uniformly expanded transition state or one with closely spaced energy levels and not with parallel folding pathways.

Biochemistry, 1996 May 28, 35(21), 6762 - 70
Low-temperature thermochromism marks a change in coordination for the metal ion in manganese superoxide dismutase; Whittaker MM et al.; We have observed thermochromism (temperature-dependent absorption) for anion complexes of manganese superoxide dismutase indicating a change in coordination number for the metal complex at low temperatures . The ligand field spectra for the Mn(III) ion, characteristic of five-coordination for the azide complex at 295 K, cleanly convert to spectra reflecting six-coordination at low temperature, with a midpoint for the transition near 200 K . The active site structure is temperature-dependent, a relatively rigid, distorted octahedral low-temperature Mn complex melting with dehydration (or displacement of one of the protein ligands) to form a five-coordinated complex under physiological conditions . Thermodynamic parameters for the transition estimated from van't Hoff analysis (delta HvH = 5 kcal/mol; delta SvH = 22 cal/mol K) are consistent with reduced chemical binding and increased fluxionality at room temperature . This thermochromism of MnSD demonstrates the existence of distinct isomeric forms of the active site metal complex, whose relative stability depends on the degree of vibrational excitation . The marginal destabilization of the six-coordinate anion complex under physiological conditions suggests that the enzyme may thermally control the stability of intermediates in a dissociative displacement mechanism for substrate binding and redox.

Biochemistry, 1996 May 28, 35(21), 6754 - 61
Analysis of the substrate-recognition mode of aromatic amino acid aminotransferase by combined use of quasisubstrates and site-directed mutagenesis: systematic hydroxy-group addition/deletion studies to probe the enzyme-substrate interactions; Hayashi H et al.; Escherichia coli aromatic amino acid aminotransferase (ArAT) catalyzes transamination reactions of both dicarboxylic amino acids and aromatic amino acids . Because both reactions are supposed to occur in a single reaction center, whether ArAT provides alternative binding sites for the two different types of substrate side chains has been an intriguing question . This was probed by spectroscopic analysis of the complexes of beta-hydroxylated substrates and the wild-type and {Tyr70-->Phe} mutant enzymes . Both L-erythro-3-hydroxyaspartate and L-erythro-3-phenylserine reacted with the wild-type ArAT to give an absorption maximum at around 500 nm, reflecting the formation of the quinonoid intermediate . When the hydroxy group of Tyr70 of ArAT was deleted by replacement of the residue with phenylalanine, the 500-nm absorption greatly decreased in either of the ArAT-beta-hydroxy amino acid complexes, showing the presence of specific interactions, which stabilize the 500-nm absorbing quinonoid intermediates, between the phenolic hydroxy group of Tyr70 and the beta-hydroxy groups of the two quasisubstrates . From these results, it was concluded that the conformations of the two quasisubstrates are essentially identical in their enzyme-bound forms . This implies that the phenyl group of the substrate phenylalanine occupies the same region as that occupied by the beta-carboxyl group of the substrate aspartate, and the region should be near Arg292, the residue that binds the beta-carboxylate group of substrates . The {Arg292-->Ala} or {Arg292-->Leu} mutation increased the Km values for aromatic amino acids 5-10 fold, and the {Arg292-->Lys} mutation increased these values 10-100-fold, without affecting the kcat values . This shows that the side chain of Arg292 is partially involved in the binding of the aromatic ring of substrates to ArAT.

Biochemistry, 1996 May 28, 35(21), 6735 - 44
Substrate binding and catalysis by ubiquitin C-terminal hydrolases: identification of two active site residues; Larsen CN et al.; Ubiquitin C-terminal hydrolases (UCH's) are a newly-defined class of thiol proteases implicated in the proteolytic processing of polymeric ubiquitin . They are important for the generation of monomeric ubiquitin, the active component of the eukaryotic ubiquitin-dependent proteolytic system . There are at least three mammalian isozymes which are tissue specific and developmentally regulated . To study the structure and functional roles of these highly homologous enzymes, we have subcloned and overexpressed two of these isozymes, UCH-L1 and UCH-L3 . Here, we report their purification, physical characteristics, and the mutagenesis of UCH-L1 . Site-directed mutagenesis of UCH-L1 reveals that C90 and H161 are involved in catalytic rate enhancement . Data from circular dichroic and Raman spectroscopy, as well as secondary structure prediction algorithms, indicate that both isozymes have a significant amount of alpha-helix (> 35%), and contain no disulfide bonds . Both enzymes are reasonably stable, undergoing a reversible thermal denaturation at 52 degrees C . These transitions are characterized by thermodynamic parameters typical of single domain globular proteins . Substrate binding affinity to UCH-L3 was directly measured by equilibrium gel filtration (Kd = 0.5 microM), and the results are similar to the kinetically determined Km for ubiquitin ethyl ester (o.6 microM) . The binding is primarily electrostatic in nature and indicates the existence of a specific and extensive binding site for ubiquitin on the surface of the enzyme.

Biochemistry, 1996 May 28, 35(21), 6727 - 34
Mutational analysis of the oligosaccharide recognition site at the active site of Escherichia coli maltodextrin phosphorylase; Drueckes P et al.; A mutagenesis approach was applied to identify specific amino acid residues that are tentatively involved in binding of the oligosaccharide substrate at the active site of Escherichia coli maltodextrin phosphorylase . From ten residues located within a proposed channel connecting the enzyme surface with the active site, nine displayed significant effects on the reaction with oligosaccharide substrates when exchanged by mutagenesis . While several mutant enzymes (N258A/D259A/N260A, N307A, E350A, and Y578F) exhibited moderate decreases in apparent binding (about 4-17-fold), two mutations, H536L and E67A, weakened apparent binding of oligosaccharide substrates by 2 orders of magnitude . Two further mutant enzymes (T346G and H310A) displayed a 10-fold increase in the apparent Km of the oligosaccharide in the degradation reaction, while binding in the synthesis direction seemed less affected, indicating partially differential binding modes of oligosaccharides in synthesis and degradation . Quite uniquely, the H310A mutant enzyme exhibits a more than 100-fold-lowered Ki for gluconolactone, indicating the existence of an inhibitor binding site similar to that expected for a carbonium ion-like transition state . This is further confirmed by the finding that glucose, which does not inhibit wild-type enzyme, became an inhibitor of the H310A mutant enzyme (Ki = 20 mM) . Since mutation of D308 did reduce kcat about 10-100-fold while Km values remained unchanged, a participation of this residue in transition state binding is probable . The insight into substrate recognition derived from this mutagenesis study corroborates a binding model where maltopentaose fits into the phosphorylase b structure in a distorted form.

Biochemistry, 1996 May 28, 35(21), 6715 - 26
The role of Glu 57 in the mechanism of the Escherichia coli MutT enzyme by mutagenesis and heteronuclear NMR; Lin J et al.; The role of the conserved residue Glu-57 in the mechanism of the MutT enzyme from Escherichia coli was investigated by mutagenesis and heteronuclear NMR methods . The enzymatic activity of the E57Q mutant is at least 10(5)-fold lower than that of the wild type enzyme . The solution structure of the E57Q mutant, based on comparisons of 1H-15N NOESY HSQC spectra and 1H-15N HSQC spectra to those of the wild type enzyme, differs in a region near Glu-57 . The dissociation constants (KD) of the E-Mg2+ and E-Mn2+ complexes increased 3.3- and 3.6-fold, respectively, in the E57Q mutant, while the KD of E-dGTP is unaltered from that of the wild type enzyme . The enhanced paramagnetic effect of enzyme-bound Mn2+ on 1/T1 of water protons is halved in the E57Q mutant indicating an altered metal-binding site . 1H-15N HSQC titrations of E57Q with MnCl2 show selective attenuation of the side chain NH signals of Gln-57 and the backbone NH signals of Gly-37, Gly-38, Lys-39, Glu-53, Glu-56, Gln-57, and Glu-98, indicating proximity of bound Nm2+ to these residues . The same resonances of the wild type and the E57Q mutant enzymes are attenuated by Mn2+, but significantly smaller paramagnetic effects (relative to the largest effect on Lys-39) are found on Gly-37, Gly-38, Val-58, and Glu-98 of the mutant, indicating an altered position of the bound divalent cation . Thus Glu-57 is probably a ligand to the enzyme-bound metal, and the profound loss of catalytic activity in the E57Q mutant results from structural and electronic changes at the site of the enzyme-bound divalent cation . 1H-15N HSQC titrations of the wild type enzyme with MgCl2 show changes in chemical shifts of 15N and NH resonances in regions closely overlapping those induced by the E57Q mutation itself, suggesting that the loss of the negative charge at Glu-57, either by mutation or by neutralization with Mg2+, induces a similar effect . In the E57Q mutant, the slow exchange of the side chain NH2 protons of Gln-57 and NOE's from the NH2 protons of Gln-57 to the beta and gamma protons of Glu-98 suggests hydrogen bonding of Gln-57 to Glu-98 in the free enzyme . 1H-15N HSQC titrations of both the wild type and mutant enzymes with dGTP show changes in 15N and NH chemicals shifts of residues in a cleft formed by beta-strands A, C, and D on one side and loop I, the end of loop IV, and the beginning of helix II on the other side, suggesting this cleft to be the nucleotide binding site . These changes in chemical shift were smaller or absent in titrations of the wild type or mutant enzymes with AMPCPP or Mg2+-AMPCPP, in accord with the strong preference of the MutT enzyme for guanine over adenine nucleotide substrates.

Biochemistry, 1996 May 28, 35(21), 6697 - 705
Tuning the equilibrium ion affinity and selectivity of the EF-hand calcium binding motif: substitutions at the gateway position; Drake SK et al.; The ion binding parameters of the EF-hand Ca2+ binding motif are carefully tuned for different biological applications . The present study examines the contribution of the ninth position of the Ca2+-coordinating EF-loop to the tuning of Ca2+ affinity and selectivity, using the model EF-loop of the Escherichia coli galactose binding protein . Eight side chains, representing the entire set of side chains commonly observed in natural EF-loop sequences, are tested at the ninth position of the model EF-loop to determine their effects on equilibrium ion binding parameters . Using the spherical metal ions of groups Ia, IIa, and IIIa and the lanthanides as probes, both the Ca2+ affinities and ionic selectivities of the engineered sites are quantitated . Neutral side chains of different size at the ninth EF-loop position {Gln (wild type), Asn, Thr, Ser, Ala, Gly} are observed to yield similar Ca2+ affinities and retain the native ability to exclude the physiological competing metal cations Na+, K+, and Mg2+ . Acidic gateway side chains (Glu, Asp) are found to reduce Ca2+ affinity and shift the ionic charge selectivity as much as 10(3)-fold toward trivalent cations . Relative to the native Gln, all engineered side chains cause a partial loss of ionic size selectivity, stemming from enhanced affinities for nonphysiological large ions . Overall, the results have implications for the molecular mechanisms used by the EF-loop to control both (i) charge selectivity, which is proposed to stem from the electrostatic repulsion between the coordinating oxygens, and (ii) size selectivity, which is theorized to involve complex interactions between multiple coordinating side chains . Finally, it has recently been shown that the ninth EF-loop position serves as a "gateway" to modulate the kinetics of Tb3+ binding and release without shifting the equilibrium affinity of this ion {Drake, S . K., & Falke, J . J . (1996) Biochemistry 35, 1753-1760} . The present results confirm that isoelectric substitutions at the gateway position have little effect on Ca2+ affinity, thereby supporting the hypothesis that the gateway side chain provides kinetic tuning of Ca2+ signaling proteins independently of their Ca2+ activation thresholds.

Biochemistry, 1996 May 28, 35(21), 6684 - 9
Influence exercised by histidine-95 on chloride transport and the photocycle in halorhodopsin; Otomo J; The anion pumping mechanism of halorhodopsin was studied using site-directed mutagenesis . Comparison of the amino acid sequence revealed that the B-C interhelix loop segment was highly homologous in all known halorhodopsins . Especially a basic residue, histidine-95, was conserved in all halorhodopsins . Using the expression-vector plasmid carrying the bop promoter, two His-95 mutants (H95R, H95A) were successfully expressed in Halobacterium salinarium . The expression levels of these halorhodopsin mutants were slightly lower than that for the wild-type halorhodopsin . In addition, these mutants were unstable under illumination compared with the wild-type . It suggested that His-95 is probably important for stabilizing the structure of halorhodopsin . The absorption maxima of these mutants are approximately 15 nm blue-shifted compared with the wild-type, suggesting that His-95 interacts with the retinal Schiff base . At low chloride concentrations, the light-induced chloride pumping activity of these mutants was more than 20 times lower than that for the wild-type . Only under physiological conditions, the chloride pumping activity was detected . Even at a high chloride concentration (1 M NaCl), the HR520 intermediate could not be detected for these mutants . These results clearly indicate that His-95 has a crucial role in the chloride transport of halorhodopsin.

Biochemistry, 1996 May 28, 35(21), 6628 - 34
A fluorescence study of single tryptophan-containing mutants of enzyme IImtl of the Escherichia coli phosphoenolpyruvate-dependent mannitol transport system; Dijkstra DS et al.; The fluorescence properties of six different single Trp mutants of the mannitol-specific transporter of Escherichia coli were studied in order to derive structural information at different locations in the enzyme . The use of pure detergent and special protein purification protocols was essential for reliable fluorescence spectra, as judged from tyrosine-like fluorescence in a tryptophan-minus mutant (Robillard et al., 1996) . The steady-state fluorescence spectra of EIImtl mutants with single tryptophan residues at positions 30, 42, 109, 117, 320, and 384 provided information concerning the polarity of the environment and the effects of mannitol binding at these positions . Tryptophan positions 42, 109, and 117 with emission maxima ranging from 337 to 340 nm are relatively polar, and position 384 with an emission maximum at 346 nm is highly polar, whereas position 30 is highly apolar with a maximum at 324 nm . The fluorescence characteristics of tryptophan 30 suggest a buried position in a hydrophobic part of the enzyme, which is confirmed by the low Stern-Volmer quenching constant for I- quenching . Positions 109 and 117 show the highest quenching constants, indicating the most exposed positions, whereas positions 320 and 42 are moderately quenched, by I- . The tryptophan residue at position 384 is, even in the absence of externally added quencher, very strongly quenched, possibly by the carboxylate from aspartate 384 or by a tyrosinate at position 458 which is nearby in the folded protein (AB et al., in preparation; van Montfort et al., in preparation) . The observed emission maxima and accessibilities of the tryptophans at the different positions are consistent with the predicted topology of the enzyme (Sugiyama et al., 1991) . When mannitol is bound to wild-type EIImtl, an increase in fluorescence emission intensity was observed (Wood, 1988) which can now be attributed primarily to increased fluorescence intensity of the tryptophan at position 30.

Biochemistry, 1996 May 28, 35(21), 6595 - 603
Autoxidation of ubiquinol-6 is independent of superoxide dismutase; Schultz JR et al.; Ubiquinone (Q) is an essential, lipid soluble, redox component of the mitochondrial respiratory chain . Much evidence suggests that ubiquinol (QH2) functions as an effective antioxidant in a number of membrane and biological systems by preventing peroxidative damage to lipids . It has been proposed that superoxide dismutase (SOD) may protect QH2 form autoxidation by acting either directly as a superoxide-semiquinone oxidoreductase or indirectly by scavenging superoxide . In this study, such an interaction between QH2 and SOD was tested by monitoring the fluorescence of cis-parinaric acid (cPN) incorporated phosphatidylcholine (PC) liposomes . Q6H2 was found to prevent both fluorescence decay and generation of lipid peroxides (LOOH) when peroxidation was initiated by the lipid-soluble azo initiator DAMP, dimethyl 2,2'-azobis (2-methylpropionate), while Q6 or SOD alone had no inhibitory effect . Addition of either SOD or catalase to Q6H2-containing liposomes had little effect on the rate of peroxidation even when incubated in 100% O2 . Hence, the autoxidation of QH2 is a competing reaction that reduces the effectiveness of QH2 as an antioxidant and was not slowed by either SOD or catalase . The in vivo interaction of SOD and QH2 was also tested by employing yeast mutant strains harboring deletions in either CuZnSOD and/or MnSOD . The sod mutant yeast strains contained the same percent Q6H2 per cell as wild-type cells . These results indicate that the autoxidation of QH2 is independent of SOD.

Biochemistry, 1996 May 28, 35(21), 6569 - 84
Heteronuclear (1H, 13C, 15N) NMR assignments and solution structure of the monocyte chemoattractant protein-1 (MCP-1) dimer; Handel TM et al.; A full high-resolution three-dimensional solution structure of the monocyte chemoattractant protein-1 (MCP-1 or MCAF) homodimer has been determined by heteronuclear multidimensional NMR . MCP-1 is a member of a family of small proteins which play a crucial role in immune surveillance by orchestrating the recruitment of specific leukocytes to areas of immune challenge . The protein was uniformly isotopically enriched with 13C and 15N by expression in Escherichia coli, and complete sequence-specific resonance assignments were obtained by a combination of heteronuclear double- and triple-resonance experiments . The secondary structure was deduced from characteristic patterns of NOEs, 13 C alpha/beta chemical shifts, measurements of 3JHNH alpha scalar couplings, and patterns of slowly exchanging amide protons . Because MCP-1 forms symmetrical homodimers, additional experiments were carried out to unambiguously establish the quaternary contacts . NOEs from these novel experiments were merged with more traditional heteronuclear separated NOE measurements in an iterative strategy to partition the restraints between explicit inter/intrasubunit contacts and a class wherein both were retained as ambiguous . With more than 30 restraints per residue, the three-dimensional structure is well-defined with a backbone rmsd of 0.37 A to the mean over residues 5-69 of the dimer . We compare the structure with those recently reported for the related chemokines MIP-1 beta and RANTES and highlight the differences in terms of receptor specificity and function as well as interpret the known biological activity data of MCP-1 mutants.

Biochemistry, 1996 May 28, 35(21), 6559 - 68
A tRNA identity switch mediated by the binding interaction between a tRNA anticodon and the accessory domain of a class II aminoacyl-tRNA synthetase; Yan W et al.; Identity elements in tRNAs and the intracellular balance of tRNAs allow accurate selection of tRNAs by aminoacyl-tRNA synthetases . The histidyl-tRNA from Escherichia coli is distinguished by a unique G-1.C73 base pair that upon exchange with other nucleotides leads to a marked decrease in the rate of aminoacylation in vitro . G-1.C73 is also a major identity element for histidine acceptance, such that the substitution of C73 brings about mischarging by glycyl-, glutaminyl-, and leucyl-tRNA synthetases . These identity conversions mediated by the G-1.C73 base pair were exploited to isolate secondary site revertants in the histidyl-tRNA synthetase from E . coli which restore histidine identity to a histidyl-tRNA suppressor carrying U73 . The revertant substitutions confer a 3-4 fold reduction in the Michaelis constant for tRNAs carrying the amber-suppressing anticodon and map to the C-terminal domain of HisRS and its interface with the catalytic core . These findings demonstrate that the histidine tRNA anticodon plays a significant role in tRNA selection in vivo and that the C-terminal domain of HisRS is in large part responsible for recognizing this trinucleotide . The kinetic parameters determined also show a small degree of anticooperativity (delta delta G = -1.24 kcal/mol) between recognition of the discriminator base and the anticodon, suggesting that the two helical domains of the tRNA are not recognized independently . We propose that these effects substantially account for the ability of small changes in tRNA binding far removed from the site of a major determinant to bring about a complete conversion of tRNA identity.

Biochemistry, 1996 May 28, 35(21), 6533 - 8
NMR studies of the effects of the 5'-phosphate group on conformational properties of 5-methylaminomethyluridine found in the first position of the anticodon of Escherichia coli tRNA(Arg)4; Sakamoto K et al.; 5-Methylaminomethyluridine (mnm5U) exists in the first position of the anticodon (position 34) of Escherichia coli tRNA4Arg for codons AGA/AGG . In the present study, the temperature dependence of the ribose-puckering equilibrium of pmnm5U was analyzed by proton NMR spectroscopy . Thus, the enthalpy difference (delta H) between the C2'-endo and C3'-endo forms was obtained at 0.65 kcal.mol-1 . By comparison of the delta H values of pU and pmnm5U, the 5-substitution was found to increase the relative stability of the C3'-endo form over the C2'-endo form significantly (by 0.56 kcal.mol-1) . Furthermore, this conformational "rigidity" was concluded to depend on the 5'-phosphate group, because nucleoside U exhibits only a negligible change in the ribose-puckering equilibrium upon the 5-methylaminomethyl substitution . Further NMR analyses and molecular dynamics calculations revealed that interactions between the 5-methylaminomethyl and 5'-phosphate groups of pmnm5U restrict the conformation about the glycosidic bond to a low anti form, enhancing steric repulsion between the 2-carbonyl and 2'-hydroxyl groups in the C2'-endo form . This intrinsic conformational rigidity of the mnm5U residue in position 34 may contribute to the correct codon recognition.

FEBS Lett, 1996 May 27, 387(1), 23 - 6
Translational fusion of heat labile enterotoxin chain B and beta-subunit of human chorionic gonadotropin: periplasmic expression in Escherichia coli and its immunogenicity; Pillai D et al.; A fusion gene was constructed consisting of heat labile enterotoxin chain B (LTB) of E . coli genetically linked at its C-terminus to the beta-subunit of human chorionic gonadotropin in translational fusion, under the control of tac promoter and LTB signal sequence . Expression of the fusion gene (about 5 microgram/ml) in E . coli was confirmed by immunoblot analysis using both anti-LTB and anti-betahCG polyclonal antibodies . The fusion protein was efficiently processed and exported to the periplasmic space . LTB in the fusion protein retained its ability to bind to GM1 ganglioside receptor . Mice immunized with the fusion protein produce antibodies that recognize recombinant betahCG and the native hCG suggesting its potential use as a contraceptive vaccine.

Neurosci Lett, 1996 May 24, 210(1), 61 - 4
Apolipoprotein E is highly susceptible to oxidation by myeloperoxidase, an enzyme present in the brain; Jolivalt C et al.; Apolipoprotein E, the most common apolipoprotein found in the brain, is linked to several pathologies like Alzheimer's disease . Apolipoprotein E directly binds to beta-amyloid with a strong affinity . Myeloperoxidase, a protein secreted by neutrophils and involved in the inflammatory process, is also present in the brain . In vitro myeloperoxidase oxidation of recombinant human apolipoprotein E leads to fragmentation of the protein with low concentrations of hydrogen peroxide and polymerization with higher concentrations . Comparison with bovine serum albumin shows a higher susceptibility of apolipoprotein E to myeloperoxidase oxidation, which may have importance in the Alzheimer's disease process.

Gene, 1996 May 24, 171(1), 71 - 3
pUCS75, a stable high-copy-number Streptomyces--Escherichia coli shuttle vector which facilitates subcloning from pUC plasmid and M13 phage vectors; Dyson PJ et al.; A new Streptomyces-Escherichia coli shuttle vector, pUCS75, has been constructed to permit facile subcloning of DNA from the multiple cloning sites of the pUC plasmid and M13 phage vectors . In contrast to other commonly used shuttle vectors, pUCS75 retains the primary site for second-strand synthesis (ssi) of the parental streptomycete replicon, pIJ101 . This sequence can not only enhance structural stability of the plasmid, but also confers on it an elevated copy number when replicated in Streptomyces . Consequently, the vector is useful for cloning sequences containing repeat structures and for allowing the high-level expression of cloned genes.

Gene, 1996 May 24, 171(1), 19 - 25
Sequence analysis and characterization of the hmw gene cluster of Mycoplasma pneumoniae; Dirksen LB et al.; Mycoplasma pneumoniae (Mp) cytadherence requires the proper anchoring of cytadhesin proteins in the mycoplasmal membrane at an attachment organelle through their interaction with a cytoskeleton-like network of accessory proteins that includes HMW1 and HMW3 . Approximately 8.25 kb of Mp DNA was sequenced, beginning at the 3' end of the hmw3 gene and continuing through hmw1 . Comparison of the resulting deduced amino acid (aa) sequence with N terminus and internal peptide aa sequences from purified HMW1 permitted definitive identification of hmw1 . HMW1 was characterized with respect to structure, hydrophobicity, possible phosphoacceptor sites and expression of the Mp recombinant protein in Escherichia coli . In addition, HMW1 membrane topography was examined for antibody accessibility on the mycoplasmal surface . hmw3 and hmw1 flank four open reading frames (ORFs) spanning approximately 4.3 kb and in the same orientation as the hmw genes . The sequences of their deduced products were evaluated for likely structural features and comparison with protein data banks . Finally, the Mp rpsD analog was identified immediately downstream from hmw1.

Gene, 1996 May 24, 171(1), 135 - 6
Gene organization in the ada-rplL region of Streptomyces virginiae; Katayama M et al.; The gene organization of a 7.4-kb region of the Streptomyces virginiae (Sv) chromosome was determined . The predicted open reading frames (ORFs) and their predicted products, in sequence order, were (i) ada, encoding adenosine deaminase {EC 3.5.4.4}, (ii) aat, encoding a protein homologous to aspartate aminotransferase {EC 2.6.1.1}, (iii) secE, encoding a protein involved in protein secretion, (iv) vbrA, encoding a NusG-like protein involved in antitermination of transcription as described by Okamoto et al . {J . Biol . Chem . 267 (1992) 1093-1098}, and (v) rplKAJL, encoding the large subunits of the ribosomal proteins L11, L1, L10 and L12 . Six of the ORFs (secE-rplL) were oriented in the same direction, but the other two (ada and aat) had the opposite orientation . The gene organization of the secE-rplL region in Sv was identical to that in Escherichia coli.

Gene, 1996 May 24, 171(1), 1 - 8
Identification of a biologically significant DNA-binding peptide motif by use of a random phage display library; Cheng X et al.; A peptide library approach was used to identify peptides that could bind to different DNA structures . A 23-mer random peptide library was displayed in the context of the pIII protein of M13 filamentous phage . Double-stranded (ds) oligodeoxyribonucleotides (oligos) were immobilized in 96-well plates using either chemical conjugation or a biotin-avidin linking method . Individual phage clones capable of binding to immobilized oligos were selected from the phage library . Using a plaque dilution assay for rapid screening of binding preferences, four groups of oligo-binding (OB) phage were tentatively identified as showing preference for: (1) single-stranded (ss) oligos irrespective of sequence; (2) ds oligos irrespective of sequence; (3) sequence-specific binding to ss oligos; and (4) weak non-specific binding to all types of oligos tested . A quantitative solution-phase competition assay was used to confirm the ability of certain phage to discriminate ss from ds oligos . A consensus motif, FGRA, was found in those phage clones that preferentially bound ss oligos; this motif has previously been noted in the binding domains of several ribonucleoproteins and ss DNA-binding proteins . Peptides based on the FGRA motif, but not scrambled controls, were able to inhibit the binding of appropriate phage clones or of Escherichia coli ss DNA-binding protein to oligos . This suggests that amino acid sequences that are capable of affecting biologically significant protein-DNA interactions can be identified from random peptide libraries using phage display techniques.

J Biol Chem, 1996 May 24, 271(21), 12372 - 9
Differential effect of precursor ribose binding protein of Escherichia coli and its signal peptide on the SecA penetration of lipid bilayer; Ahn T et al.; Digestion of vesicle-bound SecA by trypsin entrapped within the vesicles showed that refolding precursor ribose-binding protein (pRBP) of Escherichia coli retards the lipid bilayer penetration by SecA while the signal peptide enhances it . This discrepancy was found to be due to reduced SecA binding to the vesicles in the presence of the pRBP while the signal peptide induced a tight binding . Studies on the binding of 1-anilino-8-naphthalene sulfonate (ANS) to SecA indicated that SecA assumes more closed conformation upon interaction with pRBP and signal peptide induces more open structure of SecA . Kinetic studies of ANS binding to SecA upon dilution of unfolded pRBP with SecA solution showed an initial fast ANS binding, which was followed by a slow release of ANS . This suggests that first the signal peptide portion of the pRBP binds with the SecA making its structure more open and then the subsequent binding of the mature domain makes the SecA structure more compact . The pRBP enhanced the digestion of SecA added to the E . coli inverted vesicles, suggesting an inhibition of SecA penetration while the signal peptide had an opposite effect, agreeing with the results from the model systems above . When the pRBP and ATP were present together, however, the penetration of SecA increased dramatically underlining the importance of the SecY/E complex for the membrane insertion of SecA.

J Biol Chem, 1996 May 24, 271(21), 12205 - 8
Spermidine-preferential uptake system in Escherichia coli . Identification of amino acids involved in polyamine binding in PotD protein; Kashiwagi K et al.; Spermidine-binding sites on PotD protein, substrate-binding protein in periplasm, in the spermidine-preferential uptake system in Escherichia coli were studied by measuring polyamine transport activities of right-side-out membrane vesicles with mutated PotD proteins prepared by site-directed mutagenesis of the potD gene and by measuring polyamine binding activities of these mutated PotD proteins . Polyamine transport activities of the mutated PotD proteins paralleled their polyamine binding activities . It was found that Trp-34, Thr-35, Glu-36, Tyr-37, Ser-83, Tyr-85, Asp-168, Glu-171, Trp-229, Trp-255, Asp-257, Tyr-293, and Gln-327 of PotD protein were involved in the binding to spermidine . When spermidine uptake activities were measured in intact cells expressing the mutated PotD proteins, it was found that Glu-171, Trp-255, and Asp-257 were more strongly involved in the binding of spermidine to PotD protein than the other amino acids listed above . The dissociation constants of spermidine for the mutated PotD proteins at Glu-171, Trp-255, and Asp-257 increased greatly in comparison with those for the other mutated PotD proteins . Since these three amino acids clearly interact with the diaminopropane moiety of spermidine, the results are in accordance with the finding that PotD protein has a higher affinity for spermidine than for putrescine . Putrescine was found to bind at the position of the diaminobutane moiety of spermidine.

J Biol Chem, 1996 May 24, 271(21), 12141 - 4
Enzyme-DNA interactions required for efficient nucleotide incorporation and discrimination in human DNA polymerase beta; Beard WA et al.; In the crystal structure of a substrate complex, the side chains of residues Asn279, Tyr271, and Arg283 of DNA polymerase beta are within hydrogen bonding distance to the bases of the incoming deoxynucleoside 5'-triphosphate (dNTP), the terminal primer nucleotide, and the templating nucleotide, respectively (Pelletier, H., Sawaya, M . R., Kumar, A., Wilson, S . H., and Kraut, J . (1994) Science 264, 1891-1903) . We have altered these side chains through individual site-directed mutagenesis . Each mutant protein was expressed in Escherichia coli and was soluble . The mutant enzymes were purified and characterized to probe their role in nucleotide discrimination and catalysis . A reversion assay was developed on a short (5 nucleotide) gapped DNA substrate containing an opal codon to assess the effect of the amino acid substitutions on fidelity . Substitution of the tyrosine at position 271 with phenylalanine or histidine did not influence catalytic efficiency (kcat/Km) or fidelity . The hydrogen bonding potential between the side chain of Asn279 and the incoming nucleotide was removed by replacing this residue with alanine or leucine . Although catalytic efficiency was reduced as much as 17-fold for these mutants, fidelity was not . In contrast, both catalytic efficiency and fidelity decreased dramatically for all mutants of Arg283 (Ala > Leu > Lys) . The fidelity and catalytic efficiency of the alanine mutant of Arg283 decreased 160- and 5000-fold, respectively, relative to wild-type enzyme . Sequence analyses of the mutant DNA resulting from short gap-filling synthesis indicated that the types of base substitution errors produced by the wild-type and R283A mutant were similar and indicated misincorporations resulting in frequent T.dGTP and A.dGTP mispairing . With R283A, a dGMP was incorporated opposite a template thymidine as often as the correct nucleotide . The x-ray crystallographic structure of the alanine mutant of Arg283 verified the loss of the mutated side chain . Our results indicate that specific interactions between DNA polymerase beta and the template base, but not hydrogen bonding to the incoming dNTP or terminal primer nucleotide, are required for both high catalytic efficiency and nucleotide discrimination.

J Mol Biol, 1996 May 24, 258(5), 732 - 5
Inter-ring communication is disrupted in the GroEL mutant Arg13 --> Gly; Ala126 --> Val with known crystal structure; Aharoni A et al.; The crystal structures of the chaperonin GroEL Arg13 --> Gly; Ala126 --> Val double mutant, without and in complex with ATP gamma S, have been determined at atomic resolution . Here, we show that the double mutation Arg13 --> Gly; Ala126 --> Val disrupts negative co-operativity between GroEL rings, with respect to ATP, but has little effect on the positive co-operativity within each ring . Our results help to explain why the double mutation facilitated the crystallization of GroEL and why breaking of dyad symmetry between rings is not observed in crystal structures of this mutant . Our results may also help to explain why the observed structural differences between the GroEL double mutant and its ATP gamma S-bound form are small.

Nature, 1996 May 23, 381(6580), 335 - 41
X-ray and NMR structure of human Bcl-xL, an inhibitor of programmed cell death; Muchmore SW et al.; THE Bcl-2 family of proteins regulate programmed cell death by an unknown mechanism . Here we describe the crystal and solution structures of a Bcl-2 family member, Bcl-xL (ref . 2) . The structures consist of two central, primarily hydrophobic alpha-helices, which are surrounded by amphipathic helices . A 60-residue loop connecting helices alpha1 and alpha2 was found to be flexible and non-essential for anti-apoptotic activity . The three functionally important Bcl-2 homology regions (BH1, BH2 and BH3) are in close spatial proximity and form an elongated hydrophobic cleft that may represent the binding site for other Bcl-2 family members . The arrangement of the alpha-helices in Bcl-xL is reminiscent of the membrane translocation domain of bacterial toxins, in particular diphtheria toxin and the colicins . The structural similarity may provide a clue to the mechanism of action of the Bcl-2 family of proteins.

Biochim Biophys Acta, 1996 May 23, 1294(2), 147 - 52
Interactions of Escherichia coli tryptophanase with quasisubstrates and monovalent cations studied by the circular dichroism and fluorescence methods; Ben-Kasus T et al.; The reaction of tryptophanase and its W330F and W248F mutant forms with quasi-substrates forming an external pyridoxal phosphate aldimine or quinonoid is accompanied by the appearance of a positive circular dichroism (CD) peak at 290 nm . The peak seems to arise from a Tyr residue undergoing reorientation during the reaction . The peak does not appear upon formation of non-covalent Michaelis complexes of the enzyme with quasi-substrates such as indolepropionate, beta-phenyllactate and alpha-methylphenylalanine . The non-covalent complexes and external aldimines exhibit similar absorption spectra but can be distinguished by their CD and by the intensity of their fluorescence . Formation of the non-covalent complexes leads to an increase in positive CD at 420 nm while formation of the external aldimines leads to disappearance of the positive CD at 420 nm and its replacement by negative CD; it also leads to strong quenching of the coenzyme fluorescence at 500 nm . The quantum yield of fluorescence of the external aldimines is 6-times lower than that of the internal aldimine . Activating cations (K+, NH4+) strongly diminish the intensity of a negative protein CD band at 275 nm . From a comparison of the intensity of this band in the spectra of the wild-type holo- and apoenzyme and in the tryptophan mutants, it was deduced that the band belongs to a Tyr residue, which may be a part of the cation-binding site or located in its immediate vicinity.

Biochim Biophys Acta, 1996 May 21, 1290(1), 9 - 17
Fluorescence emission properties of 8-azapurines and their nucleosides, and application to the kinetics of the reverse synthetic reaction of purine nucleoside phosphorylase; Wierzchowski J et al.; An extensive study has been made of the fluorescence emission properties of the neutral and ionic forms in aqueous medium of the azapurine nucleosides, 8-azaadenosine (8-azaAdo), 8-azainosine (8-azaIno), 8-azaguanosine (8-azaGuo), and their aglycons . The fluorescence of 8-azaGuo at pH 7 originates from its anionic species (pKa = 8.05, phi= 0.55), as is also the case for 8-azaIno (pKa = 8.0, phi = 0.02), whereas 8-azaAdo is a strong emitter (phi = 0.06) as the neutral species . By contrast the corresponding free 8-azapurines are only weakly fluorescent in aqueous medium, with the exception of 8-azaguanine (8-azaG) . Examination of the emission properties of N-substituted 8-azaguanines demonstrated that the observed blue emission of the neutral form of 8-azaG (phi = 0.05 to 0.33, dependent on lambda exc) originates from a minor tautomer of the compound, the N(8)-H form, present to the extent of 10-15%; while the principal N(9)-H tautomer is virtually nonfluorescent . The 8-azapurines are substrates of purine nucleoside phosphorylase (PNP), leading to their irreversible conversion to the corresponding nucleosides in the synthetic pathway of this enzyme . The fluorescent properties of these compounds, together with spectrophotometric methods, were applied to determine the basic kinetic parameters for synthesis of 8-azapurine nucleosides by PNP from mammalian (calf spleen) and bacterial (Escherichia coli) sources . The fluorimetric method was also used to determine the kinetic parameters for the second substrate, alpha-D-ribose 1-phosphate, and for the analytical titration of the latter in solution . The pH optimum of the reverse synthetic PNP reaction with 8-azapurines as substrates is below pH 7, due to their enhanced acidity in comparison with natural purines . The 8-azapurine nucleosides, but not their aglycons, are reasonably good inhibitors of phosphorolysis of Ino and Guo by E . coli PNP . The most effective is 8-azaIno (Ki approximately 20 microM), also the only one to inhibit phosphorolysis by the calf spleen enzyme (Ki approximately 40 microM) . The nature of this inhibition is apparently uncompetitive.

Biochim Biophys Acta, 1996 May 21, 1290(1), 1 - 3
Cloning and sequencing of trehalose synthase gene from Pimelobacter sp . R48; Tsusaki K et al.; The gene encoding trehalose synthase (catalyzing the conversion of maltose into alpha, alpha-trehalose by intramolecular transglucosylation) was cloned from Pimelobacter sp . R48 . Sequence analysis revealed a 1719-bp synthase gene and a 573-residue amino-acid sequence . The 220 N-terminal residues were homologous to those of maltases from Saccharomyces carlsbergensis and Aedes aegypti.

Biochemistry, 1996 May 21, 35(20), 6500 - 7
Deletion of a B800-850 light-harvesting complex in Rhodospirillum molischianum DSM119 leads to "revertants" expressing a B800-820 complex: insights into pigment binding; Sauer PR et al.; A B800-850 light-harvesting complex (also called LH2) deficient strain of Rhodospirillum molischianum was constructed by replacing a portion of the LH2 gene cluster by a kanamycin resistance gene cartridge . The LH2 deficient strain was characterized spectroscopically and by Southern blot analysis . Surprisingly, pseudorevertants were obtained which express a B800-820 complex which could not be observed in the wild type . This B800-820 complex was isolated and characterized . It consists of an alpha- and a beta subunit with 56 and 45 amino acid residues, respectively . The amino acid sequences of both subunits are extremely similar to those of the corresponding B800-850 complex . Resonance Raman spectroscopy shows that in the B800-820 complex the two 2-acetylcarbonyl groups of the bacteriochlo-rophyll a (BChl a) molecules absorbing at 820 nm are free from hydrogen bond interactions, whereas one of the two 2-acetylcarbonyl groups of the pair of BChl a molecules absorbing at 850 nm of the B800- 850 complex is involved in hydrogen bonds . These different protein- pigment interactions are due to the replacement of alpha Trp43 in the B800-850 complex by a Phe in the B800- 820 complex . Comparison of the amino acid sequences of the B800-850 and B800-820 complexes of Rs . molischianum and Rhodopseudomonas acidophila reveals a conserved motif comprised of three amino acid residues . Molecular modeling using the known LH2 structure of Rps . acidophila Ac 10050 indicates that this motif might be important for the precise structural arrangement of the native complex and fine tuning of its spectroscopic properties.

Biochemistry, 1996 May 21, 35(20), 6470 - 82
The acid-mediated denaturation pathway of transthyretin yields a conformational intermediate that can self-assemble into amyloid; Lai Z et al.; Transthyretin (TTR) amyloid fibril formation is observed during partial acid denaturation and while refolding acid-denatured TTR, implying that amyloid fibril formation results from the self-assembly of a conformational intermediate . The acid denaturation pathway of TTR has been studied in detail herein employing a variety of biophysical methods to characterize the intermediate(s) capable of amyloid fibril formation . At physiological concentrations, tetrameric TTR remains associated from pH 7 to pH 5 and is incapable of amyloid fibril formation . Tetrameric TTR dissociates to a monomer in a process that is dependent on both pH and protein concentration below pH 5 . The extent of amyloid fibril formation correlates with the concentration of the TTR monomer having an altered, but defined, tertiary structure over the pH range of 5.0-3.9 . The inherent Trp fluorescence-monitored denaturation curve of TTR exhibits a plateau over the pH range where amyloid fibril formation is observed (albeit at a higher concentration), implying that a steady-state concentration of the amyloidogenic intermediate with an altered tertiary structure is being detected . Interestingly, 1-anilino-8-naphthalenesulfonate fluorescence is at a minimum at the pH associated with maximal amyloid fibril formation (pH 4.4), implying that the amyloidogenic intermediate does not have a high extent of hydrophobic surface area exposed, consistent with a defined tertiary structure . Transthyretin has two Trp residues in its primary structure, Trp-41 and Trp-79, which are conveniently located far apart in the tertiary structure of TTR . Replacement of each Trp with Phe affords two single Trp containing variants which were used to probe local pH-dependent tertiary structural changes proximal to these chromophores . The pH-dependent fluorescence behavior of the Trp-79-Phe mutant strongly suggests that Trp-41 is located near the site of the tertiary structural rearrangement that occurs in the formation of the monomeric amyloidogenic intermediate, likely involving the C-strand-loop-D-strand region . Upon further acidification of TTR (below pH 4.4), the structurally defined monomeric amyloidogenic intermediate begins to adopt alternative conformations that are not amyloidogenic, ultimately forming an A-state conformation below pH 3 which is also not amyloidogenic . In summary, analytical equilibrium ultracentrifugation, SDS-PAGE, far- and near-UV CD, fluorescence, and light scattering studies suggest that the amyloidogenic intermediate is a monomeric predominantly beta-sheet structure having a well-defined tertiary structure.

Biochemistry, 1996 May 21, 35(20), 6425 - 37
Domain closure in adenylate kinase; Sinev MA et al.; The method of time-resolved dynamic nonradiative excitation energy transfer (ET) was used to analyze the proposed domain closure in adenylate kinase (AK) . A highly active mutant of Escherichia coli AK, (C77S, V169W, A55C)-AK, was prepared, in which the solvent- accessible residues valine 169 and alanine 55 were replaced by tryptophan (the donor of excitation energy) and cysteine, respectively . The latter was subsequently labeled with either 5- or 4-acetamidosalicylic acid (the acceptor) . From the comparative analysis of AK crystal structures {Schulz, G.E., Muller, C.W., & Diederichs, K . (1990) J . Mol . Biol . 213, 627-630} (apo-AK,AK.AMP complex and AK.AP5A {P1,P5-di(adenosine-5') pentaphosphate} complex), "sequential formation" of the pseudoternary AK.AP5A complex is followed by two- step domain closure . The domain closure reduces interdomain distances in a two-step manner . Specifically, the distance between C alpha-atoms of the residues 169 and 55 (numbers correspond to those of E . coli AK) is decreased from 23.6 A in the apo-enzyme to 16.2 A upon the formation of the AK.AMP complex and to 12.3 A upon the further formation of the pseudoternary AK.AP5A complex . Time-resolved dynamic nonradiative excitation energy transfer was measured for the following ligand forms of the labeled derivative of the mutant enzyme: the apo-enzyme, the enzyme-MgATP complex, the enzyme.AMP complex, and the enzyme.AP5A "ternary" complex . The transfer efficiencies, which were determined in these experiments, were approximately 7.5%, 22%, 33%, and 65%, respectively . Global analyses of the time resolved ET experiments with the same ligand forms yielded intermolecular distance distributions with corresponding means of 31, 23, 19, and 12 A and full widths at half- maximum of 29, 24, 14, and 11 A . The data confirmed the proposed stepwise manner of the domain closure of the enzyme and revealed the presence of multiple conformations of E . coli AK in solution.

Biochemistry, 1996 May 21, 35(20), 6351 - 7
Interaction of cytochrome c with flavocytochrome b2; Daff S et al.; Flavocytochrome b2 from Saccharomyces cerevisiae couples L-lactate dehydrogenation to cytochrome c reduction . At 25 degrees C, 0.10 M ionic strength, and saturating L-lactate concentration, the turnover rate is 207 s-1 {per cytochrome c reduced; Miles, C . S., Rouviere, N., Lederer, F., Mathews, F . S., Reid, G . A., Black, M . T., & Chapman, S . K . (1992) Biochem . J . 285, 187-192} . The second-order rate constant for cytochrome c reduction in the pre-steady-state has been determined by stopped-flow spectrophotometry to be 34.8 (+/- 0.9) muM-1 s-1 in the presence of 10 mM L-lactate . This rate constant has been found to be dependent entirely on the rate of complex formation, the electron-transfer rate in the pre-formed complex being in excess of 1000 s-1 . Inhibition of the pre-steady-state reduction of cytochrome c by either zinc-substituted cytochrome c or ferrocytochrome c has led to the estimation of a Kd for the catalytically competent complex of 8 microM, and from this the dissociation rate constant of 280 s-1, a value much less than the actual electron-transfer rate . The inhibition observed is only partial which indicates that electron transfer from the 1:1 complex to another cytochrome c can occur and that alternative electron transfer sites exist . The cytochrome c binding site proposed by Tegoni et al . {Tegoni, M., White, S . A., Roussel, A., Mathews, F . S . & Cambillau, C . (1993) Proteins 16, 408-422} has been tested using site-directed mutagenesis . Mutations designed to affect the complex stability and putative electron-transfer pathway had little effect, suggesting that the primary cytochrome c binding site on flavocytochrome b2 lies elsewhere . The combination of tight binding and multiple electron-transfer sites gives flavocytochrome b2 a low K(m) and a high kcat, maximizing its catalytic efficiency . In the steady-state, the turnover rate is therefore largely limited by other steps in the catalytic cycle, a conclusion which is discussed in the preceding paper in this issue {Daff, S., Ingledew, W . J., Reid, G . A., & Chapman, S . K . (1996) Biochemistry 35, 6345-6350}.

Biochemistry, 1996 May 21, 35(20), 6321 - 9
Purification and characterization of two monomeric kinesin constructs; Moyer ML et al.; Steady-state and pre-steady-state kinetic methods were used to analyze two shorter Drosophila kinesin constructs (K341 and K366) in comparison to K401 . K341, K366, and K401 represent the kinesin motor domains containing the N-terminal 341, 366, or 401 amino acids, respectively . K401 is dimeric (Kd = 37 +/- 17 nM) whereas both K366 and K341 are monomeric {Correia et al . (1995) Biochemistry 34, 4898-4907} . Like native kinesin and K401, K341 and K366 demonstrate low ATPase activity in the absence of microtubules (0.03 and 0.01 s-1, respectively), and ADP release is rate-limiting during steady-state turnover . Microtubules activate the steady-state ATPase to 84 s-1 for K341 (K(m),ATP = 100 microM; K0.5,MT = 3.2 microM tubulin) and 64 s-1 for K366 (K(m),ATP = 65 microM; K0.5,MT = 2.5 microM tubulin) in comparison to K401 at 20 s-1 (K(m)ATP = 60 microM; K0.5,MT = 1 microM tubulin) . The rapid quench experiments for all three constructs show a burst of product formation during the first turnover, indicating the rate-limiting step for the microtubule-activated ATPase occurs after ATP hydrolysis . The interaction of K341 and K366 with the microtubule was analyzed by electron microscopy . The results show that K341 and K366, like K401, bind to the microtubule with an 8 nm axial periodicity . However, the addition of K366 to microtubules resulted in significant aggregation of microtubules . The pre-steady-state kinetic results show that K341 retains the kinetic and structural properties necessary to compare directly the kinetic properties of monomeric and dimeric kinesins, although the microtubule-activated ATPase is significantly faster for the monomeric constructs, suggesting possible interactions in the dimer which inhibit ATP turnover as part of the coupling to force production.

Biochim Biophys Acta, 1996 May 20, 1300(3), 219 - 25
Induction of lipid-protein mismatch by xenobiotics: kinetic cooperativity; Sandermann H Jr et al.; Lipophilic inhibitors such as general anaesthetics or drugs can conceivably act by displacing boundary lipid molecules that are required by many functional membrane proteins . The resulting lipid-protein mismatch has been analyzed previously in terms of multiple site kinetics (Sandermann H . (1993) Biochim . Biophys . Acta 1150, 130-133) . Expressions for kinetic cooperativity are now derived, and data for the inhibition of dog kidney Na+,K+-ATPase and Escherichia coli lactose permease by organic solvents are presented and analyzed . Half-maximal inhibitor concentrations were without diagnostic value because they were within the general range of critical solvent concentrations known for general anaesthesia and several membraneous and non-membraneous systems, as well as two specific liposomal parameters . The kinetic cooperativity of inhibition was of much higher diagnostic value because the cooperativity values for the solvent inhibition of Na+,K+-ATPase and lactose permease were characteristic for the lipid displacement mechanism, in contrast to cooperativity values of protein kinase C and luciferase . The latter enzymes are known not to require a boundary lipid layer, so that the degree of kinetic cooperativity provides a new diagnostic tool to distinguish between modes of action of lipophilic inhibitors.

FEBS Lett, 1996 May 20, 386(2-3), 177 - 80
RpoS-dependent regulation of genes expressed at late stationary phase in Escherichia coli; Talukder AA et al.; We have identified 6 Escherichia coli genomic genes, including 4 new genes, responsive to the stationary phase . One of them was regulated positively by RpoS at the stationary phase, and the remaining 5 negatively at a late stationary phase, all of them responding to multiple environmental stresses . Nucleotide sequences as well as such multiple responses revealed that those genes may have more than one overlapping-promoter recognized by different sigma-factors which regulate gene expressions during their cell growth.

Biochim Biophys Acta, 1996 May 20, 1274(1-2), 67 - 72
Interactions of the F1-ATPase subunits from Escherichia coli detected by the yeast two-hybrid system; Moritani C et al.; Subunit interactions among the F1-ATPase subunits were studied by the yeast two-hybrid system . Various pairwise combinations of genes encoding alpha, beta, gamma, delta and epsilon subunits of Escherichia coli H+-ATPase fused to the DNA-binding or activation domain of the yeast GAL4 gene were introduced into yeast and expression of a reporter gene encoding beta-galactosidase was detected . Combinations of the alpha and beta subunit genes, and of the epsilon and gamma subunit genes showed high levels of reporter gene expression, while those of alpha and delta, beta and delta, gamma and delta, and delta and epsilon demonstrated weak but significant reporter gene expression . However, combinations of alpha and gamma, beta and gamma, alpha and epsilon, and beta and epsilon did not induce reporter expression . None of the fused genes alone induced reporter gene expression . These results suggested that specific and strong interactions between the alpha and beta, gamma and epsilon, and weak interactions between the alpha and delta, beta and delta, and gamma and delta subunits occurred in yeast cells in the two-hybrid system . Effects of previously identified mutant beta subunits with Leu-40 to Pro . Glu-41 to Lys or Pro-332 to Gln substitutions which caused defects in molecular assembly of F1-ATPase were analyzed with regard to alpha-beta interactions . No interaction of the alpha and beta subunits was observed in this system using the beta subunit with mutation of Pro-332 to Gln . However, for the other two mutations, alpha-beta interactions were observed . This system may be useful for isolating mutants which have defects in interaction of F1-ATPase subunits.

Biochem Pharmacol, 1996 May 17, 51(10), 1373 - 8
Novenamines as inhibitors of two independent enzymes during DNA replication in a toluenized Escherichia coli cell system; Althaus IW et al.; The amphiphilic novenamines described in this report have been shown previously to be specific inhibitors of human immunodeficiency virus type 1 reverse transcriptase-associated ribonuclease, which they inhibit when they are in the micellar state but not when they are monomeric . These compounds also inhibit the bacterial enzyme DNA gyrase, which is essential for DNA replication . Hence, the present studies were initiated to determine whether the molecular species inhibiting the gyrase reaction was the monomeric or the micellar form . For this purpose, the rate of DNA replication was measured in a toluenized Escherichia coli cell system in the presence of increasing concentrations of novenamines . The resulting concentration-response curves proved anomalous, suggesting the involvement of micelles or some other, noncovalently aggregated forms of the inhibitors . The results were analyzed in terms of a variety of kinetic schemes and were found to be most consistent with the model where novenamines inhibit replicative DNA synthesis predominantly as cooperative dimers and, to a lesser extent, as monomers, but not as highly aggregated micelles . Based on this analysis and the knowledge that novobiocin and all novenamine-containing analogs are powerful gyrase inhibitors, we conclude that the target of the cooperative, dimeric inhibition is the gyrase, whereas the monomers of the novenamines inhibit another enzyme species involved in the bacterial DNA replication process.

J Biol Chem, 1996 May 17, 271(20), 11615 - 8
A phospholipid acts as a chaperone in assembly of a membrane transport protein; Bogdanov M et al.; A mutant of Escherichia coli lacking phosphatidylethanolamine (PE) and a monoclonal antibody (mAb 4B1) directed against a conformationally sensitive epitope (4B1) of lactose permease were used to establish a novel role for a phospholipid in the assembly of a membrane protein . Epitope 4B1 is readily detectable in spheroplasts and right-side-out membrane vesicles from PE-containing but not from PE-deficient cells expressing lactose permease . Lactose permease from PE-containing membranes, but not from PE-deficient membranes, subjected to sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis and Western blot analysis is also recognized by mAb 4B1 . If total E . coli phospholipids or PE (but not phosphatidylcholine, phosphatidylglycerol, or cardiolipin) are blotted on nitrocellulose sheets (Eastern blot) prior to transfer of proteins from SDS-polyacrylamide gels, the permease from PE-deficient cells regains its recognition by mAb 4B1 . Therefore, PE is required during assembly to form epitope 4B1, but, once formed, sufficient "conformational memory" is retained in the permease to either retain or reform this epitope in the absence of PE . Lactose permease lacking epitope 4B1 can be induced to form the epitope if partially denatured and then renatured in the presence of PE specifically . These results establish for the first time a role for PE as a molecular chaperone in the assembly of the lactose permease.

J Biol Chem, 1996 May 17, 271(20), 11659 - 67
A mutant RNA polymerase reveals a kinetic mechanisms for the switch between nonproductive stuttering synthesis and productive initiation during promoter clearance; Jin DJ; During transcription initiation from galP2, one of the two promoters of the Escherichia coli galactose operon with an initially transcribed sequence of pppAUUUC, RNA polymerase (RNAP) is known to engage nonproductive stuttering synthesis, which is sensitive to the concentration of UTP . This study examines the effect of this nonproductive synthesis on promoter clearance and determines other parameters that might affect stuttering synthesis by analyzing a mutant RNAP, RpoB3449, that has altered its function at this process at galP2 . RpoB3449 has dramatically diminished stuttering synthesis, and consequently, it has increased the rate of productive initiation due to its enhanced rate of promoter clearance of galP2 compared with wild-type RNAP . Thus, a direct linkage between promoter clearance and productive transcription is demonstrated . The mechanism by which the mutant RNAP has altered the switch between nonproductive stuttering synthesis and productive initiation during promoter clearance is studied . Apparently, RpoB3449 has increased its efficiency in incorporating CTP at the +5 position of the galP2 transcript leading to its reduced stuttering synthesis, indicating that the rate of an RNAP incorporating the CTP after a stretch of uridine residues is important for promoter clearance at galP2 . Because RpoB3449 demonstrates "wild-type" stuttering synthesis at the mutant galP2 promoter, which contains the 6 residue at the +5 position, it indicates that the mutant RNAP has altered in binding CTP at this context . Further experiments indicate that it is the +5 position per se of the galP2 sequence rather than a particular nucleotide at that position that is critical in determining the switch between the two alternate pathways during transcription initiation . A checkpoint model for the switch between nonproductive and productive initiations during promoter clearance is discussed.

J Biol Chem, 1996 May 17, 271(20), 11996 - 2002
The DNA binding site(s) of the Escherichia coli RecA protein; Rehrauer WM et al.; Photochemical cross-linking has been used to identify residues in the Escherichia coli RecA protein that are proximal to and may directly mediate binding of DNA . Ultraviolet irradiation promotes specific and efficient cross-linking of the RecA protein to poly(deoxythymidylic) acid . Cross-linked peptides remaining covalently attached to the polynucleotide following proteolytic digestion with trypsin correspond to amino acids 61-72, 178-183, and 233-243 of the RecA protein primary sequence . Their location and surface accessibility in the crystal structure, along with the behavior of various recA mutants, support the assignment of the cross-linked regions to the DNA binding site(s) of the RecA protein . Functional overlap of amino acids 61-72 with an element of the ATP binding site suggests a structural mechanism by which nucleotide cofactors allosterically affect the RecA nucleoprotein filament.

J Biol Chem, 1996 May 17, 271(20), 11904 - 10
Isolation of inositol 1,3,4-trisphosphate 5/6-kinase, cDNA cloning and expression of the recombinant enzyme; Wilson MP et al.; Inositol 1,3,4-trisphosphate 5/6-kinase was purified 12,900-fold from calf brain using chromatography on heparin-agarose and affinity elution with inositol hexakisphosphate . The final preparation contained proteins of 48 and 36-38 kDa . All of these proteins had the same amino-terminal sequence and were enzymatically active . The smaller species represent proteolysis products with carboxyl-terminal truncation . The Km of the enzyme for inositol 1,3,4-trisphosphate was 80 nM with a Vmax of 60 nmol of product/min/mg of protein . The amino acid sequence of the tryptic peptide HSKLLARPAGGLVGERTCNAXP matched the protein sequence encoded by a human expressed sequence tag clone (GB T09063) at 16 of 22 residues . The expressed sequence tag clone was used to screen a human fetal brain cDNA library to obtain a cDNA clone of 1991 base pairs (bp) that predicts a protein of 46 kDa . The clone encodes the amino-terminal amino acid sequence obtained from the purified calf brain preparation, suggesting that it represents its human homologue . The cDNA was expressed as a fusion protein in Escherichia coli and was found to have inositol 1,3,4-trisphosphate 5/6-kinase activity . Remarkably, both the purified calf brain and recombinant proteins produced both inositol 1,3,4,6-tetrakisphosphate and inositol 1,3,4,5-tetrakisphosphate as products in a ratio of 2.3-5:1 . This finding proves that a single kinase phosphorylates inositol in both the D5 and D6 positions . Northern blot analysis identified a transcript of 3.6 kilobases in all tissues with the highest levels in brain . The composite cDNA isolated contains 3054 bp with a poly(A) tail, suggesting that 500-600 bp of 5' sequence remains to be identified.

J Biol Chem, 1996 May 17, 271(20), 11945 - 52
Characterization of the vaccinia virus RNA 5'-triphosphatase and nucleotide triphosphate phosphohydrolase activities . Demonstrate that both activities are carried out at the same active site; Myette JR et al.; D1R1-545, an active subdomain of the large subunit of vaccinia virus mRNA capping enzyme possessing ATPase, RNA 5'-triphosphatase, and guanylyltransferase activities, was expressed in Escherichia coli and shown to be functionally equivalent to the heterodimeric enzyme (Myette, J . R., and Niles, E . G . (1996) J . Biol . Chem . 271, 11936-11944) . A detailed characterization of the phosphohydrolytic activities of D1R1-545 demonstrates that, in addition to ATPase and RNA 5'-triphosphatase activities, the capping enzyme also possesses a general nucleoside triphosphate phosphohydrolase activity that lacks a preference for the nucleoside base or sugar . Nucleoside triphosphate and mRNA saturation kinetics are markedly different, with RNA exhibiting a Km and turnover number 100- and 10-fold less, respectively, than those values measured for any NTP . The linear competitive inhibition of RNA 5'-triphosphatase activity by ATP, and the relative manner by which both ATPase and RNA 5'-triphosphatase activities are inhibited by specific oligonucleotides, kinetically demonstrate that each activity is carried out at a common active site . Direct UV photo-cross-linking of either 32P-radiolabeled ATP or 23-mer triphosphorylated RNA, followed by cyanogen bromide cleavage of the photo-linked enzyme, localizes the major binding site for both ATP and RNA to a region between amino acids 1 and 221 . The inability of ATP to competitively inhibit either E approximately GMP formation or the transfer of GMP to RNA kinetically differentiates the phosphohydrolase active site from the guanylyltransferase active site.

J Biol Chem, 1996 May 17, 271(20), 11936 - 44
Domain structure of the vaccinia virus mRNA capping enzyme . Expression in Escherichia coli of a subdomain possessing the RNA 5'-triphosphatase and guanylyltransferase activities and a kinetic comparison to the full-size enzyme; Myette JR et al.; The RNA 5'-triphosphatase, nucleoside triphosphate phosphohydrolase, and guanylyltransferase activities of the vaccinia virus mRNA capping enzyme were previously localized to an NH2-terminal 60-kDa domain of the D1R subunit . Measurement of the relative ATPase and guanylyltransferase activities remaining in D1R carboxyl-terminal deletion variants expressed in Escherichia coli BL21(DE3)plysS localizes the carboxyl terminus of the active domain to between amino acids 520 and 545 . Failure to obtain a deletion mutant with the loss of one activity indicates that the catalysis of both reactions requires a common domain structure . Based on these results, a truncated D1R protein terminating at amino acid 545 was expressed in E . coli and purified to homogeneity . D1R1-545 was found to be kinetically equivalent to the holoenzyme in regard to ATPase, RNA 5'-triphosphatase, and guanylyltransferase activities . Measurement of RNA binding by mobility shift and UV photo-cross-linking analyses also demonstrates the ability of this domain to bind RNA independent of the methyltransferase domain, comprised of the carboxyl terminus of D1R from amino acids 498-844 and the entire D12L subunit . RNA binding to D1R1-545 is substantially weaker than binding to either the methyltransferase domain or the holoenzyme . Binding is inhibited by 5'-OH RNA and to a lesser extent by DNA oligonucleotides in a concentration dependent manner which correlates with the inhibition of RNA 5'-triphosphatase activity by these same oligonucleotides . We conclude that D1R1-545 represents a functionally independent domain of the mRNA capping enzyme, fully competent in substrate binding and catalysis at both the triphosphatase and guanylyltransferase active sites.

J Biol Chem, 1996 May 17, 271(20), 11652 - 8
Recombinant N-terminal nucleotide-binding domain from mouse P-glycoprotein . Overexpression, purification, and role of cysteine 430; Dayan G et al.; Varying length cDNAs encoding the N-terminal nucleotide-binding domain (NBD1) from mouse mdr1 P-glyco- protein were prepared on the basis of structure predictions . Corresponding recombinant proteins were overexpressed in Escherichia coli, and the shortest one containing amino acids 395-581 exhibited the highest solubility . Insertion of an N-terminal hexahistidine tag allowed domain purification by nickel-chelate affinity chromatography . NBD1 efficiently interacted with nucleotides . Fluorescence methods showed that ATP bound at millimolar concentrations and its 2',3'-O-(2,4,6-trinitrophenyl) derivative at micromolar concentrations, while the 2'(3')-N-methylanthraniloyl derivative had intermediate affinity . Photoaffinity labeling was achieved upon irradiation with 8-azido-ATP . The domain exhibited ATPase activity with a Km for MgATP in the millimolar range, and ATP hydrolysis was competitively inhibited by micromolar 2',3'-O-(2,4,6-trinitrophenyl)-ATP . NBD1 contained a single cysteine residue, at position 430, that was derivatized with radiolabeled N-ethylmaleimide . Cysteine modification increased 6-fold the Kd for 2'(3')-N-methylanthraniloyl-ATP and prevented 8-azido-ATP photolabeling . ATPase activity was inhibited with a 5-fold increase in the Km for MgATP . The results suggest that chemical modification of Cys-430 is involved in the N-ethylmaleimide inhibition of whole P-glycoprotein by altering substrate interaction.

J Biol Chem, 1996 May 17, 271(20), 11750 - 5
A new glycosaminoglycan from the giant African snail Achatina fulica; Kim YS et al.; A new glycosaminoglycan has been isolated from the giant African snail Achatina fulica . This polysaccharide had a molecular weight of 29,000, calculated based on the viscometry, and a uniform repeating disaccharide structure of -->4)-2-acetyl,2-deoxy-alpha-D-glucopyranose (1-->4)-2-sulfo-alpha-L-idopyranosyluronic acid (1--> . This polysaccharide represents a new, previously undescribed glycosaminoglycan . It is related to the heparin and heparan sulfate families of glycosaminoglycans but is distinctly different from all known members of these classes of glycosaminoglycans . The structure of this polysaccharide, with adjacent N-acetylglucosamine and 2-sulfo-iduronic acid residues, also poses interesting questions about how it is made in light of our current understanding of the biosynthesis of heparin and heparan sulfate . This glycosaminoglycan represents 3-5% of the dry weight of this snail's soft body tissues, suggesting important biological roles for the survival of this organism, and may offer new means to control this pest . Snail glycosaminoglycan tightly binds divalent cations, such as copper(II), suggesting a primary role in metal uptake in the snail . Finally, this new polysaccharide might be applied, like the Escherichia coli K5 capsular polysaccharide, to the study of glycosaminoglycan biosynthesis and to the semisynthesis of new glycosaminoglycan analogs having important biological activities.

Mutat Res, 1996 May 17, 360(1), 51 - 74
A semi-automated, microplate version of the SOS Chromotest for the analysis of complex environmental extracts; White PA et al.; Environmental monitoring for genotoxicity requires that a large number of measurements be made across space and time . This requirement demands a rapid and efficient bioassay system . The SOS Chromotest is a rapid, efficient bacterial system for the detection of DNA damaging agents . Over 100 publications have described its use on a variety of samples . Relatively few studies have used the test to examine complex mixtures . Effective testing of complex samples poses a variety of problems . Although solutions have been proposed, few have validated the resulting protocol . In this work we present a semi-automated microplate version of the SOS Chromotest for the examination of complex mixtures . Experiments were conducted to determine the optimal cell concentration, exposure time, substrate conversion time and S9 enzyme concentration . The performance of the method was evaluated using 6 reference genotoxins and 3 complex mixtures . The complex mixtures examined are extracts of diesel particulate matter, urban dust and coal tar . The results obtained indicate that optimal responses often require fewer cells (approximately equal to 5-10 x 10(6) CFU/ml) and a longer exposure (3 h) than that recommended in the original protocol . Interfering effects of colored and turbid samples are removed using centrifugation and initial optical density readings taken 60 min after cell resuspension and lysis . The performance of the established protocol was evaluated using mitomycin C and benzo{a}pyrene results for 207 microplates and solvent control results for 293 microplates . The results indicate that the established method is accurate, sensitive and precise . Coefficient of variation on mean SOSIP values for mitomycin C and benzo{a}pyrene are < 5% . Solvent control data indicate that the standard threshold for determination of a positive response (induction factor > 1.5) is excessively conservative . All liquid transfers were automated using the Biomek automated laboratory workstation . Automation permits a throughput of up to 72 samples per day and maintains excellent precision and accuracy.

Cell, 1996 May 17, 85(4), 573 - 83
The 70 kDa S6 kinase complexes with and is activated by the Rho family G proteins Cdc42 and Rac1; Chou MM et al.; The 70 kDa ribosomol S6 kinase (pp70S6k) plays an important role in the progression of cells through G1 phase of the cell cycle . However, little is known of the signaling molecules that mediate its activation . We demonstrate that Rho family G proteins regulate pp70S6k activity in vivo . Activated alleles of Cdc42 and Rac1, but not RhoA, stimulate pp70S6k activity in multiple cell types . Activation requires an intact effector domain and isoprenylation of Cdc42 and Rac1 . Coexpression of Dbl, an exchange factor for Cdc42, also activates pp70S6k . Growth factor-induced activation of pp70S6k is abrogated by dominant negative alleles of Cdc42 and Rac1 . In addition, Cdc42 and Rac1 form GTP-dependent complex with the catalytically inactive form of pp70S6k in vitro and in vivo, suggesting a mechanism by which these G proteins activate pp70S6k.

J Mol Biol, 1996 May 17, 258(4), 614 - 26
Cloning, characterization and properties of plasmids containing CGG triplet repeats from the FMR-1 gene; Shimizu M et al.; The FMR-1 gene for the human fragile-chi syndrome, a mental retardation disease inherited by non-Mendelian transmission, contains a genetically unstable CGG region in the 5' non-translated region . The severity of the disease is correlated with the length of the CGG tract . The cloning of 28 stable plasmids containing (CGG)n inserts (where n = 6 to 240) with different extents and types of sequence interruptions (polymorphisms), and in different orientations was accomplished by three strategies in Escherichia coli . Some shorter tracts were prepared by the direct cloning of synthetic oligonucleotides, and longer runs were clones of multimers of (CGG)61, (CGG)11AGG(CGG)60CAG(CGG)8, from a cDNA from a fragile-chi patient or from expansions or deletions of these sequences in E . coli . The genetic stability of the inserts, especially for the longer tracts, was dependent on the sequence length, the presence of polymorphisms, the host cell genotypes, the orientation of the inserts in the vector and the position of cloning in a vector . Two-dimensional agarose gel electrophoresis studies on fully methylated and on non-methylated plasmids as well as chemical probe studies revealed the absence of underwound structures or accessible base-pairs . These DNAs enable a range of genetic and biochemical investigations into the molecular basis of the fragile-chi syndrome.

J Mol Biol, 1996 May 17, 258(4), 600 - 13
Mapping in three dimensions of regions in a catalytic RNA protected from attack by an Fe(II)-EDTA reagent; Westhof E et al.; The accessibility of the ribose groups in the phosphodiester chain of M1 RNA, the catalytic subunit of ribonuclease P from Escherichia coli, has been probed with an Fe(II)-EDTA reagent when the RNA is alone in solution, when it is in a complex with a tRNA precursor substrate, and when it is in the holoenzyme complex with its cofactor, C5 protein . The regions found to be protected under these various conditions, as well as those previously identified in other chemical probing experiments, have been mapped on a three-dimensional working model of M1 RNA and are generally compatible with the previously proposed placement of the substrate on the enzyme and with previous data and inferences regarding the interactions of C5 protein with M1 RNA . On the basis of the accessibilities of the C(4') atoms, refinements have been introduced in the model to accommodate the Fe(II)-EDTA protection data . The protein cofactor makes contact with several helical regions of the catalytic RNA on the opposite side of the surface to which substrates bind.

J Mol Biol, 1996 May 17, 258(4), 588 - 99
Suppression of temperature-sensitive defects of polypeptide release factors RF-1 and RF-2 by mutations or by an excess of RF-3 in Escherichia coli; Matsumura K et al.; The termination of protein synthesis in bacteria requires two codon-specific polypeptide-release factors, RF-1 and RF-2 . A third factor, RF-3, stimulates the RF-1 and RF-2 activities in vitro . To clarify the in vivo role of RF-3 for the RF-2 dependent termination, we isolated and characterized suppressor mutations for the temperature-sensitive RF-2 mutation prfB286 . One of the intergenic suppressor mutations, srb-1, acquired an up-promoter alteration in the RF-3 gene, which enhanced the RF-3 expression four- to fivefold . Consistently a threefold increase in the RF-3 level by a promoter-controlled expression plasmid suppressed prfB286 . On the other hand, a temperature-sensitive mutation in RF-1, prfA1, was suppressed only slightly by the high-level expression of wild-type RF-3 . The RF-3 mutations that suppress prfA1 were isolated and named sra . They were classified into four specific alleles; two each in the N and C-terminal regions . These altered RF-3 proteins restored the RF-1-dependent termination at UAG in prfA1 cells . Moreover, they enhanced the RF-2-dependent UGA termination in both wild-type and prfB286 cells . The termination-stimulating activity of RF-3 was further additively increased by the double sra mutations, suggesting that they affected two distinct protein domains that modulate the termination reaction . Taking these and other results into consideration, RF-3 is likely to interact functionally and cooperatively with the release factors RF-1 and RF-2 in Escherichia coli.

J Mol Biol, 1996 May 17, 258(4), 548 - 54
Deletion analysis of the che operon in the archaeon Halobacterium salinarium; Rudolph J et al.; Halobacterium salinarium is a chemo- and phototactic archaeon whose signal transduction pathway includes the classical two-component system made up of CheA and CheY . Deletion analysis of the che operon in H . salinarium has been undertaken . Following the removal of the entire operon, the importance of each of the four individual members, cheY, cheB, cheA, and the novel member cheJ, was evaluated by their replacement in combinations of three . The mutant strains were investigated for their motility, their chemo- and phototactic signalling, and the rotational bias of their flagella . Loss of cheA, cheY or cheB led to the complete loss of chemo- and phototaxis, whereas the absence of cheJ caused a reduction in chemo- and phototactic ability . Reverse swimming and counterclockwise rotation of the flagella required the presence of cheA and CheY . The wild-type 50:50 distribution of forward and reverse swimming was observed in the strain lacking cheB, whereas this distribution was perturbed to 88:12 in the strain lacking cheJ . These results are compared with the corresponding deletion strains in Escherichia coli and provide new insights into the eu- and archeabacterial flagellar switch.

J Mol Biol, 1996 May 17, 258(4), 543 - 7
CTG triplet repeats from the myotonic dystrophy gene are expanded in Escherichia coli distal to the replication origin as a single large event; Kang S et al.; The expansion and contraction of CTG and CGG trinucleotide repeat sequences have been associated with several heritable genetic diseases . We developed a system for investigating the expansion of triplet repeat sequences in Escherichia coli in order to elucidate molecular mechanisms . Analysis of expanded regions using the interrupting CTA triplet sequence as a location marker within the CTG tract revealed that the expansion of large CTG repeats is one event rather than an accumulation of multiple small expansions and that the expansions occur more frequently in the region distal from the replication origin . Also, we showed that a loss of interruptions increases the expansion frequency . Thus, the instability of large triplet repeats in hereditary diseases occurs by a mechanism different from the instability in microsatellite sequences caused by defects in mismatch repair systems for certain sporadic cancers and hereditary non-polyposis colorectal cancers.

Nature, 1996 May 16, 381(6579), 245 - 8
Localization of dopamine D4 receptors in GABAergic neurons of the primate brain; Mrzljak L et al.; Dopamine receptors are the principal targets of drugs used in the treatment of schizophrenia . Among the five mammalian dopamine-receptor subtypes, the D4 subtype is of particular interest because of its high affinity for the atypical neuroleptic clozapine . Interest in clozapine stems from its effectiveness in reducing positive and negative symptoms in acutely psychotic and treatment-resistant schizophrenic patients without eliciting extrapyramidal side effects . We have produced a subtype-specific antibody against the D4 receptor and localized it within specific cellular elements and synaptic circuits of the central nervous system . The D4-receptor antibody labelled GABAergic neurons in the cerebral cortex, hippocampus, thalamic reticular nucleus, globus pallidus and the substantia nigra (pars reticulata) . Labelling was also observed in a subset of cortical pyramidal cells . Our findings suggest that clozapine's beneficial effects in schizophrenia may be achieved, in part, through D4-mediated GABA modulation, possibly implicating disinhibition of excitatory transmission in intrinsic cortical, thalamocortical and extrapyramidal pathways.

J Biotechnol, 1996 May 15, 46(3), 255 - 63
Impact of plasmid presence and induction on cellular responses in fed batch cultures of Escherichia coli; Andersson L et al.; Fed batch cultivations of plasmid-free and recombinant Escherichia coli were employed in order to determine cellular responses and effects of plasmid presence and induction on the host cell physiology . While plasmid presence was shown to have minor influence on overall biomass yield, induction with 0.1 mM IPTG led to a marked reduction . The number of dividing cells, measured as colony forming ability, was influenced by plasmid presence and to a larger extent by induction . The latter caused a decline in the number of dividing cells to less than 10% of the population within 10 h . However, this cell segregation did not affect the specific rate of product formation, which was approximately constant throughout the cultivations . Analysis of the in vivo degradation rate of the product indicated that it was proteolytically stable . The cellular content of the stringent response signal substance, ppGpp, peaked immediately after transition from batch to fed batch mode to stabilise at a higher value than in the batch phase . When the specific growth rate declined below 0.06 h-1 an additional rise in ppGpp concentration was observed.

Biochem Biophys Res Commun, 1996 May 15, 222(2), 576 - 83
Characterization of a recombinant chimeric plasminogen activator composed of Gly-Pro-Arg-Pro tetrapeptide and truncated urokinase-type plasminogen activator expressed in Escherichia coli; Hua ZC et al.; A chimeric plasminogen activator, GPRP-u-PA (144-411), consisting of the Gly-Pro-Arg-Pro tetrapeptide fused to the N-terminal of a truncated urokinase-type plasminogen activator (comprising Leu 144 through Leu 411), was produced by expression of the corresponding chimeric cDNA in Escherichia coli cells . After renaturation, the chimera was purified to homogeneity with specific amidolytic activity of 100,000 IU/mg protein . The chimera showed 6-fold greater affinity for fibrin clots than native low molecular weight urokinase (LUK) and 1.5-fold greater affinity than a chemical conjugate, GPRP-LUK, generating via coupling Gly-Pro-Arg-Pro tetrapeptide to native low molecular weight urokinase . The chimera had 2 to 3 fold greater fibrinolytic potency than native LUK in vitro . Fibrinogen had no influence on fibrinolysis of the chimera . The chimera consumed much less fibrinogen than native LUK.

Biochem Biophys Res Commun, 1996 May 15, 222(2), 472 - 7
Subdomain structure of the matrix attachment region located within the mouse immunoglobulin kappa gene intron; Okada S et al.; Using the matrix attachment region (MAR) derived from Ig kappa gene intron, we assessed the importance of internal subregions required for the specific binding to the nuclear matrix . Relative affinities of MAR subfragments were compared in an in vitro binding reaction with isolated matrix . Cleavage at the near-centric MboII site generated two subfragments retaining a significant binding affinity . Dimerization of these subfragments greatly increased the affinity . Only a partial segment (130 bp) of the 3' fragment was necessary to restore the binding . The dimerization effect was lost when the monomer units were separated by nonMAR spacers of 500 bp < . This bipartite organization of Ig kappa MAR could be a general feature of AT-rich MARs, regardless of their genomic locations.

Biochem Biophys Res Commun, 1996 May 15, 222(2), 460 - 5
A sensitive RNase protection assay to detect transcripts from potentially functional human endogenous L1 retrotransposons; Woodcock DM et al.; A high background of read-through transcripts from degenerate human L1 retrotransposons is present in almost all human cell types . This prevents the detection of RNA transcripts from potentially functional elements . To overcome this, we have developed an RNase protection assay based on the reconstructed consensus sequence for the 5' end of the major L1 family . In the human Ntera2D1 teratocarcinoma cell line, this assay readily detected L1 transcripts that were located primarily in the cytoplasm and where 20% were in filterable particles . By this assay, potentially functional L1 elements are also transcriptionally active in lymphocytes from some but not all normal individuals . Together with the full length protection product, there were three other discrete L1 RNAs, two of which (305 and 275 bases) were transcribed from the 5' end of the L1 element . These smaller L1 RNAs do not appear to be derived from transcripts from divergent L1 families but are either discrete shorter transcripts or specifically processed products from longer initial transcripts.

Biochem Biophys Res Commun, 1996 May 15, 222(2), 439 - 44
High-level expression of mouse inducible nitric oxide synthase in Escherichia coli requires coexpression with calmodulin; Wu C et al.; We report a method to generate and purify large quantities of fully active mouse iNOS from E . coli, and show that calmodulin coexpression is essential to generate the active iNOS . E . coli were transformed with a plasmid containing mouse iNOS with a six-histidine tag on its N-terminus or were cotransformed with piNOS and a distinct plasmid that contained human calmodulin . Protein expression was induced by IPTG followed by culture at room temperature . Coexpression with calmodulin enabled production of active iNOS (20 mg/L culture), of which half could be recovered in pure form by sequential metal chelate and 2', 5' ADP Sepharose chromatography . The calmodulin-replete iNOS was dimeric, contained normal quantities of heme, flavins, and tightly bound calmodulin, and had high NO synthesis activity (0.7 - 1.2 mumol NO/min per mg) . In contrast, calmodulin-deficient iNOS was monomeric, devoid of flavins and heme, and had no NO synthesis activity . We conclude that calmodulin is essential to fold and stabilize mouse iNOS.

Biochem Biophys Res Commun, 1996 May 15, 222(2), 249 - 55
Expression of biologically active isoforms of the tumor angiogenesis factor VEGF in Escherichia coli; Siemeister G et al.; Vascular endothelial growth factor (VEGF) was identified as an endothelial cell specific mitogen that induces angiogenesis and vascular permeability in vivo . VEGF is a homodimeric protein which contains three intramolecular and two intermolecular disulfide bridges . Here, we report on an efficient procedure for recombinant production of VEGF isoforms VEGF121 and VEGF165 in Escherichia coli . The proteins were solubilized from inclusion bodies, refolded, and purified by chromatographic methods . The final protein products were almost completely in the dimeric conformation, bound to VEGF receptor FLT1 with a Kd of 30 pM, stimulated proliferation of human umbilical vein endothelial cells half-maximally at a concentration of 30 pM, and induced in vivo neovascularization and vascular permeability on the chicken chorioallantoic membrane.

Eur J Biochem, 1996 May 15, 238(1), 192 - 7
Conformational requirements of a recombinant ferredoxin-NADP+ reductase precursor for efficient binding to and import into isolated chloroplasts; Ceccarelli EA et al.; The cytosolic precursor of the chloroplast flavoprotein ferredoxin-NADP+ reductase was expressed in Escherichia coli rendering a soluble protein that contained bound FAD and could be imported by isolated chloroplasts . The mechanism of plastid translocation was studied under defined conditions using this recombinant precursor holoprotein and intact pea chloroplasts . The first step in the import pathway, namely, binding of the reductase precursor to isolated chloroplasts, was saturable at about 2000 molecules/plastid, and showed a high-affinity interaction with a dissociation constant Kd of approximately 5 nM . Binding was not affected by the addition of soluble leaf extracts or by prior denaturation of the precursor with urea . Analysis of the initial import rates at different precursor concentrations indicated the existence of a single translocation system for this protein . Inclusion of leaf extracts in the assay resulted in a three-fold increase of the maximal import rates to 14,000 molecules . min-(1).chloroplast-(1), with a concomitant decrease in the apparent Km for the recombinant precursor, from 1 microM to 100-150 nM . Comparison of Km and Kd values under various conditions indicated that the binding step of the translocation process is largely irreversible, favouring import and processing . In the absence of extract, a denatured precursor obtained by incubation with urea was a better substrate for plastid import than the holoprotein . Treatment of the precursor with either extract or urea resulted in similar increases in import efficiency (V/Km), suggesting that stimulation by leaf extracts is probably related to unfolding of the precursor prior to translocation.

Eur J Biochem, 1996 May 15, 238(1), 112 - 20
Reconstitution and pigment-binding properties of recombinant CP29; Giuffra E et al.; The minor light-harvesting chlorophyll-a/b-binding protein CP29 (Lhcb4), overexpressed in Escherichia coli, has been reconstituted in vitro with pigments . The recombinant pigment-protein complexes show biochemical and spectral properties identical to the native CP29 purified from maize thylakoids . The xanthophyll lutein is the only carotenoid necessary for reconstitution, a finding consistent with the structural role of two lutein molecules/polypeptide suggested by the crystallographic data for the homologous protein light-harvesting chlorophyll-a/b-binding protein of photosystem II (LHCII) . The CP29 protein scaffold can accommodate different chromophores . This conclusion was deduced by the observation that the pigment composition of the reconstituted protein depends on the pigments present in the reconstitution mixture . Thus, in addition to a recombinant CP29 identical to the native one, two additional forms of the complex could be obtained by increasing chlorophyll b content . This finding is typical of CP29 because the major LHCII complex shows an absolute selectivity for chromophore binding {Plumley, F . G . & Schmidt, G . W . (1987) Proc . Natl Acad . Sci . USA 84, 146-150; Paulsen, H., Rumler, U . & Rudiger, W . (1990) Planta (Heidelb.) 181, 204-211}, and it is consistent with the higher stability of CP29 during greening and in chlorophyll b mutants compared with LHCII.

EMBO J, 1996 May 15, 15(10), 2547 - 55
Multiple roles for divalent metal ions in DNA transposition: distinct stages of Tn10 transposition have different Mg2+ requirements; Junop MS et al.; Tn10 transposition takes place by a non-replicative mechanism in which the transposon is excised from donor DNA and integrated into a target site . Mg2+ is an essential cofactor in this reaction . We have examined the Mg2+ requirements at various steps in Tn10 transposition . Results presented here demonstrate that Tn10 excision can occur efficiently at a 16-fold lower Mg2+ concentration than strand transfer and that, at Mg2+ concentrations in the range of 60-fold below the wildt-ype optimum, double strand cleavage events at the two transposon ends are completely uncoupled . These experiments identify specific breakpoints in Tn10 transposition which are sensitive to Mg2+ concentration . Whereas the uncoupling of double strand cleavage events at the two transposon ends most likely reflects the inability of two separate IS10 transposase monomers in the synaptic complex to bind Mg2+, the uncoupling of transposon excision from strand transfer is expected to reflect either a conformational change in the active site or the existence of an Mg2+ binding site which functions specifically in target interactions . We also show that Mn2+ relaxes target specificity in Tn10 transposition and suppresses a class of mutants which are blocked specifically for integration . These observations can be explained by a model in which sequence-specific target site binding is tightly coupled to a conformational change in the synaptic complex which is required for catalysis of strand transfer.

EMBO J, 1996 May 15, 15(10), 2488 - 95
A sigma factor that modifies the circadian expression of a subset of genes in cyanobacteria; Tsinoremas NF et al.; We isolated mutants affected in the circadian expression of the psbAI gene in Synechococcus sp . strain PCC 7942 using a strategy that tags the genomic locus responsible for the mutant phenotype . The search identified one short period (22 h) mutant (M2) and two low amplitude mutants, one of which showed apparent arhythmia (M11) and one that was still clearly rhythmic (M16) . We characterized the disrupted locus of the low amplitude but still rhythmic mutant (M16) as the rpoD2 gene, a member of a gene family that encodes sigma70-like transcription factors in Synechococcus . We also inactivated rpoD2 in a number of reporter strains and showed that the circadian expression of some genes is not modified by the loss of this sigma factor . Therefore, we conclude that rpoD2 is a component of an output pathway of the biological clock that affects the circadian expression of a subset of genes in Synechococcus . This work demonstrates a direct link between a transcription factor and the manifestation of circadian gene expression.

EMBO J, 1996 May 15, 15(10), 2356 - 64
The channel domain of colicin A is inhibited by its immunity protein through direct interaction in the Escherichia coli inner membrane; Espesset D et al.; A bacterial signal sequence was fused to the colicin A pore-forming domain: the exported pore-forming domain was highly cytotoxic . We thus introduced a cysteine-residue pair in the fusion protein which has been shown to form a disulfide bond in the natural colicin A pore-forming domain between alpha-helices 5 and 6 . Formation of the disulfide bond prevented the cytotoxic activity of the fusion protein, presumably by preventing the membrane insertion of helices 5 and 6 . However, the cytotoxicity of the disulfide-linked pore-forming domain was reactivated by adding dithiothreitol into the culture medium . We were then able to co-produce the immunity protein with the disulfide linked pore-forming domain, by using a co-immunoprecipitation procedure, in order to show that they interact . We showed both proteins to be co-localized in the Escherichia coli inner membrane and subsequently co-immunoprecipitated them . The interaction required a functional immunity protein . The immunity protein also interacted with a mutant form of the pore-forming domain carrying a mutation located in the voltage-gated region: this mutant was devoid of pore-forming activity but still inserted into the membrane . Our results indicate that the immunity protein interacts with the membrane-anchored channel domain; the interaction requires a functional membrane-inserted immunity protein but does not require the channel to be in the open state.

EMBO J, 1996 May 15, 15(10), 2331 - 42
Molecular identification of zeaxanthin epoxidase of Nicotiana plumbaginifolia, a gene involved in abscisic acid biosynthesis and corresponding to the ABA locus of Arabidopsis thaliana; Marin E et al.; Abscisic acid (ABA) is a plant hormone which plays an important role in seed development and dormancy and in plant response to environmental stresses . An ABA-deficient mutant of Nicotiana plumbaginifolia, aba2, was isolated by transposon tagging using the maize Activator transposon . The aba2 mutant exhibits precocious seed germination and a severe wilty phenotype . The mutant is impaired in the first step of the ABA biosynthesis pathway, the zeaxanthin epoxidation reaction . ABA2 cDNA is able to complement N.plumbaginifolia aba2 and Arabidopsis thaliana aba mutations indicating that these mutants are homologous . ABA2 cDNA encodes a chloroplast-imported protein of 72.5 kDa, sharing similarities with different mono-oxigenases and oxidases of bacterial origin and having an ADP-binding fold and an FAD-binding domain . ABA2 protein, produced in Escherichia coli, exhibits in vitro zeaxanthin epoxidase activity . This is the first report of the isolation of a gene of the ABA biosynthetic pathway . The molecular identification of ABA2 opens the possibility to study the regulation of ABA biosynthesis and its cellular location.

Genomics, 1996 May 15, 34(1), 97 - 106
A human chromosome 22 fosmid resource: mapping and analysis of 96 clones; Birren BW et al.; We have created a resource for chromosome 22 consisting of 96 unique, well-characterized Fosmids . The Fosmid vector permits efficient cloning of DNA fragments averaging 40 kb in a single-copy vector based on the F factor of Escherichia coli . We have found that Fosmid clones from human chromosome 22 show remarkable stability and are useful for a wide variety of applications in genome analysis . These 96 clones have been localized by FISH, using high-resolution fluorescent banding and multicolor mapping techniques, and their position on the chromosome was correlated with their content of a number of common repeated sequence elements . We identified a subset of clones likely to contain genes by restriction analysis using the enzymes NotI, MluI, SacII, and BssHII . This collection of cytogenetically anchored clones, representing nearly 7% of the chromosome, is of immediate value for detecting chromosomal rearrangements, for use in gene isolation, and as a framework for physical mapping.

Ann N Y Acad Sci, 1996 May 15, 782, 441 - 55
Implementation in an expert system of a selection rationale for purification processes for recombinant proteins; Leser EW et al.; This work presents the development of an expert system for selecting the best sequence of operations for the downstream processing of proteins . The core of the discussion is how the rationale for the selection of a sequence of processes for purification was developed . It consists of a system that compares extensive data on the product to be purified and on the main contaminant proteins found in the expression system to be used (e.g . E . coli) . Definitions of parameters that translate (i) the rules employed in separations and (ii) the selection between high-resolution purification operations (chromatography) using the databases of properties of proteins in a rational quantitative manner to guide the selection are described . After each separation step, there is a reduction in the amount of contaminant proteins . The amount of each "contaminant" eliminated is determined using an algorithm developed for this purpose, based on simplified interpretations of chromatograms, that indicates the new concentrations after each step . The number of steps must be sufficient to achieve a defined level of purity . The comparison of the concentration of the product after the separation with the defined level of purity indicates whether an additional step is necessary.

Ann N Y Acad Sci, 1996 May 15, 782, 182 - 90
Optimization of recombinant gene expression in Escherichia coli; Mattanovich D et al.; The major targets for improvement of recombinant expression efficiency in Escherichia coli are gene dosage, transcription and, to some extent, translation . In order to evaluate the relative importance of these factors, the kinetics of specific mRNA compared to product formation was studied for different widely used expression systems, producing recombinant human superoxide dismutase . For a system employing phage T7 RNA polymerase, where a high level of recombinant protein expression puts a high metabolic burden on the cells, it was shown that transcription is not the limiting factor . To improve the translation rate of a common vector based on the tac promoter, the Shine-Dalgarno (SD) sequence was mutated towards stronger homology to the anti-SD sequence of the E . coli 16S rRNA . A 12.2-fold increase in protein yield was accompanied by a 4.3-fold increase in specific mRNA, indicating that transcription of the recombinant gene is coupled to translation . As this coupling amplifies the detrimental effect of a low-efficiency ribosomal binding site, much attention should be paid to translation initiation when optimizing a recombinant protein production system . Finally, reasons for the high expression level before induction are discussed, and first results towards reducing it are presented.

Nucleic Acids Res, 1996 May 15, 24(10), 1950 - 3
Apurinic/apyrimidinic (AP) endonuclease from Dictyostelium discoideum: cloning, nucleotide sequence and induction by sublethal levels of DNA damaging agents; Freeland TM et al.; We have cloned an AP endonuclease gene (APEA) from Dictyostelium discoideum, along with 1.8 kb of the 5' flanking region . There are no introns . The sequence predicts a protein of 361 amino acids, showing high homology to the major human/Escherichia coli exonuclease III family of AP endonucleases . There is 47% identity and 64% similarity to the Ape endonuclease of human cells using the C-terminal 257 amino acids of the Dictyostelium protein . The 104 amino acids on the N-terminus show only low homology with other AP endonucleases . Instead, this region shows high homology with the acid-rich regions of proteins associated with chromatin, such as nucleolins and HMG proteins . The gene is transcriptionally activated up to 7-fold after treatment of cells with sublethal levels of DNA damaging agents, including ultraviolet light, MNNG and bleomycin . Induction does not occur following blocking of replication fork polymerases with aphidicolin . It is not eliminated by treatment with kinase or phosphatase inhibitors . Four DNA damage-sensitive mutants all retained the DNA damage-induced up-regulation.

Nucleic Acids Res, 1996 May 15, 24(10), 1895 - 900
A nuclear matrix-specific factor that binds a specific segment of the negative regulatory element (NRE) of HIV-1 LTR and inhibits NF-kappa(B) activity; Hoover T et al.; The negative regulatory element (NRE) of human immunodeficiency virus type-1 (HIV-1) long terminal repeat (LTR) is a defined region that has been reported to downregulate LTR-directed HIV gene expression . However, information on the precise role of this region in regulating HIV gone transcription is lacking . We have investigated the possibility that these NRE sequences regulate HIV transcription by a mechanism mediated through a nuclear matrix-specific DNA-protein interaction . We find a nuclear matrix attachment region (MAR) present within the NRE of the HIV-1 LTR that recognizes a sequence-specific DNA-binding protein present in the nuclear matrix of HIV infected cells . Moreover, we also show that the purified DNA-binding nuclear matrix protein (NMP) specifically represses the DNA-binding activity of NF-kappaB . It is likely that the MAR and MAR-enriched specific DNA-binding NMP are brought into juxtaposition by the non-chromatin scaffolding of the nucleus, thus influencing NF-kappaB (and other nuclear proteins) DNA-binding activity through protein-protein and protein-DNA interactions . Our date suggest that one possible role of the NRE could be to act as a matrix attachment site in the nuclear matrix, thus, allowing interaction with a sequence-specific trans-acting factor . The negative effect on NF-kappaB activity due to this MAR-NMP-specific interaction provides a mechanism by which the NRE downregulates HIV gene expression.

Nucleic Acids Res, 1996 May 15, 24(10), 1855 - 64
The basic domain/leucine zipper protein hXBP-1 preferentially binds to and transactivates CRE-like sequences containing an ACGT core; Clauss IM et al.; The transcription factor hXBP-1 belongs to the family of basic region/leucine zipper (bZIP) proteins and interacts with the cAMP responsive element (CRE) of the major histocompatibility complex (MHC) class II A alpha, DR alpha and DP beta genes . However, the developmental expression of hXBP-1 as revealed by in situ hybridization in mouse embryos, has suggested that it interacts with the promoter of additional genes . To identify other potential target genes of this factor, we performed binding site selection experiments with recombinant hXBP-1 protein . The results indicated that hXBP-1 binds preferably to the CRE-like element GAT-GACGTG(T/G)NNN(A/T)T, wherein the core sequence ACGT is highly conserved, and that it also binds to some TPA response elements (TRE) . hXBP-1 can transactivate multimers of the target sequences to which it binds in COS cells, and the level of transactivation directly correlates with the extent of binding as observed in gel retardation experiments . One target sequence that is strongly bound by hXBP-1 is the 21 bp repeat in the HTLV-1 LTR, and we demonstrate here that hXBP-1 can transactivate the HTLV-1 LTR . Further, the transactivation domain of hXBP-1 encompasses a large C-terminal region of the protein, containing domains rich in glutamine, serine and threonine, and proline and glutamine residues, as shown in transient transfection experiments using hXBP-1-GAL4 fusion proteins and a reporter gene under the control of GAL4-binding sites.

Nucleic Acids Res, 1996 May 15, 24(10), 1841 - 8
Oligodeoxynucleoside phosphoramidates (P-NH2): synthesis and thermal stability of duplexes with DNA and RNA targets; Peyrottes S et al.; Syntheses of non ionic oligodeoxynucleoside phosphoramidates (P-NH2) and mixed phosphoramidate- phosphodiester oligomers were accomplished on automated solid supported DNA synthesizer using both H-phosphonate and phosphoramidite chemistries, in combination with t-butylphenoxyacetyl for N-protection of nucleoside bases, an oxalyl anchored solid support and a final treatment with methanolic ammonia . Thermal stabilities of the hybrids formed between these new analogues and their DNA and RNA complementary strands were determined and compared with those of the corresponding unmodified oligonucleotides, as well as of the phosphorothioate and methylphosphonate derivatives . Dodecathymidines containing P-NH2 links form less stable duplexes with DNA targets, d(C2A12C2) (deltaTm/modification -1.4 degrees C) and poly dA (deltaTm/modification -1.1 degrees C) than the corresponding phosphodiester and methylphosphonate analogues, but the hybrids are slightly more stable than the one obtained with phosphorothioate derivative . The destabilization is more pronounced with poly rA as the target (deltaTm/modification -3 degrees C) and could be compared with that found with the dodecathymidine methylphosphonate . The modification is less destabilizing in an heteropolymer-RNA duplex (deltaTm/modification -2 degrees C) . As expected, the P-NH2 modifications are highly resistant towards the action of various nucleases . It is also demonstrated that an all P-NH2 oligothymidine does not elicit Escherichia coli RNase H hydrolysis of the poly rA target but that the modification may be exploited in chimeric oligonucleotides combining P-NH2 sections with a central phosphodiester section.

Nucleic Acids Res, 1996 May 15, 24(10), 1837 - 40
Mutagenicity of a unique thymine-thymine dimer or thymine-thymine pyrimidine pyrimidone (6-4) photoproduct in mammalian cells; Gentil A et al.; The mutagenic properties of UV-induced photoproducts, both the cis-syn thymine-thymine dimer (TT) and the thymine-thymine pyrimidine pyrimidone (6-4) photoproduct {T(6-4)T} were studied in mammalian cells using shuttle vectors . A shuttle vector able to replicate in both mammalian cells and bacteria was produced in its single-stranded DNA form . A unique photoproduct was inserted at a single restriction site and after recircularization of the single-stranded DNA vector, this latter was transfected into simian COS7 cells . After DNA replication the vector was extracted from cells and used to transform bacteria . Amplified DNA was finally analyzed without any selective screening, DNA from randomly picked bacterial colonies being directly sequenced . Our results show clearly that both lesions are mutagenic, but at different levels . Mutation frequencies of 2 and 60% respectively were observed with the TT dimer and the T(6-4)T . With the TT dimer the mutations were targeted on the 3'-T . With the T(6-4)T a large variety of mutations were observed . A majority of G-->T transversions were semi-targeted to the base before the 5'-T of the photoproduct . These kinds of mutations were not observed when the same plasmid was transfected directly into SOS-induced JM105 bacteria or when the T(6-4)T oligonucleotide inserted in a different plasmid was replicated in SOS-induced SMH10 Escherichia coil bacteria . These semi-targeted mutations are therefore the specific result of bypass of the T(6-4)T lesion in COS7 cells by one of the eukaryotic DNA polymerases.

Biochem J, 1996 May 15, 316 ( Pt 1), 43 - 8
Recombinant bovine conglutinin, lacking the N-terminal and collagenous domains, has less conglutination activity but is able to inhibit haemagglutination by influenza A virus; Eda S et al.; Conglutinin is a bovine serum protein which was first described as a vertebrate lectin . This protein belongs to the family of C-type lectins . These lectins are composed of four characteristic domains: (1) an N-terminal cysteine-rich domain, (2) a collagen-like domain, (3) a neck domain and (4) a carbohydrate recognition domain (CRD) . Recently lectins have been shown to function as immunoglobulin-independent defence molecules due to a complement-mediated mechanism or opsonization . Our previous study showed that bovine conglutinin can inhibit haemagglutination by influenza A viruses and act by directly neutralizing them due to its lectin properties . In order to elucidate the biological role of the collagen-like domain, a recombinant partial conglutinin lacking this collagen-like domain was produced in an Escherichia coli system and its biological activities were examined . A 497 bp sequence, consisting of a short collagen region (two repeats of G-X-Y amino acid sequences), the neck domain, and the CRD of conglutinin cDNA, was amplified by the reverse-transcriptase PCR technique . The cDNA was transferred to a bacterial expression vector system (pRSET-A) and stable transfectants with a high level of conglutinin production were obtained . SDS/PAGE and Western blotting analyses showed a recombinant fusion protein of 27 kDa . Results of a cross-linking study and gel-filtration assay indicated that the recombinant conglutinin can form a trimeric structure and that it has sugar binding activity and specificity similar to that of native conglutinin . The recombinant conglutinin was also found to inhibit haemagglutination caused by influenza A virus as well as to possess less conglutination activity . These results suggest that in order for conglutinin to inhibit haemagglutination caused by the influenza virus, as well as to have sugar binding activity or to form trimers, it does not require the N-terminal and collagenous domains; however, they are essential for full conglutination activity.

Biochem J, 1996 May 15, 316 ( Pt 1), 337 - 43
Isoleucine 368 is involved in low-affinity binding of N6-modified cAMP analogues to site B of the regulatory subunit of cAMP-dependent protein kinase I; Huq I et al.; The regulatory (R) subunit of cAMP-dependent protein kinase has a well-defined domain structure including the two in-tandem cAMP-binding sites that constitute the C-terminus of the protein . The N-terminal binding site (A) has a considerably higher affinity for analogues of cAMP that are substituted with bulky and hydrophobic substituents at the 6-amino group of the adenine ring compared to the affinity observed at the second site (B) . On the basis of the crystal structure of the catabolite gene activator protein from Escherichia coli, molecular modelling of the binding domains suggested that a tyrosine (Y244) in site A could be involved in a high-affinity hydrophobic interaction, whereas a corresponding isoleucine (I368) in domain B could lead to steric hindrance in the binding of bulky N6-substituted analogues . Site-directed mutagenesis was used to construct mutations in Y244 and I368 . Binding displacement experiments showed that replacing the tyrosine in site A with isoleucine (Y244I) did not affect the interaction of either N6-substituted or otherwise modified analogues with this site . However, replacing I368 with tyrosine (I368Y) led to a 3-4-fold increase in affinity for those N6-modified analogues that had a hydrophobic group attached directly or close to the 6-amino molecule . We conclude that I368 is involved in the molecular interaction between binding domain B and the 6-amino group of the adenine moiety of cAMP and that this residue is partly responsible for the reduced affinity of N6-substituted cAMP analogues for this site.

Biochem J, 1996 May 15, 316 ( Pt 1), 157 - 60
Expression of ferredoxin-NADP+ reductase in heterocysts from Anabaena sp; Razquin P et al.; The expression of ferredoxin-NADP+ reductase (FNR) from Anabaena sp . PCC 7119 in heterocysts and vegetative cells has been quantified . Specific reductase activity in heterocysts was approximately 10 times higher than in vegetative cells, corresponding to the increased FNR protein content . This was confirmed by immunoquantification of the FNR protein from whole filaments of Anabaena sp . PCC 7120 grown in media with and without combined nitrogen . Transcription of the petH gene was markedly enhanced in the absence of combined nitrogen . This suggests that the increased RNA level is mainly responsible for the up-regulation of FNR in heterocysts . As has been observed for nif genes, iron deficiency also increased transcription of petH . Characterization of the FNR purified from isolated heterocysts showed no detectable differences from the enzyme from vegetative cells . Although nitrogen stress was a key regulatory factor, localization of the petH gene in the genomic map of Anabaena PCC 7120 showed that this gene is not physically associated with the nif cluster.

Biochem J, 1996 May 15, 316 ( Pt 1), 131 - 6
Heterologous expression, purification and characterization of rat class theta glutathione transferase T2-2; Jemth P et al.; Rat glutathione transferase (GST) T2-2 of class Theta (rGST T2-2), previously known as GST 12-12 and GST Yrs-Yrs, has been heterologously expressed in Escherichia coli XLI-Blue . The corresponding cDNA was isolated from a rat hepatoma cDNA library, ligated into and expressed from the plasmid pKK-D . The sequence is the same as that of the previously reported cDNA of GST Yrs-Yrs . The enzyme was purified using ion-exchange chromatography followed by affinity chromatography with immobilized ferric ions, and the yield was approx . 200 mg from a 1 litre bacterial culture . The availability of a stable recombinant rGST T2-2 has paved the way for a more accurate characterization of the enzyme . The functional properties of the recombinant rGST T2-2 differ significantly from those reported earlier for the enzyme isolated from rat tissues . These differences probably reflect the difficulties in obtaining fully active enzyme from sources where it occurs in relatively low concentrations, which has been the case in previous studies . 1-Chloro-2,4-dinitrobenzene, a substrate often used with GSTs of classes Alpha, Mu and Pi, is a substrate also for rGST T2-2, but the specific activity is relatively low . The Km value for glutathione was determined with four different electrophiles and was found to be in the range 0.3 mM-0.8 mM . The Km values for some electrophilic substrates were found to be in the micromolar range, which is low compared with those determined for GSTs of other classes . The highest catalytic efficiency was obtained with menaphthyl sulphate, which gave a Kcat/Km value of 2.3 x 10(6) s-1.M-1 and a rate enhancement over the uncatalysed reaction of 3 x 10(10).

Mutat Res, 1996 May 15, 363(1), 15 - 25
Amino acid residues affecting the activity and stability of human O6-alkylguanine-DNA alkyltransferase; Crone TM et al.; Amino acid residues in the human O6-alkylguanine-DNA alkyltransferase (AGT) were mutated and seventeen of the mutant proteins expressed in the ada- ogt-E . coli strain GWR 109 which is very sensitive to killing by methylating agents because of the absence of endogenous alkyltransferases . Thirteen of the mutations tested (delta-10, delta 1-19, R128A, N137A, H146A, R147A, delta N157, Y158A, E172Q, delta 92-97, Y114E, C145A and E172stop) reduced activity to below detectable levels when crude cell extracts were tested for the ability to remove O6-{3H}methylguanine from 3H-methylated DNA . However, only 4 of these mutations (delta 92-97, Y114E, C145A and E172stop) led to a complete loss of activity when tested for the ability to protect the cells from killing by MNNG . This suggests that the other nine mutations do not lead to the complete inactivation of AGT but produce protein with a reduced activity or in reduced amounts . These results show that none of the residues altered in these mutations (delta 1-10, delta 1-19, R128A, N137A, delta N157, H146A, R147A, Y158A and E172Q) are absolutely essential for AGT activity in protection against killing by MNNG . The stability of the mutant AGT proteins was determined by measuring the half-life of the protein synthesis was blocked . These results indicated that five of mutants that lacked AGT activity when tested in the crude extracts (Y114E, R128A, C145A, delta N157 and Y158A) were stable in the cell showing that the alteration of these residues does greatly reduce AGT activity . The other eight mutants lacking activity in crude extracts (delta 1-10, delta 92-97, E172Q, E172stop, delta 1-19, N137A, H146A and R147A) produced a large decrease in the stability of the AGT protein . This may account for the inability to detect AGT activity in vitro despite the ability to protect from MNNG toxicity in vivo . It is of particular interest that mutation of residues His146, Arg147, Asn137 and Glu172 resulted in unstable AGT proteins active in vivo but not in vitro . The crystal structure of the related Ada-C alkyltransferase suggests the involvement of these residues with the Cys145 acceptor site in a hydrogen bond network that may stabilize the protein and aid in the reaction mechanism . The data presented here support the existence of such an interaction existing in the human AGT and stress its importance in maintaining the configuration of the protein.

Cancer Res, 1996 May 15, 56(10), 2375 - 81
Mechanisms for the involvement of DNA methylation in colon carcinogenesis; Schmutte C et al.; C --> T transitions at CpG sites are the most prevalent mutations found in the p53 tumor suppressor gene in human colon tumors and in the germline (Li-Fraumeni syndrome) . All of the mutational hot spots are methylated to 5-methylcytosine, and it has been hypothesized that the majority of these mutations are caused by spontaneous hydrolytic deamination of this base to thymine . We have previously reported that bacterial methyltransferases induce transition mutations at CpG sites by increasing the deamination rate of C --> U when the concentration of the methyl group donor S-adenosylmethionine (AdoMet) drops below its Km, suggesting an alternative mechanism to create these mutations . Unrepaired uracil pairs with adenine during replication, completing the C --> T transition mutation . To determine whether this mechanism could contribute to the development of human colon cancer, we examined the level of DNA (cytosine-5)-methyltransferase (MTase) expression, the concentration of AdoMet, and the activity of uracil-DNA glycosylase in human colon tissues, and searched for the presence of mutations in the MTase gene . Using reverse transcription-PCR methods, we found that average MTase mRNA expression levels were only 3.7-fold elevated in tumor tissues compared with surrounding normal mucosa from the same patient . Also, no mutations were found in conserved regions of the gene in 10 tumors sequenced . High-performance liquid chromatographic analysis of extracts from the same tissues showed that AdoMet concentrations were not reduced below the Km value for the mammalian enzyme, and the concentration ratio of AdoMet:S-adenosylhomocysteine, the breakdown product of AdoMet and the competitive MTase inhibitor, did not differ significantly . Finally, extracts from the tumor tissue efficiently removed uracil from DNA . Therefore, biochemical conditions favoring a mutagenic pathway of C --> U --> T were not found in a target tissue known to undergo a high rate of C --> T transitions at CpG sites.

Carbohydr Res, 1996 May 14, 285, 141 - 50
Substrate specificity of native dTDP-D-glucose-4,6-dehydratase: chemo-enzymatic syntheses of artificial and naturally occurring deoxy sugars; Naundorf A et al.; Incubation of dTDP-glucose with the enzyme dTDP-glucose-4,6-dehydratase {EC 4.2.1.46} from wild type E . coli B yielded a mixture of 3- and 4-keto-6-deoxy sugars after work-up . Model experiments with chemically synthesized methyl 6-deoxy-4-keto-glucoside (9) revealed that dTDP-6-deoxy-alpha-D-ribo-hexopyran-3-ulose (3) is formed by keto-enol tautomerization during the isolation procedure from initially formed dTDP-6-deoxy-alpha-D-xylo-hexopyran-4-ulose (2) . dTDP-3-deoxyglucose (4) and dTDP-3-azido-3-deoxyglucose (6) were substrates and showed Michaelis-Menten kinetics (4: KM = 200 microM and V(max) = 130 mumol/h mg; 6: KM = 300 microM and V(max) = 90 mumol/h mg) . In 100-mg-scale experiments, both non-natural substrates gave the respective 6-deoxy-4-keto compounds, dTDP-3,6-dideoxy-alpha-D-erythro-hexopyran-4-ulose (5) and dTDP-3-azido-3,6-dideoxy-alpha-D-xylo-hexopyran-4-ulose++ + (7), in yields ranging from 24 to 40%.

Proc Natl Acad Sci U S A, 1996 May 14, 93(10), 5141 - 5
Preferential induction of a Th1 immune response and inhibition of specific IgE antibody formation by plasmid DNA immunization; Raz E et al.; We compared the antigen-specific antibody isotypes and lymphokine secretion by CD4+ T cells in BALB/c mice immunized intradermally with either Escherichia coli beta-galactosidase (beta-gal) or plasmid DNA (pDNA) encoding beta-gal in a cytomegalovirus-based expression vector (pCMV-LacZ) . pCMV-LacZ induced mainly IgG2a, whereas beta-gal in saline or alum induced IgG1 and IgE beta-gal-specific antibodies . In addition, splenic CD4+ T helper (Th) cells isolated from pDNA-immunized mice secreted interferon-gamma but not interleukin (IL)-4 and IL-5, whereas Th cells from beta-gal-injected mice secreted IL-4 and IL-5 but not interferon-gamma after in vitro stimulation with antigen . Together these data demonstrate that pDNA immunization induced a T helper type 1 (Th1) response, whereas protein immunization induced a T helper type 2 (Th2) response to the same antigen . Interestingly, priming of mice with pCMV-LacZ prevented IgE antibody formation to a subsequent i.p . beta-gal in alum injection . This effect was antigen-specific, because priming with pCMV-LacZ did not inhibit IgE anti-ovalbumin antibody formation . Most importantly, intradermal immunization with pCMV-LacZ (but not pCMV-OVA) of beta-gal in alum-primed mice caused a 66-75% reduction of the IgE anti-beta-gal titer in 6 weeks . Also, pCMV-LacZ induced specific IgG2a antibody titers and interferon-gamma secretion by Th cells in the beta-gal in alum-primed mice . The data demonstrate that gene immunization induces a Th1 response that dominates over an ongoing protein-induced Th2 response in an antigen-specific manner . This suggests that immunization with pDNA encoding for allergens may provide a novel type of immunotherapy for allergic diseases.

Proc Natl Acad Sci U S A, 1996 May 14, 93(10), 5014 - 8
Functional complementation of xeroderma pigmentosum complementation group E by replication protein A in an in vitro system; Kazantsev A et al.; Xeroderma pigmentosum (XP) is caused by a defect in nucleotide excision repair . Patients in the complementation group E (XP-E) have the mildest form of the disease and the highest level of residual repair activity . About 20% of the cell strains derived from XP-E patients lack a damaged DNA-binding protein (DDB) activity that binds to ultraviolet-induced (6-4) photoproducts with high affinity . We report here that cell-free extracts prepared from XP-E cell strains that either lacked or contained DDB activity were severely defective in excising DNA damage including (6-4) photoproducts . However, this excision activity defect was not restored by addition of purified DDB that, in fact, inhibited removal of (6-4) photoproducts by the human excision nuclease reconstituted from purified proteins . Extensive purification of correcting activity from HeLa cells revealed that the correcting activity is inseparable from the human replication/repair protein A {RPA (also known as human single stranded DNA binding protein, HSSB)} . Indeed, supplementing XP-E extracts with recombinant human RPA purified from Escherichia coli restored excision activity . However, no mutation was found in the genes encoding the three subunits of RPA in an XP-E (DDB-) cell line . It is concluded that RPA functionally complements XP-E extracts in vitro, but it is not genetically altered in XP-E patients.

Proc Natl Acad Sci U S A, 1996 May 14, 93(10), 4937 - 41
Two-dimensional protein crystallization via metal-ion coordination by naturally occurring surface histidines; Frey W et al.; A powerful and potentially general approach to the targeting and crystallization of proteins on lipid interfaces through coordination of surface histidine residues to lipid-chelated divalent metal ions is presented . This approach, which should be applicable to the crystallization of a wide range of naturally occurring or engineered proteins, is illustrated here by the crystallization of streptavidin on a monolayer of an iminodiacetate-Cu(II) lipid spread at the air-water interface . This method allows control of the protein orientation at interfaces, which is significant for the facile production of highly ordered protein arrays and for electron density mapping in structural analysis of two-dimensional crystals . Binding of native streptavidin to the iminodiacetate-Cu lipids occurs via His-87, located on the protein surface near the biotin binding pocket . The two-dimensional streptavidin crystals show a previously undescribed microscopic shape that differs from that of crystals formed beneath biotinylated lipids.

Proc Natl Acad Sci U S A, 1996 May 14, 93(10), 4787 - 92
Targeted replacement of the mycocerosic acid synthase gene in Mycobacterium bovis BCG produces a mutant that lacks mycosides; Azad AK et al.; A single gene (mas) encodes the multifunctional enzyme that catalyzes the synthesis of very long chain multiple methyl branched fatty acids called mycocerosic acids that are present only in slow-growing pathogenic mycobacteria and are thought to be important for pathogenesis . To achieve a targeted disruption of mas, an internal 2-kb segment of this gene was replaced with approximately the same size hygromycin-resistance gene (hyg), such that hyg was flanked by 4.7- and 1.4-kb segments of mas . Transformation of Mycobacterium bovis BCG with this construct in a plasmid that cannot replicate in mycobacteria yielded hygromycin-resistant transformants . Screening of 38 such transformants by PCR revealed several transformants representing homologous recombination with single crossover and one with double crossover . With primers representing the hyg termini and those representing the mycobacterial genome segments outside that used to make the transformation construct, the double-crossover mutant yielded PCR products expected from either side of hyg . Gene replacement was further confirmed by the absence of the vector and the 2-kb segment of mas replaced by hyg from the genome of the mutant . Thin-layer and radio-gas chromatographic analyses of the lipids derived from {1-14C}propionate showed that the mutant was incapable of synthesizing mycocerosic acids and mycosides . Thus, homologous recombination with double crossover was achieved in a slow-growing mycobacterium with an intron-containing RecA . The resulting mas-disrupted mutant should allow testing of the postulated roles of mycosides in pathogenesis.

Proc Natl Acad Sci U S A, 1996 May 14, 93(10), 4754 - 9
Sequence-specific DNA-binding dominated by dehydration; Lundback T et al.; Fluorescence spectroscopy and isothermal titration calorimetry were used to study the thermodynamics of binding of the glucocorticoid receptor DNA-binding domain to four different, but similar, DNA-binding sites . The binding sites are two naturally occurring sites that differ in the composition of one base pair, i.e., an A-T to G-C mutation, and two sites containing chemical intermediates of these base pairs . The calorimetrically determined heat capacity change (Delta C(p)o(obs)) for glucocorticoid receptor DNA-binding domain binding agrees with that calculated for dehydration of solvent-accessible surface areas . A dominating effect of dehydration or solvent reorganization on the thermodynamics is also consistent with an observed linear relationship between observed enthalpy change (Delta Ho(obs)) and observed entropy change (Delta So(obs)) with a slope close to the experimental temperature . Comparisons with structural data allow us to rationalize individual differences between Delta Ho(obs) (and Delta So(obs)) for the four complexes . For instance, we find that the removal of a methyl group at the DNA-protein interface is enthalpically favorable but entropically unfavorable, which is consistent with a replacement by an ordered water molecule.

Proc Natl Acad Sci U S A, 1996 May 14, 93(10), 4644 - 8
Peroxo-iron and oxenoid-iron species as alternative oxygenating agents in cytochrome P450-catalyzed reactions: switching by threonine-302 to alanine mutagenesis of cytochrome P450 2B4; Vaz AD et al.; Among biological catalysts, cytochrome P450 is unmatched in its multiplicity of isoforms, inducers, substrates, and types of chemical reactions catalyzed . In the present study, evidence is given that this versatility extends to the nature of the active oxidant . Although mechanistic evidence from several laboratories points to a hypervalent iron-oxenoid species in P450-catalyzed oxygenation reactions, Akhtar and colleagues {Akhtar, M., Calder, M . R., Corina, D . L . & Wright, J . N . (1982) Biochem . J . 201, 569-580} proposed that in steroid deformylation effected by P450 aromatase an iron-peroxo species is involved . We have shown more recently that purified liver microsomal P450 cytochromes, including phenobarbital-induced P450 2B4, catalyze the analogous deformylation of a series of xenobiotic aldehydes with olefin formation . The investigation presented here on the effect of site-directed mutagenesis of threonine-302 to alanine on the activities of recombinant P450 2B4 with N-terminal amino acids 2-27 deleted {2B4 (delta2-27)} makes use of evidence from other laboratories that the corresponding mutation in bacterial P450s interferes with the activation of dioxygen to the oxenoid species by blocking proton delivery to the active site . The rates of NADPH oxidation, hydrogen peroxide production, and product formation from four substrates, including formaldehyde from benzphetamine N-demethylation, acetophenone from 1-phenylethanol oxidation, cyclohexanol from cyclohexane hydroxylation, and cyclohexene from cyclohexane carboxaldehyde deformylation, were determined with P450s 2B4, 2B4 (delta2-27), and 2B4 (delta2-27) T302A . Replacement of the threonine residue in the truncated cytochrome gave a 1.6- to 2.5-fold increase in peroxide formation in the presence of a substrate, but resulted in decreased product formation from benzphetamine (9-fold), cyclohexane (4-fold), and 1-phenylethanol (2-fold) . In sharp contrast, the deformylation of cyclohexane carboxaldehyde by the T302A mutant was increased about 10-fold . On the basis of these findings and our previous evidence that aldehyde deformylation is supported by added H202, but not by artificial oxidants, we conclude that the iron-peroxy species is the direct oxygen donor . It remains to be established which of the many other oxidative reactions involving P450 utilize this species and the extent to which peroxo-iron and oxenoid-iron function as alternative oxygenating agents with the numerous isoforms of this versatile catalyst.

Proc Natl Acad Sci U S A, 1996 May 14, 93(10), 4638 - 43
Expression cloning of a cDNA for human ceramide glucosyltransferase that catalyzes the first glycosylation step of glycosphingolipid synthesis; Ichikawa S et al.; We have isolated a cDNA encoding human ceramide glucosyltransferase (glucosylceramide synthase, UDP-glucose:N-acylsphingosine D-glucosyltransferase, EC 2.4.1.80) by expression cloning using as a recipient GM-95 cells lacking the enzyme . The enzyme catalyzes the first glycosylation step of glycosphingolipid synthesis and the product, glucosylceramide, serves as the core of more than 300 glycosphingolipids . The cDNA has a G+C-rich 5' untranslated region of 290 nucleotides and the open reading frame encodes 394 amino acids (44.9 kDa) . A hydrophobic segment was found near the N terminus that is the potential signal-anchor sequence . In addition, considerable hydrophobicity was detected in the regions close to the C terminus, which may interact with the membrane . A catalytically active enzyme was produced from Escherichia coli transfected with the cDNA . Northern blot analysis revealed a single transcript of 3.5 kb, and the mRNA was widely expressed in organs . The amino acid sequence of ceramide glucosyltransferase shows no significant homology to ceramide galactosyltransferase, which indicates different evolutionary origins of these enzymes.

Proc Natl Acad Sci U S A, 1996 May 14, 93(10), 4617 - 22
The platelet-derived growth factor alpha-receptor is encoded by a growth-arrest-specific (gas) gene; Lih CJ et al.; Using the Escherichia coli lacZ gene to identify chromosomal loci that are transcriptionally active during growth arrest of NIH 3T3 fibroblasts, we found that an mRNA expressed preferentially in serum-deprived cells specifies the previously characterized alpha-receptor (alphaR) for platelet-derived growth factor (PDGF), which mediates mitogenic responsiveness to all PDGF isoforms . Both PDGFalphaR mRNA, which was shown to include a 111-nt segment encoded by a DNA region thought to contain only intron sequences, and PDGFalphaR protein accumulated in serum-starved cells and decreased as cells resumed cycling . Elevated PDGFalphaR gene expression during serum starvation was not observed in cells that had been transformed with oncogenes erbB2, src, or raf, which prevent starvation-induced growth arrest . Our results support the view that products of certain genes expressed during growth arrest function to promote, rather than restrict, cell cycling . We suggest that accumulation of the PDGFalphaR gene product may facilitate the exiting of cells from growth arrest upon mitogenic stimulation by PDGF, leading to the state of "competence" required for cell cycling.

Biochemistry, 1996 May 14, 35(19), 6100 - 6
Substrate binding causes movement in the ATP binding domain of Escherichia coli adenylate kinase; Bilderback T et al.; Crystallographic evidence suggests that there is a large hinged domain motion associated with substrate binding in adenylate kinase . To test this hypothesis, resonance energy transfer measurements of substrate binding were initiated . Adenylate kinase from Escherichia coli consists of three domains: the main body of the enzyme with alpha-helical and beta-sheet secondary structure, and domains that close over the AMP and ATP binding sites . Four single tryptophan mutants were constructed to map distances . Two tryptophan mutants were positioned at residues 133 (Y133W) and 137 (F137W), which are in the domain that closes over the ATP binding site . Mutant F86W that is located at the AMP binding site, and mutant S41W that is in the loop that close over AMP, complete the mapping library . Energy transfer was measured between each of these tryptophans and 5-{{2-(acetylamino)ethyl}amino}naphthalene-1-sulfonic acid (AEDANS) covalently bound to the single cysteine residue at position 77, which is located in the main body of adenylate kinase . The distance between the tryptophan of the F137W mutant adenylate kinase and the AEDANS-labeled Cys-77 decreased by 12.1 A upon the binding of the bisubstrate inhibitor P1, P5-bis(5'-adenosyl) pentaphosphate (AP5A) . There were only small alterations in the tryptophan to Cys-77-AEDANS distances in the Y133W, F86W, and S41W mutants upon the binding of AP5A, ATP, or AMP, implying that movement of residues 133, 86, and 41 in relation to the Cys-77 residue was minimal . These results suggest that there is significant closure of the ATP binding domain upon the binding of ATP or AP5A . Unexpectedly, exposure of the enzyme to AMP also introduced a partial closure of the ATP hinged domain.

Biochemistry, 1996 May 14, 35(19), 6089 - 99
Binding of Ixr1, a yeast HMG-domain protein, to cisplatin-DNA adducts in vitro and in vivo; McA'Nulty MM et al.; Ixr1 is a yeast HMG-domain protein that binds specifically to DNA adducts formed by the antitumor drug cisplatin . Interruption of the IXR1 gene in yeast desensitizes cells to cisplatin . This effect is unrelated to a natural function of Ixr1, which is to repress the transcription of COX5b . Ixr1 interacts specifically and preferentially with DNA modified by cisplatin . In the present work, Ixr1 was purified from a clone expressed in Escherichia coli . The dissociation constant for Ixr1 binding site-specifically to a 92-bp probe containing a single cis-{Pt(NH3)2 inverted question markd(GpG)-N7(1) -N7(2) inverted question mark} intrastrand cross-link was measured to be 2.5 (+/- 0.1) x 10(-7) M, similar to that found for HMG1 . Ixr1 binds at least an order of magnitude more tightly to cisplatin-DNA adducts than to unmodified DNA . Hydroxyl radical footprinting revealed that Ixr1 protects an area of platinated DNA that is approximately 15 bp in size and centered at the platinum adduct . The binding of HMG-domain proteins to cisplatin-DNA adducts has been proposed to divert these proteins from their natural DNA-binding sites, disrupting transcription . This hypothesis was tested for Ixr1 in yeast . The protein was not titrated away from the Cox5b promoter sufficiently well to disrupt transcription either of Cox5b mRNA from genomic DNA or of the beta-galactosidase gene under control of the promoter in a plasmid DNA transformed into yeast.

Biochemistry, 1996 May 14, 35(19), 6010 - 9
Crystal structure of the rat liver fructose-2,6-bisphosphatase based on selenomethionine multiwavelength anomalous dispersion phases; Lee YH et al.; The crystal structure of the recombinant fructose-2,6-bisphosphatase domain, which covers the residues between 251 and 440 of the rat liver bifunctional enzyme, 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase, was determined by multiwavelength anomalous dispersion phasing and refined at 2.5 A resolution . The selenomethionine-substituted protein was induced in the methionine auxotroph, Escherichia coli DL41DE3, purified, and crystallized in a manner similar to that of the native protein . Phase information was calculated using the multiwavelength anomalous dispersion data collected at the X-ray wavelengths near the absorption edge of the K-shell alpha electrons of selenium . The fructose-2,6-bisphosphatase domain has a core alpha/beta structure which consists of six stacked beta-strands, four parallel and two antiparallel . The core beta-sheet is surrounded by nine alpha-helices . The catalytic site, as defined by a bound phosphate ion, is positioned near the C-terminal end of the beta-sheet and close to the N-terminal end of an alpha-helix . The active site pocket is funnel-shaped . The narrow opening of the funnel is wide enough for a water molecule to pass . The key catalytic residues, including His7, His141, and Glu76, are near each other at the active site and probably function as general acids and/or bases during a catalytic cycle . The inorganic phosphate molecule is bound to an anion trap formed by Arg6, His7, Arg56, and His141 . The core structure of the Fru-2,6-P2ase is similar to that of the yeast phosphoglycerate mutase and the rat prostatic acid phosphatase . However, the structure of one of the loops near the active site is completely different from the other family members, perhaps reflecting functional differences and the nanomolar range affinity of Fru-2,6-P2ase for its substrate . The imidazole rings of the two key catalytic residues, His7 and His141, are not parallel as in the yeast phosphoglycerate mutase . The crystal structure is used to interpret the existing chemical data already available for the bisphosphatase domain . In addition, the crystal structure is compared with two other proteins that belong to the histidine phosphatase family.

Biochemistry, 1996 May 14, 35(19), 5999 - 6009
X-ray structures of a designed binding site in trypsin show metal-dependent geometry; Brinen LS et al.; The three-dimensional structures of complexes of trypsin N143H, E151H bound to ecotin A86H are determined at 2.0 A resolution via X-ray crystallography in the absence and presence of the transition metals Zn2+, Ni2+, and Cu2+ . The binding site for these transition metals was constructed by substitution of key amino acids with histidine at the trypsin-ecotin interface in the S2'/P2' pocket . Three histidine side chains, two on trypsin at positions 143 and 151 and one on ecotin at position 86, anchor the metals and provide extended catalytic recognition for substrates with His in the P2' pocket . Comparisons of the three-dimensional structures show the different geometries that result upon the binding of metal in the engineered tridentate site and suggest a structural basis for the kinetics of the metal-regulated catalysis . Of the three metals, the binding of zinc results in the most favorable binding geometry, not dissimilar to those observed in naturally occurring zinc binding proteins.

Biochemistry, 1996 May 14, 35(19), 5992 - 8
Delocalizing trypsin specificity with metal activation; Willett WS et al.; Recognition for proteolysis by trypsin depends almost exclusively on tight binding of arginine or lysine side chains by the primary substrate specificity pocket . Although extended subsite interactions are important for catalysis, the majority of binding energy is localized in the P1 pocket . Analysis of the interactions of trypsin with the P1 residue of the bound inhibitors ecotin and bovine pancreatic trypsin inhibitor suggested that the mutation D189S would improve metal-assisted trypsin N143H, E151H specificity toward peptides that have a Tyr at P1 and a His at P2' . In the presence of transition metals, the catalytic efficiency of the triple mutant Tn N143H, E151H, D189S improved toward the tyrosine-containing peptide AGPYAHSS . Trypsin N143H, E151H, D189S exhibits a 25-fold increase in activity with nickel and a 150-fold increase in activity with zinc relative to trypsin N143H, E151H on this peptide . In addition, activity of trypsin N143H, E151H, D189S toward an arginine-containing peptide, YLVGPRGHFYDA, is enhanced by copper, nickel, and zinc . With this substrate, copper yields a 30-fold, nickel a 70-fold, and zinc a 350-fold increase in activity over background hydrolysis without metal . These results demonstrate that the engineering of multiple substrate binding subsites in trypsin can be used to delocalize protease specificity by increasing relative substrate binding contributions from alternate engineered subsites.

Biochemistry, 1996 May 14, 35(19), 5963 - 70
Inosine-uridine nucleoside hydrolase from Crithidia fasciculata . Genetic characterization, crystallization, and identification of histidine 241 as a catalytic site residue; Gopaul DN et al.; Protozoa depend on purine salvage for nucleic acid synthesis . An abundant salvage enzyme in Crithidia fasciculata is the inosine-uridine nucleoside hydrolase (IU-nucleoside hydrolase) . The enzyme was cloned by polymerase chain reaction techniques using primers corresponding to the amino acid sequences of tryptic fragments and to the miniexon of C . fasciculata . The full-length cDNA was expressed in Escherichia coli and the protein purified to > 99% homogeneity . The open reading frame encodes a protein of 315 amino acids . Enzyme purified from C . fasciculata was missing the N-terminal Met and gave a major mass peak of 34 194 amu by mass spectrometry . Predicted mass from the DNA sequence for the Met-processed enzyme was 34 196 . A pET3d-IUNH construct expressed in E . coli introduced MetAla instead of MetPro at the N-terminus . Enzyme purified from this construct also had a processed N-terminus and gave predicted and observed masses of 34 168 and 34 170 amu, respectively . The amino acid sequence for IU-nucleoside hydrolase has no close relatives among the known proteins . A cDNA clone of unknown function from Leishmania major shows near identity in the N-terminal deduced amino acid sequence . Open reading frames near 1 and 47 min on the E . coli chromosome and from two yeast genomes encode for proteins of similar size with substantial amino acid identity . Mutation of His241Ala caused a 2100-fold loss in k(cat) for inosine but a 2.8-fold increase in k(cat) with p-nitrophenyl beta-D-ribofuranoside, establishing the location of the catalytic site and implicating His241 as a proton donor for leaving group activation . IU-nucleoside hydrolase from C . fasciculata and the protein expressed in E . coli were crystallized and diffract to 2.5 and 2.1 A resolution, respectively . Both belong to the P2(1)2(1)2 orthorhombic space group with unit cell parameters a = 63.5 A, b = 131.9 A, c = 90.1 A, and alpha = beta = gamma = 90 degrees . Two subunits of the tetrameric enzyme are present in the asymmetric unit . The following paper reports the X-ray crystal structure for this enzyme.

Mol Cell Biochem, 1996 May 10, 158(1), 57 - 63
Src homology domains of v-Src stabilize an active conformation of the tyrosine kinase catalytic domain; Xu B et al.; To examine the interactions between Src homology domains and the tyrosine kinase catalytic domain of v-Src, various combinations of domains have been expressed in bacteria as fusion proteins . Constructs containing the isolated catalytic domain, SH2 + catalytic domain, and SH3 + SH2 + catalytic domains were active in autophosphorylation assays . For the catalytic domain of v-Src, but not for v-Abl, addition of exogenous Src SH3-SH2 domains stimulated the autophosphorylation activity . In contrast to results for autophosphorylation, constructs containing Src homology domains were more active towards a synthetic peptide substrate than the isolated catalytic domain . The ability of the SH2 and SH3 domains of v-Src to stabilize an active enzyme conformation was also confirmed by refolding after denaturation in guanidinium hydrochloride . Collectively the data suggest that, in addition to their roles in intermolecular protein-protein interactions, the Src homology regions of v-Src exert a positive influence on tyrosine kinase function, potentially by maintaining an active conformation of the catalytic domain.

J Mol Biol, 1996 May 10, 258(3), 420 - 32
The dynamic structure of EF-G studied by fusidic acid resistance and internal revertants; Johanson U et al.; We have previously identified 20 different fusidic acid-resistant alleles of fusA, encoding mutant forms of the ribosomal translocase EF-G . One of these, P413L, is used here as the starting point in selections for internal revertants, identifying 20 different pseudo-wild-type forms of EF-G . We have also identified two alleles of fusA previously isolated as suppressors of 4.5 S RNA deficiency . All of these mutants are analysed in terms of their effects on the structural dynamics of EF-G . Most mutation conferring fusidic acid-resistance interfere with conformational changes of EF-G, but some may be located at a possible fusidic acid binding site . Revertants of the P413L mutations restore the function of EF-G with or without affecting the level of resistance to fusidic acid . The revertant mutations probably restore the balance between the GDP and GTP conformations of EF-G off the ribosome, and most of them are located close to the interface between the G domain and domain II . The procedure for the isolation of pseudo-wild-type forms of EF-G can be used to direct evolution progressively away from the wild-type while still maintaining the essential functions of EF-G.

Science, 1996 May 10, 272(5263), 868 - 72
Homologous DNA pairing promoted by a 20-amino acid peptide derived from RecA; Voloshin ON et al.; The molecular structure of the Escherichia coli RecA protein in the absence of DNA revealed two disordered or mobile loops that were proposed to be DNA binding sites . A short peptide spanning one of these loops was shown to carry out the key reaction mediated by the whole RecA protein: pairing (targeting) of a single-stranded DNA to its homologous site on a duplex DNA . In the course of the reaction the peptide bound to both substrate DNAs, unstacked the single-stranded DNA, and assumed a beta structure . These events probably recapitulate the underlying molecular pathway or mechanism used by homologous recombination proteins.

J Biol Chem, 1996 May 10, 271(19), 11532 - 40
DNA binding by the coliphage 186 repressor protein CI; Dodd IB et al.; The cI gene of coliphage 186 maintains lysogeny and confers immunity to 186 infection by repressing the major early promoter, p(R), and the promoter for the late transcription activator gene, p(B) . Gel mobility shirt and DNase I footprinting show that CI protein binds to the DNA at p(R) and p(B) and also to sites approximately 300 base pairs upstream and downstream of p(R), called FL and FR . Mutations which cause virulence reduce CI binding to p(R) . The biochemical and genetic data identify three CI operators at p(R), two at p(B), and single operators at FL and FR . The operators at the p(B), FL, FR, and central p(R) sites are inverted repeat sequences, separated by 5 base pairs (Type A) or, in the case of p(R), by 4 base pairs (Type A') . A different inverted repeat operator sequence (Type B) is proposed for the binding sites on each side of the central site at p(R) . Thus, CI appears to recognize two distinct DNA sequences . CI binds cooperatively to adjacent operators, and binding at p(R) is strongly dependent on these cooperative interactions . A high order CI multimer appears to be the active DNA binding species, even at single operators.

J Biol Chem, 1996 May 10, 271(19), 11525 - 31
Purification and self-association equilibria of the lysis-lysogeny switch proteins of coliphage 186; Shearwin KE et al.; The CI repressor protein, responsible for maintenance of the lysogenic state, and the Apl protein, required for efficient prophage induction, are the two control proteins of the lysis-lysogeny transcriptional switch of coliphage 186 . These proteins have been overexpressed, purified, and their self-association behavior examined by sedimentation equilibrium . Phage 186 CI dimers self-associate in solution through tetramers to octamers in a concerted process . The Apl protein of 186 is an unusual example of a helix-turn-helix protein which is monomeric in solution.

J Biol Chem, 1996 May 10, 271(19), 11518 - 24
Purification and properties of HuD, a neuronal RNA-binding protein; Chung S et al.; HuD is a human neuronal specific RNA-binding protein . In this study we have purified HuD and examined its RNA binding properties in detail . HuD binds to mRNAs that contain an AU-rich element with high affinity . In the case of the c-fos AU-rich element, HuD binds to a 27-nucleotide core element comprising AUUUA, AUUUUA, and AUUUUUA motifs . Mutation in any two of these motifs abrogates binding . HuD contains two tandem RNA recognition motifs (RRM), a basic domain, and a third RRM . Deletion analysis has shown that only the first and second RRMs are essential for RNA binding . Thus, these specific RNA binding properties support the idea that the HuD regulates gene expression at the posttranscriptional level.

J Biol Chem, 1996 May 10, 271(19), 11462 - 7
Endothelial nitric-oxide synthase . Expression in Escherichia coli, spectroscopic characterization, and role of tetrahydrobiopterin in dimer formation; Rodriguez-Crespo I et al.; Bovine endothelial nitric-oxide synthase (eNOS) expressed in Escherichia coli does not have the post-translational modifications found in the native enzyme and is free of tetrahydrobiopterin (BH4) . In the presence of BH4, eNOS has an absorption maximum at 400 nm that shifts to 395 nm when the substrate L-arginine is added . The low-spin component of the spectrum of the BH4-free protein is decreased by the addition of BH4 without a corresponding increase in the high-spin component . Addition of BH4 decreases the low-spin population of eNOS even in the presence of excess L-arginine . These results indicate that BH4 directly modulates the heme environment . BH4-free eNOS is completely inactive, but catalytic activity is recovered when BH4 (EC50 approximately 200 nM) is added . The spectroscopically determined binding constants for L-arginine are approximately 1.9 microM in the presence and approximately 4.0 microM in the absence of BH4 . The BH4-supplemented enzyme has an activity of 90-120 nmol of citrulline.min-1.mg-1 and Km values of 3 and 14 microM for L-arginine and N-hydroxy-L-arginine, respectively . Of particular interest is the finding by SDS-polyacrylamide gel electrophoresis that BH4-free eNOS exists in a monomer-dimer equilibrium very similar to that observed with the BH4-reconstituted protein . Addition of BH4, increases the percent of the dimer by only approximately 5% . The results establish that BH4 influences the heme environment and stabilizes the protein with respect to heme loss but is not required for dimer formation.

J Biol Chem, 1996 May 10, 271(19), 11309 - 16
Isolation and characterization of a disulfide-linked human stem cell factor dimer . Biochemical, biophysical, and biological comparison to the noncovalently held dimer; Lu HS et al.; Distinct from the noncovalently linked recombinant human stem call factor (rhSCF) dimer, we report here the isolation and identification of an SDS-nondissociable dimer produced during folding/oxidation of rhSCF . Experimental evidence using various cleavage strategies and analyses shows that the isolated dimer is composed of two rhSCF monomers covalently linked by four disulfide bonds . The cysteines are paired as in the noncovalently associated dimer except that all pairings are intermolecular rather than intramolecular . Other structural models, involving intertwining of intramolecular disulfide loops, are ruled out . The molecule behaves similarly to the noncovalently associated dimer during ion-exchange or gel permeation chromatography . However, the disulfide-linked dimer exhibits increased hydrophobicity in reverse-phase columns and in the native state does not undergo spontaneous dimer dissociation-association as seen for the noncovalent dimer . Spectroscopic analyses indicate that the disulfide-linked and noncovalently associated rhSCF dimers have grossly similar secondary and tertiary structures . In vitro, the disulfide-linked dimer exhibits approximately 3-fold higher biological activity in supporting growth of a hematopoietic cell line and stimulating hematopoietic cell colony formation from enriched human CD34+ cells . The molecule binds to the rhSCF receptor, Kit, with an efficiency only half that of the noncovalently associated dimer . Formation of intermolecular disulfides in the disulfide-linked dimer with retention of biological activity has implications for the three-dimensional structure of noncovalently held dimer and disulfide-linked dimer.

J Biol Chem, 1996 May 10, 271(19), 11301 - 8
Refolding and oxidation of recombinant human stem cell factor produced in Escherichia coli; Jones MD et al.; Oxidative folding of recombinant human stem cell factor (rhSCF) produced in Escherichia coli was investigated in vitro . Folding of denatured and reduced rhSCF involves at least five intermediate forms, I-1 to I-5, detectable by their differences in hydrophobicity using reverse-phase high performance liquid chromatography . Both I-1 and I-2 contain a native-like disulfide bond, Cys4-Cys89 and Cys43-Cys138, respectively, and I-3 forms a mispaired disulfide, Cys43-Cys89 . These forms appear to reach steady state equilibrium and are important folding intermediates . I-1 was found to be the prominent intermediate that directly folds into native rhSCF (N); and the thermodynamically less stable I-2 favors rearrangment into I-1 . I-3 may serve as an intermediate for disulfide rearrangment between I-1 and I-2 . I-4 and I-5, which are disulfide-linked dimers, are in equilibrium with reduced rhSCF and other intermediates and may not play an important role in rhSCF folding . Both trifluoroacetic acid-trapped I-1 and I-2, after isolation by high performance liquid chromatography, proceed with the remaining oxidative folding process after reconstitution . Iodoacetate-trapped I-1 and I-2 contain low alpha-helical content and some tertiary structure, while I-3 and reduced rhSCF have little ordered structure . Gel filtration/light-scattering experiments indicate that reduced rhSCF and iodoacetate-trapped I-1, I-2, and I-3 exist as dimeric forms, indicating that rhSCF dimerization precedes formation of disulfide bonds . I-1, I-2, I-3, and the C43,138A analog lacking Cys43-Cys138 bond are not biologically active or exhibit significantly lower activity . The two disulfide bonds in rhSCF seem to be essential for the molecule to maintain an active conformation required for its receptor binding and biological activities.

J Biol Chem, 1996 May 10, 271(19), 11228 - 35
Thrombin-induced platelet aggregation is inhibited by the heptapeptide Leu271-Ala277 of domain 3 in the heavy chain of high molecular weight kininogen; Kunapuli SP et al.; The ability of kininogens to modulate thrombin-induced aggregation of human platelets has been assigned to domain 3 (D3) in the common heavy chain coded for by exons 7, 8, and 9 of kininogen gene . We expressed each of the exons 7, 8, and 9, and various combinations as glutathione S-transferase fusion proteins in Escherichia coli . Each of the exon products 7 (Lys236-Gln292), 9 (Val293-Gly328), and 8 (Gln329-Met357), and their combinations were evaluated for the ability to inhibit thrombin induced platelet aggregation . Only products containing exon 7 inhibited platelet aggregation induced by thrombin with an IC50 of > 20 microM . A deletion mutant of exon 7 product, polypeptide 7A product (Lys236-Lys270) did not block thrombin-induced platelet aggregation, while 7B product (Thr255-Gln292) and 7C product (Leu271-Gln292) inhibited aggregation . These findings indicated that the inhibitory activity is localized to residues Leu271-Gln292 . Peptides Phe279-Ile283 and Phe281-Gln292 did not block thrombin, and Asn275-Phe279 had only minimal inhibitory activity . A heptapeptide Leu271-Ala277 inhibited thrombin-induced aggregation of platelets with an IC50 of 65 microM . The effect is specific for the activation of platelets by thrombin but not ADP or collagen . No evidence for a thrombin-kininogen complex was found, and neither HK nor its derivatives directly inhibited thrombin activity . Knowledge of the critical sequence of kininogen should allow design of compounds that can modulate thrombin activation of platelets.

J Biol Chem, 1996 May 10, 271(19), 11209 - 13
D4-GDI, a substrate of CPP32, is proteolyzed during Fas-induced apoptosis; Na S et al.; Apoptosis (programmed cell death) is a fundamental process for normal development of multicellular organisms, and is involved in the regulation of the immune system, normal morphogenesis, and maintenance of homeostasis, ICE/CED-3 family cysteine proteases have been implicated directly in apoptosis, but relatively few of the substrates through which their action is mediated have been identified . Here we report that D4-GDI, an abundant hematopoietic cell GDP dissociation inhibitor for the Ras-related Rho family GTPases, is a substrate of the apoptosis protease CPP32/Yama/Apopain . D4-GDI was rapidly truncated to a 23-kDa fragment in Jurkat cells with kinetics that parallel the onset of apoptosis following Fas cross-linking with agonistic antibody or treatment with staurosporine . Fas- and staurosporine-induced apoptosis as well as cleavage of D4-GDI were inhibited by the ICE inhibitor, YVAD-cmk . D4-GDI was cleaved in vitro by recombinant CPP32 expressed in Escherichia coli to form a 23-kDa fragment . The CPP32-mediated cleavage of D4-GDI was completely inhibited by 1 microM DEVD-CHO, a reported selective inhibitor of CPP32 . In contrast, the ICE-selective inhibitors, YVAD-CHO or YVAD-cmk, did not inhibit CPP32-mediated D4-GDI cleavage at concentrations up to 50 microM . N-terminal sequencing of the 23-kDa D4-GDI fragment demonstrated that D4-GDI was cleaved between Asp19 and Ser20 of the poly(ADP-ribose) polymerase-like cleavage sequence DELD19S . These data suggest that regulation by D4-GDI of Rho family GTPases may be disrupted during apoptosis by CPP32-mediated cleavage of the GDI protein.

J Biol Chem, 1996 May 10, 271(19), 11076 - 82
Involvement of the switch 2 domain of Ras in its interaction with guanine nucleotide exchange factors; Quilliam LA et al.; While Ras proteins are activated by stimulated GDP release, which enables acquisition of the active GTP-bound state, little is known about how guanine nucleotide exchange factors (GEFs) interact with Ras to promote this exchange reaction . Here we report that mutations within the switch 2 domain of Ras (residues 62-69) inhibit activation of Ras by the mammalian GEFs, Sos1, and GRF/CDC25Mm . While mutations in the 62-69 region blocked upstream activation of Ras, they did not disrupt Ras effector functions, including transcriptional activation and transformation of NIH 3T3 cells . Biochemical analysis indicated that the loss of GEF responsiveness of a Ras(69N) mutant was due to a loss of GEF binding, with no change in intrinsic nucleotide exchange activity . Furthermore, structural analysis of Ras(69N) using NMR spectroscopy indicated that mutation of residue 69 had a very localized effect on Ras structure that was limited to alpha-helix 2 of the switch 2 domain . Together, these results suggest that the switch 2 domain of Ras forms a direct interaction with GEFs.

Biochemistry, 1996 May 7, 35(18), 5893 - 901
Determination of regions in the dihydrofolate reductase structure that interact with the molecular chaperonin GroEL; Clark AC et al.; Dihydrofolate reductase (DHFR) from Escherichia coli does not interact with the molecular chaperonin GroEL regardless of whether the interaction is initiated from the native or the unfolded state . In contrast, murine DHFR shows a strong interaction with GroEL . Using the structure of human DHFR as a model for the murine protein, a superimposition of the two structures shows that there are three distinct external loops in the eukaryotic DHFR that are not present in the E . coli protein . Removal of one loop (residues 99-108) from the eukaryotic murine DHFR has no effect on the interaction with GroEL . On the basis of the differences in structures, we inserted either of two surface loops of murine DHFR into the corresponding regions of E . coli DHFR . In the first mutant (EcDHFR-i(9)36), residues 36 and 37 (L-N) of E . coli DHFR were replaced with the nine amino acid sequence T-T-S-S-V-E-G-K-Q . In the second mutant (EcDHFR-i(7)136), residues 136-139 (V-F-S-E) of E . coli DHFR were replaced with the seven amino acid sequence L-P-E-Y-P-G-V . Both E . coli DHFR mutants formed a complex with GroEL starting from either the native or the unfolded states of DHFR . The binding was specific since the presence of MgATP caused the release of the proteins from GroEL . As with murine DHFR, nonnative conformations of EcDHFR-i(9)36 and EcDHFR-i(7)136 are bound to GroEL . Fluorescence titration techniques were used to quantitate the interaction between GroEL and these proteins . A simple chromatographic procedure was developed to remove contaminating tryptophan containing peptides from GroEL samples . The mutant EcDHFR-i(7)136 binds to GroEL with a stoichiometry of 4-5 mol of DHFR per mol of GroEL tetradecamer, while murine DHFR binds to GroEL with a stoichiometry of 2 mol of DHFR per mol of GroEL tetradecamer . Both murine DHFR and EcDHFR-i(7)136 bind to GroEL very tightly, with equilibrium dissociation constants of less than 85 nM.

Biochemistry, 1996 May 7, 35(18), 5883 - 92
Analysis of substrate-induced electronic, catalytic, and structural changes in inducible NO synthase; Sennequier N et al.; Inducible nitric oxide synthase (iNOS) catalyzes the NADPH-dependent formation of nitric oxide (NO) and citrulline from L-arginine and O2 . In addition to serving as substrate, L-arginine alters the enzyme's heme iron spin equilibrium, increases its NADPH oxidation, and promotes assembly of active dimeric iNOS from inactive monomers . To understand what structural aspects of L-arginine are important for causing these effects, we have studied the interactions of iNOS with several L-arginine and guanidine analogs . Very few analogs supported NO synthesis even when bound to iNOS at saturating or near-saturating levels . In contrast, almost all analogs shifted the heme iron spin equilibrium and either increased or decreased NADPH oxidation by iNOS . The guanidine analogs displayed the same pattern of effects as their amino acid counterparts but exhibited a lower affinity except for analogs containing S-alkylisothiourea or aminoguanidine groups . Most analogs also promoted iNOS dimerization, with hydroxyguanidine and S-ethylisothiourea promoting more dimerization than L-arginine itself . Although the analog concentrations required to promote dimerization of monomers were somewhat higher than those required for binding to dimeric iNOS, they followed the same rank order . The degree of dimerization promoted by each analog did not correlate to its binding affinity, its causing a high- or low-spin shift in heme iron spin state, or to its increasing or decreasing NADPH oxidation . Together, we conclude that the enzyme's high degree of substrate specificity only applies to NO synthesis, in that a number of "inactive" structural analogs still bind to iNOS and affect its heme chemistry and structure in the absence of supporting NO synthesis . These latter affects are mediated through binding of the guanidinium portion of L-arginine and its analogs to a single site within iNOS and are relatively independent of the amino acid portion of the molecule.

Biochemistry, 1996 May 7, 35(18), 5838 - 46
Inhibition of human cytomegalovirus UL80 protease by specific intramolecular disulfide bond formation; Baum EZ et al.; A symmetrically substituted disulfide compound, CL13933, was identified as a potent inhibitor of human cytomegalovirus UL80 protease . Two types of inhibited protease were observed, depending on inhibitor concentration . At high concentrations, CL13933 formed a covalent adduct with the protease on Cys residues . At lower concentrations, this compound induced specific intramolecular disulfide formation between Cys84 and Cys87, and between Cys138 and Cys161 . In contrast, Cys202 did not form disulfide bonds . Inhibition was reversed upon reduction of the protease . Each of the five cysteines of the UL80 protease was individually mutated to Ala . Each of the mutant proteases retained enzymatic activity, but mutants C138A and C161A were resistant to inhibition by CL13933, suggesting that disulfide bond formation between Cys138 and Cys161 is responsible for inhibition . This disulfide is apparently not induced by air oxidation . Examination of the CL13933 loading patterns of wild type and the five mutant proteases by mass spectrometry revealed that residues Cys87, Cys138, and Cys161 react with CL13933, and that the disulfide pair partner of each (Cys84, Cys161, and Cys138, respectively) is able to displace the compound via thiol-disulfide exchange . The possible significance of these reactive thiols in the protease is discussed.

Biochemistry, 1996 May 7, 35(18), 5810 - 6
Factor XIIIa-catalyzed cross-linking of recombinant alpha C fragments of human fibrinogen; Matsuka YV et al.; Direct measurements of the structure and function of the COOH-terminal portion of the A alpha chain (residues 220-610) of human fibrinogen have been hampered by the difficulty of isolating intact fragments derived from this protease-sensitive region . Here, we overcame this problem by expressing two fragments, alpha C45K (A alpha 221-610) and a truncated version of it, alpha C30K (A alpha 368-610), in Escherichia coli . Both proteins were purified to homogeneous state, and their integrity was confirmed at protein level by sequencing . Upon treatment with factor XIIIa, the alpha C45K fragment but not the alpha C30K fragment formed polymers similar to those derived from fibrin clots . Sequence analysis of cross-linked alpha C45K polymers revealed involvement in the cross-linking reaction of at least three Gln residues (221, 237, 328) in the NH2-terminal region of the fragment and four Lys residues (539, 556, 580, 601) located in the COOH-terminal part of the molecule . In addition, a fraction of alpha C45K fragment was found in an intramolecular cross-linked form, suggesting a high level of flexibility of its polypeptide chain and consistent with the location of its donor and acceptor residues in clusters near the ends of the molecule . The alpha C30K fragment, lacking the NH2-terminal Gln residues, was not able to form polymers or internally cross-linked monomers . Thus, the C-terminal part of the A alpha chain comprises an autonomous, functionally active, and flexible region that plays a key role in alpha polymer formation and stabilization of fibrin clots by factor XIIIa.

Biochemistry, 1996 May 7, 35(18), 5741 - 6
Carboranyl oligonucleotides . 3 . Biochemical properties of oligonucleotides containing 5-(o-carboranyl-1-yl)-2'-deoxyuridine; Lesnikowski ZJ et al.; Boronated oligonucleotides are potential candidates for boron neutron capture therapy, antisense technology, and as tools in molecular biology . The biological properties of dodecathymidylic acids containing one or more 5-(o-carboran-1-yl)-2'-deoxyuridine residues at different locations within the oligonucleotide chain were studied . 5-(o-Carboran-1-yl)-2'-deoxyuridine containing oligonucleotides manifested marked increased lipophilicity and resistance to 3'- or 5'-phosphodiesterases compared to the corresponding unmodified oligomer . They were substrates for T4 polynucleotide kinase and primers for Escherichia coli polymerase I and human immunodeficiency virus type 1 reverse transcriptase but not for human DNA polymerase alpha and beta . They also formed heteroduplexes that were substrates for E . coli RNase H, an essential property for antisense technology . These studies indicate that the carboranyl-containing oligonucleotides have desirable properties that need to be exploited further in the design of novel biopharmaceuticals.

Biochemistry, 1996 May 7, 35(18), 5726 - 34
ATPase activity of Escherichia coli Rep helicase is dramatically dependent on DNA ligation and protein oligomeric states; Wong I et al.; The Escherichia coli Rep helicase catalyzes the unwinding of duplex DNA using the energy derived from ATP binding and hydrolysis . Rep functions as a dimer but assembles to its active dimeric form only on binding DNA . Each promoter of a dimer contains a DNA binding site that can bind either single-stranded (S) or duplex (D) DNA . The dimer can bind up to two oligodeoxynucleotides in five DNA-ligation states: two half-ligated states, P2S and P2D, and three fully-ligated states, P2S2, P2D2, and P2SD . We have previously shown that the relative stabilities of these ligation states are allosterically regulated by the binding and hydrolysis of ATP and have proposed an "active rolling" model for DNA unwinding where the enzyme cycles through a series of these ligation states in a process that is coupled to the catalytic cycle of ATP hydrolysis {Wong, I., & Lohman, T.M., (1992), Science 256, 350-355} . THe basal ATPase activity of Rep protein is stimulated by ss DNA binding and by protein dimerization . We have measured the steady-state ATPase activities of Rep bound to dT(pT)15 in each distinct ss DNA ligation state (PS, P2S, and P2S2) to compare with our previous measurements with unligated Rep monomer (P) {Moore, K.J.M., & Lohman, T.M . (1994) Biochemistry 33, 14550} . We find the ATPase activity of Rep is influenced dramatically by both dimerization and ss DNA ligation state, with the following kcat values for ATP hydrolysis increasing by over 4 orders of magnitude: 2.1 x 10(-3) s(-1) for P, 2.17 +/- 0.04 s(-1) for PS, 16.5 +/- 0.2 s(-1) for P2S, and 71 +/- 2.5 s(-1) for P2S2 (20 mM Tris-HCl, pH 7.5, 6mM NaCl, 5 mM MgCl2, 10% glycerol, 4 degrees C) . The apparent KM's for ATP hydrolysis are 2.05 +/- 0.1 microM for PS and 2.7 +/- 0.2 microM for P2S . These widely different ATPase activities reflect the allosteric effects of DNA ligation and demonstrate that cooperative communication occurs between the ATP and DNA site of both subunits of the Rep dimer . These results further emphasize the need to explicitly consider the population distribution of oligomerization and DNA ligation states of the helicase when attempting to infer information about elementary processes such as helicase translocation based solely on macroscopic steady-state ATPase measurements.

Biochemistry, 1996 May 7, 35(18), 5679 - 83
Conserved C-terminus of the phosphatase CheZ is a binding domain for the chemotactic response regulator CheY; Blat Y et al.; CheZ is the phosphatase of the chemotactic response regulator, CheY . There are three conserved domains on CheZ . Here we determined the function of the C-terminal domain (residues 202-214) . A truncated form of CheZ, missing residues 202-214, hardly bound to the phosphorylated form of CheY . Conversely, a peptide composed of the last 19 amino acid residues of the CheZ (residues 196-214), generated by tryptic digestion, bound specifically to the phosphorylated form of CheY . This was demonstrated by both fluorescence depolarization of the peptide (labeled with fluorescein) and cross-linking . It is concluded that the conserved C-terminus of CheZ functions as a CheY-binding domain.

Biochemistry, 1996 May 7, 35(18), 5670 - 8
Determinants of cofactor specificity in isocitrate dehydrogenase: structure of an engineered NADP+ --> NAD+ specificity-reversal mutant; Hurley JH et al.; The 7-fold mutation Cys201Met/Cys332Tyr/Lys344Asp/Tyr345Ile/Val35 1Ala/Tyr391Lys/Arg395Ser converts the cofactor specificity of Escherichia coli isocitrate dehydrogenase from a 7000-fold preference for NADP+ to a 200-fold preference for NAD+, with overall activity comparable to that of wild-type NAD+-dependent isocitrate dehydrogenases . The structure of the NAD+-dependent mutant has been determined and refined to a working R-factor of 0.186 at 1.9 A resolution . The structure shows that NADP+ affinity is destroyed by removing favorable interactions between the 2'-phosphate and Tyr345, Tyr391, and Arg395 and by adding a repulsive interaction with Asp344 . NAD+ affinity is enhanced by adding hydrogen bonds between Asp344 and the free 2'-hydroxyl . The favorable Asp344-2'-OH interaction requires a change in the pucker of the ribose to C2' endo and a shift in the adenine ring . The ring shift is made possible by a series of changes in steric packing interactions . The linchpin for repacking in the adenosine binding site is residue 351 . The side chain of this "second layer" residue dictates packing of the surrounding "first layer" residues which interact with the 2' moiety and, in turn, directly determine specificity.

Biochemistry, 1996 May 7, 35(18), 5641 - 6
Mutation of position 52 in ERK2 creates a nonproductive binding mode for adenosine 5'-triphosphate; Robinson MJ et al.; Among the protein kinases, an absolutely conserved lysine in subdomain II is required for high catalytic activity . This lysine is known to interact with the substrate ATP, but otherwise its role is not well understood . We have used biochemical and structural methods to investigate the function of this lysine (K52) in phosphoryl transfer reactions catalyzed by the MAP kinase ERK2 . The kinetic properties of activated wild-type ERK2 and K52 mutants were examined using the oncoprotein TAL2, myelin basic protein, and a designed synthetic peptide as substrates . The catalytic activities of K52R and K52A ERK2 were lower than that of wild-type ERK2, primarily as a consequence of reductions in kcat . Further, there was little difference in Km for ATP, but the Km,app for peptide substrate was higher for the K52 mutants . The three-dimensional structure of unphosphorylated K52R ERK2 in the absence and presence of bound ATP was determined and compared with the structure of unphosphorylated wild-type ERK2 . ATP adopted a well-defined but distinct binding mode in K52R ERK2 compared to the binding mode in the wild-type enzyme . The structural and kinetic data show that mutation of K52 created a nonproductive binding mode for ATP and suggest that K52 is essential for orienting ATP for catalysis.

Biochemistry, 1996 May 7, 35(18), 5633 - 40
Structure and dynamics of a CheY-binding domain of the chemotaxis kinase CheA determined by nuclear magnetic resonance spectroscopy; McEvoy MM et al.; The Escherichia coli histidine autokinase CheA plays an important role in coupling signals received from membrane-bound receptors to changes in the swimming behavior of the cells in order to respond appropriately to environmental signals . Here we describe the structure of the 14 kDa fragment of the chemotaxis kinase CheA, residues 124--257, which binds to the downstream targets of phosphorylation, the response regulators CheY and CheB . This protein fragment contains the CheY-binding domain flanked on each side by regions that correspond to domain linkers in the intact protein . The structure of the domain was determined from 1429 restraints derived from heteronuclear multidimensional NMR experiments . Hybrid distance geometry--dynamical simulated annealing methods were used to calculate a family of structures that satisfy the experimental distance restraints and torsion angle restraints . The root mean square deviation of the 69 ordered residues in the domain is 0.52 A for the backbone heavy atoms and 0.99 A for all heavy atoms . The residues that have been implicated as important for CheY binding form a face consisting of several partially buried hydrophobic residues, framed by charged residues . The dynamic properties of this protein fragment were measured and analyzed using both isotropic and anisotropic models of molecular motion . The linker regions are very flexible and disordered, as evidenced by the very dynamics properties as compared to the CheY-binding domain . The CheY-binding domain of CheA is structurally similar to the histidine-containing phosphocarrier, HPr, which is a protein involved in the phosphoenolpyruvate:sugar phosphotransferase (PTS) pathway . This structural similarity suggests a possible evolutionary relationship of the PTS and chemotaxis pathways.

Biochemistry, 1996 May 7, 35(18), 5624 - 32
Kinetics of halide release of haloalkane dehalogenase: evidence for a slow conformational change; Schanstra JP et al.; Haloalkane dehalogenase converts haloalkanes to their corresponding alcohols and halides . The reaction mechanism involves the formation of a covalent alkyl-enzyme complex which is hydrolyzed by water . The active site is a hydrophobic cavity buried between the main domain and the cap domain of the enzyme . The enzyme has a broad substrate specificity, but the kcat values of the enzyme for the best substrates 1,2-dichloroethane and 1,2-dibromoethane are rather low (3 and 3.5 s-1, respectively) . Stopped-flow fluorescence experiments with substrate under single-turnover conditions indicated that halide release could limit the overall kcat . Furthermore, at 5mM 1,2-dibromoethane the observed rate of substrate binding to free enzyme was faster than 700 s-1 (within the dead time of the stopped-flow instrument) whereas displacement of halide by 5mM 1,2-dibromoethane occurred at a rate of only 8 s-1 . The binding of bromide and chloride to free enzyme was also studied using stopped-flow fluorescence, and the dependence of kobs on the halide concentration suggested that there were two parallel routes for halide binding . One route, in which a slow enzyme isomerization is followed by rapid halide binding, was predominant at low halide concentrations . The other route involves rapid binding into an initial collision complex followed by a slow enzyme isomerization step and prevailed at higher halide concentrations . The overall rate of halide release was low and limited by a slow enzyme isomerization preceding actual release (9 and 14.5 s-1 for bromide and chloride, respectively) . We propose that this slow isomerization is a conformational change in the cap domain that is necessary to allow water to enter and solvate the halide ion . A solvent kinetic isotope effect of 2H2O was found both on kcat and on the rate of halide release . 2H2O mainly affected the rate of the conformational change, which is in agreement with this step being rate-limiting and the overall stabilizing effect of 2H2O on the conformation of proteins.

FEBS Lett, 1996 May 6, 385(3), 165 - 70
Stable monomeric form of an originally dimeric serine proteinase inhibitor, ecotin, was constructed via site directed mutagenesis; Pal G et al.; Ecotin, a homodimer protein of E . coli, is a unique member of canonical serine proteinase inhibitors, since it is a potent agent against a variety of serine proteinases having different substrate specificity . Monomers of ecotin are held together mostly by their long C-terminal strands that are arranged as a two-stranded antiparallel beta-sheet in the functional dimer . One ecotin dimer can chelate two proteinase molecules, each of them bound to both subunits of ecotin at two different sites, namely the specific primary and the non-specific secondary binding sites . In this study the genes of wild type ecotin and its Met84Arg P1 site mutant were truncated resulting in new forms of ecotin that lack 10 amino acid residues at their C-terminus . These mutants do not dimerize spontaneously, though in combination with trypsin they assemble into the familiar heterotetramer . Our data suggest that this heterotetramer exists even in extremely diluted solutions, and the interaction, which is responsible for the dimerization of ecotin, contributes to the stability of the heterotetrameric complex.

Biochem Biophys Res Commun, 1996 May 6, 222(1), 58 - 63
Generation of active immunotoxins containing recombinant restrictocin; Rathore D et al.; Restrictocin, a toxin produced by the fungus Aspergillus restrictus, is a potent inhibitor of eukaryotic protein synthesis . Recombinant restrictocin was made in Escherichia coli and purified to homogeneity in large amounts . The recombinant protein was found to be poorly immunogenic in mice with low toxicity, when injected intraperitoneally . Two immunotoxins were constructed by coupling the recombinant restrictocin to an antibody to the human transferrin receptor, using a cleavable and a stable linkage . The immunotoxins so generated showed specific cytotoxic activity toward receptor bearing cells in tissue culture . Immunotoxin with a cleavable linkage, however, was more active than that containing a stable linkage . Restrictocin appears to be a promising candidate to be developed as a chimeric toxin for targeted therapy.

J Biol Chem, 1996 May 3, 271(18), 10996 - 1000
Inhibition of beta-ketoacyl-acyl carrier protein synthase III (FabH) by acyl-acyl carrier protein in Escherichia coli; Heath RJ et al.; beta-Ketoacyl-acyl carrier protein (ACP) synthase III (the fabH gene product) condenses acetyl-CoA with malonyl-ACP to initiate fatty acid biosynthesis in the dissociated, type II fatty acid synthase systems typified by Escherichia coli . The accumulation of malonyl-acyl carrier protein (ACP) following the inhibition of a reconstituted fatty acid synthase system by acyl-ACP implicated synthase III (FabH) as a target for acyl-ACP regulation (Heath, R . J., and Rock, C . O . (1996) J . Biol . Chem . 271, 1833-1836); therefore, the FabH protein was purified and its biochemical and regulatory properties examined . FabH exhibited a Km of 40 microM for acetyl-CoA and 5 microM for malonyl-ACP . FabH also accepted other acyl-CoAs as primers with the rank order of activity being acetyl-CoA approximately propionyl-CoA >> butyryl-CoA . FabH utilized neither hexanoyl-CoA nor octanoyl-CoA . Acyl-ACPs suppressed Fabh activity, and their potency increased with increasing acyl chain length between 12 and 20 carbon atoms . Nonesterified ACP was not an inhibitor . Acyl-ACP inhibition kinetics were mixed with respect to acetyl-CoA, but were competitive with malonyl-ACP, indicating that acyl-ACPs decrease FabH activity by binding to either the free enzyme or the acyl-enzyme intermediate . These data support the concept that the inhibition of chain initiation at the beta-ketoacyl-ACP synthase III step contributes to the attenuation of fatty acid biosynthesis by acyl-ACP.

J Biol Chem, 1996 May 3, 271(18), 10767 - 74
Purification of a soluble UmuD'C complex from Escherichia coli . Cooperative binding of UmuD'C to single-stranded DNA; Bruck I et al.; The Escherichia coli UmuD' and UmuC proteins play essential roles in SOS-induced mutagenesis . Previous studies investigating the molecular mechanisms of mutagenesis have been hindered by the lack of availability of a soluble UmuC protein . We report the extensive purification of a soluble UmuD'C complex and its interactions with DNA . The molecular mass of the complex is estimated to be 70 kDa, suggesting that the complex consists of one UmuC (46 kDa) and two UmuD' (12 kDa) molecules . In contrast to its inability to bind to double-stranded DNA, UmuD'C binds cooperatively to single-stranded DNA as measured by agarose gel electrophoresis and confirmed by steady-state fluorescence depolarization . A Hill coefficient, n = 3, characterizes the binding of UmuD'C to M13 DNA and to a 600 nucleotide DNA oligomer, suggesting that at least three protein complexes may interact cooperatively when binding to DNA . The apparent equilibrium binding constant of UmuD'C to single-stranded DNA is approximately 300 nM . Binding of the complex to a short, 80 nucleotide, DNA oligonucleotide was detectable by fluorescence depolarization, but it did not appear to be cooperative . Binding of UmuD'C to single-stranded M13 DNA causes an acceleration of the protein-DNA complex, suggesting that the longer DNA may undergo compaction . The UmuD'C complex associates with RecA-coated DNA, and the UmuD'C complex remains bound to DNA in the presence of RecA.

J Biol Chem, 1996 May 3, 271(18), 10681 - 9
The MalT-dependent and malZ-encoded maltodextrin glucosidase of Escherichia coli can be converted into a dextrinyltransferase by a single mutation; Peist R et al.; malZ is a member of the mal regulon . It is controlled by MatT, the transcriptional activator of the maltose system . MalZ has been purified and identified as an enzyme hydrolyzing maltotriose and longer maltodextrins to glucose and maltose . MalZ is dispensable for growth on maltose or maltodextrins . Mutants lacking amylomaltase (encoded by malQ), the major maltose utilizing enzyme, cannot grow on maltose, maltotriose, or maltotetraose, despite the fact that they contain an effective transport system and MalZ . From such a malQ mutant a pseudorevertant was isolated that was able to grow on maltose . The suppressor mutation was mapped in malZ . The mutant gene was cloned . It contained a Trp to Cys exchange at position 292 of the deduced protein sequence . Surprisingly, the purified mutant enzyme was still unable to hydrolyze maltose as was the wild type enzyme, while both were able to release glucose from maltodextrins . However, the mutant enzyme had gained the ability to transfer dextrinyl moieties to glucose, maltose, and other maltodextrins . Thus, it had gained an activity associated with amylomaltase . It was the MalZ292-associated transferase reaction that allowed the utilization of maltose . In addition, we discovered that mutant and wild type enzyme alike were highly active as gamma-cyclodextrinases.

J Biol Chem, 1996 May 3, 271(18), 10538 - 44
Programming the Rous sarcoma virus protease to cleave new substrate sequences; Ridky TW et al.; The Rous sarcoma virus protease displays a high degree of specificity and catalyzes the cleavage of only a limited number of amino acid sequences . This specificity is governed by interactions between side chains of eight substrate amino acids and eight corresponding subsite pockets within the homodimeric enzyme . We have examined these complex interactions in order to learn how to introduce changes into the retroviral protease (PR) that direct it to cleave substrates . Mutant enzymes with altered substrate specificity and wild-type or greater catalytic rates have been constructed previously by substituting single key amino acids in each of the eight enzyme subsites with those residues found in structurally related positions of human immunodeficiency virus (HIV)-1 PR . These individual amino acid substitutions have now been combined into one enzyme, resulting in a highly active mutant Rous sarcoma virus (RSV) protease that displays many characteristics associated with the HIV-1 enzyme . The hybrid protease is capable of catalyzing the cleavage of a set of HIV-1 viral polyprotein substrates that are not recognized by the wild-type RSV enzyme . Additionally, the modified PR is inhibited completely by the HIV-1 PR-specific inhibitor KNI-272 at concentrations where wild-type RSV PR is unaffected . These results indicate that the major determinants that dictate RSV and HIV-1 PR substrate specificity have been identified . Since the viral protease is a homodimer, the rational design of enzymes with altered specificity also requires a thorough understanding of the importance of enzyme symmetry in substrate selection . We demonstrate here that the enzyme homodimer acts symmetrically in substrate selection with each enzyme subunit being capable of recognizing both halves of a peptide substrate equally.

J Biol Chem, 1996 May 3, 271(18), 10470 - 6
Molecular cloning and expression of cDNA encoding rat brain cytosolic acyl-coenzyme A thioester hydrolase; Broustas CG et al.; The cDNA encoding rat brain cytosolic acyl-CoA thioester hydrolase (ACT) has been cloned and sequenced, and the primary structure of the enzyme has been deduced . A partial amino acid sequence (38 amino acids) of the enzyme was determined using the peptides generated after CNBr digestion of the purified enzyme . Primers synthesized on the basis of this information were used to isolate two cDNA clones, each encoding the full length of the enzyme . The nucleotide sequences of these clones contained an open reading frame encoding a 358-amino acid polypeptide with a calculated molecular mass of 39.7 kDa, similar to that determined for the purified enzyme (40.9 kDa) . The deduced ACT sequence showed no homology to the known sequences of any other thioesterases nor to any other known protein sequence . However, there was a strong homology to a number of expressed sequence tag human brain cDNA clones . The identity of the ACT cDNA was confirmed by the expression of ACT activity in Escherichia coli . There was a 10-15-fold increase in ACT-specific activity in the bacterial extracts after induction with isopropyl thiogalactoside, and the properties of the expressed enzyme (fusion protein) were the same as those of the purified rat brain ACT . Northern blot analysis showed that a 1.65-kilobase ACT transcript was present in rat brain and testis but not in any other rat tissues examined . However, the ACT mRNA was induced in the liver of rats that were fed Wy-14,643, a peroxisome proliferator and inducer of rodent liver cytosolic acyl-CoA thioesterase . These results indicate that the induced rat liver ACT is homologous to the constitutive rat brain ACT.

J Mol Biol, 1996 May 3, 258(2), 308 - 21
Structure of a hexanucleotide RNA hairpin loop conserved in ribosomal RNAs; Huang S et al.; The structure of 5'-pppGGAC(GUAAUA)GUCC has been deduced from NMR data . The six-nucleotide hairpin loop is highly conserved in large subunit ribosomal RNA and confers unusual stability on RNA hairpins . Standard assignment methods, including through-bond strategies for backbone protons, were used to assign all 31P resonances and all but two of the non-exchangeable protons . The model calculated by restrained molecular dynamics shows that the loop adopts a specific structure stabilized by five non-canonical hydrogen bonds . An edge-to-edge G-A pair (hydrogen bonds G5 N3-A10 NH6, G5 H2-A1O N7 and A10 NH6-G5 O2') closes the hairpin loop . A trans 5' P-O bond at A7 reverses the direction of the backbone and is stabilized by two base-backbone hydrogen bonds (U6 NH3-U9 OP and U6 O2'-A8 N7) . The only unstructured part of the loop is U9, which is completely unstacked and also the only phylogenetically variable position . The U6-A7-A8 "U-turn" reproduces hydrogen bonds and backbone torsion seen in the tRNA anticodon loop and TTpsiC loop, and in an internal loop of the hammerhead ribozyme . G-A is a common closing mismatch in ribosomal RNA hairpin loops . The U-turn and G-A pair found in this hexaloop structure may therefore be common structural units of RNA hairpin loops.

J Mol Biol, 1996 May 3, 258(2), 213 - 23
Identifying interacting regions in the beta subunit of Escherichia coli RNA polymerase; Tavormina PL et al.; Numerous physical and genetic approaches have identified residues in the alpha, beta, beta' and sigma subunits of Escherichia coli RNA polymerase that are involved in transcriptional processes; in contrast, relatively little data exist to demonstrate interacting regions within or between the subunits themselves . As a means of identifying regions in the beta subunit that may interact, we have sought intragenic suppressor mutations of a class of elongation-defective and termination-proficient inviable rpoB alleles that affect highly conserved residues . We obtained intragenic allele-specific suppressors of GD566 (located in conserved region D) and AV676 (located in conserved region E) . With one exception, these allele-specific suppressors also map to highly conserved regions of the beta subunit . Allele specific suppression is a genetic criterion for protein-protein interaction . Moreover, the functional properties of the mutants suggests that suppression is likely to result from protein-protein interaction rather than from functional compensation . Our suppression studies provide evidence for the interaction of conserved regions B and D as well as conserved regions E and H of the beta polypeptide . We suggest that these, as well as other conserved regions of the beta polypeptide, may interact with each other to provide a framework for the function of the enzyme.

Biochim Biophys Acta, 1996 May 2, 1294(1), 77 - 82
High substrate specificity and induction characteristics of trimethylamine-N-oxide reductase of Escherichia coli; Iobbi-Nivol C et al.; Using a wide variety of N- and S-oxide compounds we have shown by kinetic analysis that only two N-oxides, trimethylamine-N-oxide and 4-methylmorpholine-N-oxide, can be considered good substrates for trimethylamine-N-oxide (TMAO) reductase on the basis of their kcat/Km ratio . This result demonstrates that TMAO reductase possesses a high substrate specificity . Induction of the torCAD operon using the same S- and N-oxide compounds was also analyzed . We demonstrate that there is no correlation between the ability for a compound to be reduced by TMAO reductase and to induce TMAO reductase synthesis.

Biochim Biophys Acta, 1996 May 2, 1294(1), 55 - 62
A type I ovine interferon with limited similarity to IFN-alpha, IFN-omega and IFN-tau: gene structure, biological properties and unusual species specificity; Liu L et al.; A gene encoding a 195 amino-acid (a.a.) polypeptide with a putative 23 a.a . signal sequence that had about 60% a.a . sequence identity to ovine interferon-omega (OvIFN-omega) and 55% or less identity to BoIFN-tau, OvIFN-tau and all known IFN-alpha and -beta has been identified from an ovine genomic DNA library . Surprisingly, it shared almost complete identity to genes for rabbit IFN-omega within its coding sequence and proximal promoter region, although the two were different in their 3'-ends . This IFN (tentatively termed ovine IFN-omega variant, OvIFN-omegav), purified in recombinant form from E . coli, had normal antiviral activity when tested on sheep fetal tongue and brain cells and rabbit kidney cells, but very low activity towards bovine, goat and human cells . It competed with 125I-labeled BoIFN-tau for binding to IFN receptors on ovine cells . Expression of OvIFN-omegav was not detected by reverse transcription-PCR either in ovine peripheral blood leukocytes infected with Sendai virus, or in any other tissues examined . OvIFN-omegav may represent a previously unrecognized, non-virally inducible type I subtype distinct from IFN-alpha, -beta, -omega and -tau . The presence of a conserved gene in rabbit and sheep could reflect a recent interspecies transfer.

J Bone Miner Res, 1996 May, 11(5), 578 - 86
Phosphorylation of the cytoplasmic tail of the PTH/PTHrP receptor; Blind E et al.; Activation of the G protein-coupled receptor for parathyroid hormone (PTH)/PTH-related protein (PTHrP) produces homologous desensitization of receptor signaling . We have shown recently that the opossum PTH/PTHrP receptor stably expressed in human embryonic kidney (HEK) 293 cells is phosphorylated upon agonist binding and upon activation of serine/threonine protein kinases (PKA and PKC), an event which for some G protein-coupled receptors has been linked to desensitization . To locate the sites of phosphorylation, mutated forms of the opossum PTH/PTHrP receptor were stably expressed in HEK 293 cells, and ligand-stimulated receptor phosphorylation was evaluated . The five serine and threonine residues of the third cytoplasmic loop of the receptor were not required for receptor phosphorylation . Basal and ligand-induced phosphorylation were, however, completely abolished upon deletion of all but the 16 juxtamembrane residues of the cytoplasmic C-terminal tail of the receptor, even though this truncated receptor resembled the wild-type receptor in its level of expression based on Western blotting and radioligand binding . To identify further the phosphorylation sites, the 129 amino acid C-terminal tail of the rat PTH/PTHrP receptor was expressed in E . coli as a recombinant glutathione S-transferase fusion protein . Elimination of a single PKA consensus site in the tail (serine 491) resulted in > or = 90% loss of PKA-mediated phosphorylation, identifying this as the preferential site for PKA, with two other sites (serine 473 and/or 475) being minor sites . Phosphorylation by PKC occurred largely in the proximal portion of the tail, whereas beta-adrenergic receptor kinase 1 (beta ARK1) phosphorylated more distally in the tail . The ability of these kinases to phosphorylate the PTH/PTHrP receptor at distinct sites on the cytoplasmic tail may allow differential regulation of receptor signaling and trafficking.

Immunol Invest, 1996 May, 25(3), 185 - 90
Neutrophil function in elderly persons assessed by flow cytometry; Esparza B et al.; It is well known that the immune response declines with senescence and it is suggested that these changes render an individual susceptible to infection, autoimmune phenomena and cancer . Bacterial and viral infections are a major cause of illness and death amongst aged subjects, and once infection is established, the elderly also have a diminished capacity to prevent its spread (1) . The cellular and molecular basis for this age-related decline in immunocompetence are still unknown and, possibly, are related to an alteration in cell transduction mechanisms (2).

Shock, 1996 May, 5(5), 349 - 59
Regional arteriovenous differences in P(CO2) and pH can reflect critical organ oxygen delivery during endotoxemia; Zhang H et al.; The goal of this study was to assess whether serial measurements of regional veno-arterial PcoC2 (VAPco2) and arteriovenous pH (AVpH) differences reflect the onset of tissue hypoxia in various organs during endotoxemia . In 12 anesthetized, mechanically ventilated dogs, ultrasonic flow probes were placed around superior mesenteric, renal, and femoral arteries to measure regional blood flow . The corresponding veins were cannulated for blood sampling . Oxygen uptake (V02) was determined from exhaled gas analysis, and oxygen delivery (D02) was calculated as the product of thermodilution cardiac output and arterial oxygen content . Six dogs served as controls, and six received Escherichia coli endotoxin . Cardiac tamponade was induced to reduce D02 . Systemic, mesenteric, and femoral critical D02 (DO2crit) were higher in the endotoxic than in the control group (systemic: 12.1 + or - 2.2 vs . 7.9 + or - 2.6 mL/kg min; mesenteric: 8.2 + or - 2.5 vs . 4.1 + or - .6 mL/100 g tissue-min; femoral: 8.3 + or - 2.3 vs . 4.6 + or - .9 mL/min; all p < .05) . Systemic and regional critical oxygen extraction ratio (O2ERcrit) were lower in the endotoxic than in the control group (systemic: 45.1 + or - 9.7 vs . 74.1 + or - 9.1%; mesenteric: 37.1 + or - 15.4 vs . 71.1 + or - 7.4%; renal: 30.7 + or - 24.6 vs . 53.9 + or - 28.7%; femoral: 48.1 + or - 9.2 vs . 75.3 + or - 6.9%; all p < .05) . With and without endotoxin, systemic and regional DO2crit calculated from V02, VAPco2, or AVpH were similar . In conclusion, systemic and regional VAPco2 and AVpH gradients can reflect hypoxic threshold in the presence, as in the absence, of endotoxemia.

Shock, 1996 May, 5(5), 344 - 8
Hepatic release of tumor necrosis factor in the endotoxin-treated conscious dog; McGuinness OP et al.; The effects of a 4 h intraportal infusion of Escherichia coli lipopolysaccharide (LPS, .21 mu g/kg/min) on the release of tumor necrosis factor (TNF) by hepatic and nonhepatic splanchnic tissues was assessed in the chronically catheterized conscious dog (n = 7) using arteriovenous difference techniques . TNF levels were measured using both a WEHI-164 cytotoxicity assay (WEHI) and a h-TNF-alpha EIA kit (ELISA; Biosource, Camarillo, CA) . Using WEHI, arterial TNF levels increased from 10 + or - 6 pg/mL to a peak of 4667 + or - 1442 pg/mL 100 min after LPS and fell to 443 + or - 199 pg/mL by 240 min . Using ELISA, arterial TNF levels increased from 5 + or - 5 pg/mL to a peak of 12,234 + or - 2046 pg/mL at 100 min and fell to 3511 + or - 991 pg/mL by 240 min . WEHI could not be used to assess organ TNF release due to excessive assay variability . Based upon ELISA, net hepatic TNF output increased from undetectable release at basal to 23.0 + or - 10.7 ng/kg/min at 60 min and returned toward basal by 240 min (4.7 + or - 3.8 ng/kg/min) . Net release of TNF by the nonhepatic splanchnic bed was not observed . One compartment analysis of the arterial TNF response indicated that net release of TNF by the liver accounted for the majority of the increase in the arterial TNF levels . In summary, after intraportal LPS infusion, it was determined that 1) both assays predict similar qualitative TNF response, while the quantitative response differs, 2) the liver is the major site of TNF production, and 3) the nonhepatic splanchnic bed is not a net producer of TNF.

Genes Cells, 1996 May, 1(5), 443 - 51
The directionality of RuvAB-mediated branch migration: in vitro studies with three-armed junctions; Hiom K et al.; BACKGROUND: The Escherichia coli RuvA and RuvB proteins promote the branch migration of 4-way (Holliday) junctions during genetic recombination . The active complex is a tripartite structure in which RuvA protein is bound to the crossover and is sandwiched between two hexameric rings of RuvB . Branch migration requires ATP hydrolysis and occurs as the DNA passes through each RuvB ring . RESULTS: In this work, we have investigated the mechanism by which RuvAB catalyses the branch migration of a three-armed (Y) junction . Using synthetic DNA structures, we observed the formation of DNA products, a partial duplex DNA molecule and a single-stranded oligonucleotide, indicative of a branch migration reaction that occurred with unique polarity . Analysis of the RuvAB-junction complex by DNase footprinting showed that RuvA bound asymmetrically to the junction and targeted a single hexameric RuvB ring to one arm of DNA . CONCLUSION: Branch migration of a three-armed junction occurs in a unidirectional manner that is determined by the assembly of a single RuvB ring onto one arm of the DNA . The asymmetry of the complex and observed directionality of branch migration indicate that strand passage occurs as the DNA is pulled into the RuvB ring structure, a reaction likely to be coupled with DNA unwinding.

Genes Cells, 1996 May, 1(5), 437 - 42
Implications of the zinc-finger motif found in the DNA-binding domain of the human XPA protein; Morita EH et al.; BACKGROUND: The XPA (xeroderma pigmentosum group A) protein specifically recognizes the UV-or chemically damaged DNA lesions, and triggers the nucleotide excision repair process . This XPA protein contains the functional domain which is crucial to the recognition of damaged DNA . Its primary structure suggests that this DNA binding domain may contain a zinc-finger motif . To gain a more detailed insight into this zinc-finger motif, we have measured the 113Cd-NMR spectra of the DNA binding domains derived from the wild-type and mutant XPA proteins . RESULTS: 113Cd-NMR analysis, combined with atomic absorption and site-directed mutagenesis, revealed that the DNA binding domain contains one zinc ion, coordinated with four cysteine residues (Cys105, Cys108, Cys126, and Cys129), that is essential for correct protein folding in vivo and in vitro . CONCLUSIONS: The four ligand cysteine residues form a Cys-X2-Cys-X17-Cys-X2-Cys motif, which is reminiscent of the (Cys)4 type zinc-finger motif found in numerous transcriptional regulatory proteins . However, the secondary structure prediction and the 3D-1D compatibility analysis demonstrate that there is no structural similarity in the vicinity of the zinc-finger motif between the XPA protein and other zinc-finger containing proteins . We conclude that the XPA protein contains a new type of zinc-finger motif.

Genes Cells, 1996 May, 1(5), 421 - 7
Glutaminyl-tRNA synthetase: from genetics to molecular recognition; Ibba M et al.; Accurately aminoacylated tRNAs are an a priori requirement for translation of the genetic code . They are synthesized by the aminoacyl-tRNA synthetases which select both the correct amino acid and tRNA from a total of more than 400 possible combinations . Genetic, biochemical and structural studies have begun to reveal the mechanisms by which this specificity is achieved by Escherichia coli glutaminyl-tRNA synthetase (GlnRS) . Sequence-specific interactions between GlnRS and tRNA(Gln) determine both the accuracy of tRNA selection and the efficiency of aminoacylation . Thus, amino acid recognition is tRNA-dependent . Consequently, while a noncognate tRNA may be recognized by GlnRS, the resulting tRNA-enzyme complex displays a considerably reduced affinity for glutamine compared to wild-type . This mechanism now provides a ready explanation as to why the majority of tRNA mischarging events, including those originally described over 25 years ago for GlnRS, impair cellular viability only to a limited degree.

J Appl Bacteriol, 1996 May, 80(5), 511 - 6
Effect of the pre-treatments for milk samples filtration on direct viable cell counts; Fernandez-Astorga A et al.; Escherichia coli O25:H-42 was selected to study the effect of pre-treatments on the enumeration of direct viable cells from milk samples . Before and after inducing cell elongation by cellular division inhibitors, three pre-treatments for milk-filtration were used . One involved a pre-treatment with trypsin (1.5 min at 50 degrees C), addition of hot Triton X-100 after heating and filter rinses with phosphate saline buffer . The other two involved pre-treatment with trypsin and Triton X-100 (10 min at 50 degrees C), filter rinses with hot Triton X-100 and organic solvents . Pre-treatments applied after inducing cell elongation had an effect on cell recovery from milk samples depending on the pre-treatment used . The most suitable, on the basis of the number and percentage of enlarged cells obtained was the first described . The others selectively affected recovery of elongated cells . Pre-treatments applied before inducing the cell elongation, negatively affected viability with enumeration in milk samples being significantly (P < 0.001) lower than those found in controls . However, the negative effects of first pre-treatment on viability was lower than that produced by the pre-treatments involving organic solvents.

FEMS Microbiol Lett, 1996 May 1, 138(2-3), 245 - 50
The rpoD gene functions as a multicopy suppressor for mutations in the chaperones, CbpA, DnaJ and DnaK, in Escherichia coli; Shiozawa T et al.; The CbpA protein is an analog of the DnaJ molecular chaperone of Escherichia coli . The dnaJ- cbpA- double-null mutant exhibits severe defects in cell growth, namely, a very narrow temperature range for growth . To gain insight into the functions of CbpA as well as DnaJ, we isolated a multicopy suppressor gene that permits this dnaJ- cbpA- mutant to grow normally at low temperatures . The suppressor gene was identified as rpoD, the gene that encodes the major sigma 70 . The biological implications of this finding are examined and discussed.

FEMS Microbiol Lett, 1996 May 1, 138(2-3), 185 - 9
The Escherichia coli K99 periplasmic chaperone FanE is a monomeric protein; Mol O et al.; The monomeric or dimeric nature of the K99 periplasmic chaperone FanE was examined . The gene encoding FanE was subcloned in a pINIIIA1 derivative expression vector . A complementation experiment showed that the subcloned FanE was biologically functional . The protein was purified from the periplasm of cells harbouring the constructed plasmid . Automated Edman degradation experiments confirmed the predicted N-terminal amino acid sequence of FanE . A polyclonal mouse antiserum was raised against the FanE chaperone . The monomeric or oligomeric nature of the protein in the periplasm was studied by gel filtration, immunoblotting and chemical cross-linking experiments . The results indicated that FanE is a monomeric protein, in contrast to the K88 periplasmic chaperone.

FEMS Microbiol Lett, 1996 May 1, 138(2-3), 161 - 5
Identification of iron superoxide dismutase and a copper/zinc superoxide dismutase enzyme activity within the marine cyanobacterium Synechococcus sp . WH 7803; Chadd HE et al.; Three constitutive forms of superoxide dismutase activity have been demonstrated in the cyanobacterial marine picoplankter Synechococcus sp . WH 7803 using polyacrylamide gel activity staining techniques . A protein which gave a positive non-haem iron stain on native polyacrylamide gels exhibited N-terminal similarity to both the iron superoxide dismutase and the manganese superoxide dismutase of Escherichia coli . The metal prosthetic group of each of the three activity bands was characterised by analysing their differential sensitivities to 5 mM H2O2, 2 mM cyanide and 2 mM of the copper chelator diethyldithiocarbamate . Three distinct superoxide dismutase activities were observed, an iron superoxide dismutase, a copper/zinc superoxide dismutase and a third form which has not been identified . Growth of Synechococcus cells in ASW medium containing no added iron resulted in no alteration in the activity of the iron superoxide dismutase . Growth of cultures in the absence of copper or zinc resulted in differential changes in the activities of the copper/zinc superoxide dismutase and the unidentified superoxide dismutase.

FEMS Microbiol Lett, 1996 May 1, 138(2-3), 129 - 34
Phosphorylation of GroEL, DnaK and other proteins from Thiobacillus ferrooxidans grown under different conditions; Seeger M et al.; The levels of phosphorylation of the chaperones DnaK and GroEL and other proteins varied when cells of Thiobacillus ferrooxidans were subjected to phosphate starvation . The phosphorylated amino acid of GroEL was found to be threonine . Our results show that not only heat shock, but also a nutrient starvation stress leads to phosphorylation of chaperones and, in addition, support the possible role of phosphorylation of these proteins in the sensing and regulation of stress responses in bacteria.

Gen Comp Endocrinol, 1996 May, 102(2), 173 - 82
Heterologous expression of the spiny dogfish shark (Squalus acanthias) cytochrome P450c17 (17 alpha-hydroxylase) in Escherichia coli; Trant JM; Cytochrome P450c17 is a key steroidogenic enzyme for the production of sex steroids in gonadal tissue and for cortisol production in adrenal tissue . This protein possesses two enzymatic activities . The 17-alpha-hydroxylase activity introduces a hydroxyl group into the steroid nucleus . The resultant 17-alpha-hydroxylated, C(21) pregnenes can be converted to a C(19) androgen by the C(17,20)-lyase activity . The cDNA encoding the spiny dogfish shark (Squalus acanthias) testicular form of cytochrome P450c17 was used to direct heterologous expression in Escherichia coli . The wild-type P450c17 protein was not conducive to expression in E . coli using either a pET21 or a pCwori+ vector . However, modification of the amino terminus permitted overexpression of inactive shark protein with the pFT21 vector . This protein was fused with a hexahistidinyl peptide to facilitate purification by column chromatography using a Ni(2+)-chelated resin . Transformed bacteria yielded an average of 1.8 mg of purified, concentrated P450c17 protein per 100-ml culture . This modified protein was used to raise antisera in rabbits and the resultant antisera was used at a working titer of 1:10,000 for Western blot analysis . Culture conditions that result in the accumulation of bioactive recombinant bovine P450c17 failed to demonstrate expression of either the native or the modified form of shark P450c17 using the pCWori+ and pET21 vectors . These results suggest that the amino terminus of the native shark P450c17 was not conducive to synthesis in E . coli . However, modifications of the amino terminus permitted synthesis of an inactive protein that was protected from degradation within inclusion bodies.

J Vasc Res, 1996 May-Jun, 33(3), 249 - 57
Lack of endotoxin-induced hyporesponsiveness to U46619 in isolated neonatal porcine pulmonary but not mesenteric arteries; Perez-Vizcaino F et al.; The effects of endotoxin from Escherichia coli on the vasoconstrictor responses to noradrenaline (10 nM-100 microM) and the thromboxane A2 analog U46619 (100 pM-1 microM) were evaluated on isolated pulmonary and mesenteric arteries from neonatal piglets . Incubation for 20 h with endotoxin (1 microgram ml-1) induced a decrease in the contractile responses to noradrenaline in both arteries (p < 0.05) which was inhibited by NG-nitro-L-arginine-methyl ester (L-NAME, 100 microM) . Endotoxin-treated mesenteric arteries also showed a reduction of the maximal contractions induced by U46619 (p < 0.05) and this effect was inhibited by L-NAME . In contrast, the contractile responses to U46619 were similar in control and endotoxin-treated pulmonary arteries . In endothelium-denuded pulmonary rings, endotoxin was also unable to modify the contractile responses to U46619 . In pulmonary rings, the contractions induced by U46619 (100 nM) were much less sensitive to sodium nitroprusside, 8-bromo-cyclic GMP or dipyridamole than those induced by 10 microM noradrenaline . In conclusion, endotoxin-treated pulmonary arteries exhibited decreased responses to noradrenaline due to enhanced nitric oxide release but not to the thromboxane A2 analog U46619 . This lack of hyporesponsiveness to U46619 in pulmonary arteries may be attributed to a relative insensitivity to nitric oxide . The absence of pulmonary hyporesponsiveness to U46619 may explain why pulmonary hypertension occurs in septic shock despite Ca(2+)-independent nitric oxide synthase induction in the lung.

DNA Cell Biol, 1996 May, 15(5), 401 - 6
Multi-drug delivery system using streptavidin-transforming growth factor-alpha chimeric protein; Ohno K et al.; Tissue-specific delivery of a variety of molecules has been a valuable technique for biological and medical research . Therefore, we have constructed a recombinant plasmid containing the coding regions for streptavidin core and mature human transforming growth factor-alpha (TGF-alpha) . The recombinant plasmid has been expressed in Escherichia coli to produce a chimeric protein with both streptavidin and TGF-alpha activity . The streptavidin-TGF-alpha chimeric protein (ST-TGF-alpha) could efficiently transfer biotinylated beta-galactosidase into A431 cells via the epidermal growth factor receptor . More than 99% of the cells contained the enzyme transferred . Furthermore, ST-TGF-alpha complexed with biotinylated-glucose oxidase had a significant cytotoxic effect when incubated with A431 cells . These findings suggest that the ST-TGF-alpha chimeric protein could be used to deliver proteins of interest into target cells without the need for chemical linkage or genetic construction . Essentially, ST-TGF-alpha serves as a high-modular "molecular bridge" for the passage of a wide variety of effector molecules into target cells.

Pept Res, 1996 May-Jun, 9(3), 127 - 35
Tandem use of PCR and synthetic peptides to map helper T-cell epitopes on 27-kDa sexual stage antigen of Plasmodium falciparum; Koski GK et al.; Monoclonal antibodies recognizing two overlapping linear epitopes (amino acid residues 10 to 25) on the 27-kDa sexual stage antigen of Plasmodium falciparum (Pfg 27) effectively reduce the infectivity of the parasites to mosquitoes . Although malaria transmission-blocking immunity is largely antibody-mediated, T cells play critical roles in the regulation of antibody-secreting B cells . In order to facilitate the development of a malaria transmission-blocking subunit vaccine, studies were undertaken to map epitopes on Pfg 27 recognized by T-helper lymphocytes . Pfg27-specific T-cell hybridoma clones were produced from rPfg27-immunized BALB/c (H-2d) and C57BL/6 (H-2b) mice, and used in studies to map antigenic determinants using PCR-generated Pfg27 gene fragments expressed in E . coli and synthetic peptides based on the Pfg27 sequence . We identified and mapped five distinct T-cell epitopes that are recognized by major histocompatibility complex (MHC) class II-restricted T-cell hybridoma clones . A single peptide (21 residues) was shown to contain two tandem or partially overlapping epitopes recognized by T-cell hybridomas in the context of I-Ad and I-Ab, respectively . Synthetic peptides representing epitopes recognized by T-cell hybridoma clones elicited strong IgG responses in immunized mice, suggesting that T-cells of the helper phenotype were stimulated in vivo by these peptides . These studies represent the first detailed T-cell epitope analysis of a malaria sexual-stage antigen.

Trends Biochem Sci, 1996 May, 21(5), 174 - 7
Iron-sulfur clusters as biosensors of oxidants and iron; Rouault TA et al.; Iron-sulfur clusters are prosthetic groups commonly found in proteins that participate in oxidation-reduction reactions and catalysis . Here, we focus on two proteins that contain iron-sulfur clusters, the fumarate nitrate reduction (FNR) protein of Escherichia coli and mammalian iron-responsive-element-binding protein 1 (IRP1), both of which function as direct sensors of oxygen and iron levels . Assembly and disassembly of iron-sulfur clusters is the key to sensing in these proteins and we speculate that iron-sulfur clusters might be found in other regulatory proteins that sense levels of iron and/or oxygen.

Protein Expr Purif, 1996 May, 7(3), 275 - 80
A method of screening for mutant proteins containing cysteine residues using fluorescein-5-maleimide; McLachlin DT et al.; A method of screening transformed bacterial colonies for introduction of a cysteine residue into an overexpressed protein is described . After treating SDS extracts of induced bacterial cells with fluorescein-5-maleimide, the proteins containing cysteine were visible on SDS-PAGE gels under ultraviolet light as fluorescent bands . If the wild-type protein contains no endogenous cysteine residues, then mutant proteins containing cysteine may be easily identified by their fluorescence . In addition, a shift in electrophoretic mobility of modified proteins was observed, with mutant proteins containing cysteine at more than one site exhibiting incremental decreases in electrophoretic mobility . This effect permits the detection of cysteine mutations even when endogenous cysteines are present . The described method allows the rapid screening of a large number of transformants.

Protein Expr Purif, 1996 May, 7(3), 323 - 8
Site-directed mutagenesis techniques in the study of Escherichia coli serine hydroxymethyltransferase; Iurescia S et al.; The 3340-bp fragment containing the Escherichia coli glyA gene coding for serine hydroxymethyltransferase was reduced in size by PCR, and the 1600-bp fragment obtained was cloned into the vector pBR322 in both orientations (5'-3', and 3'-5') . This DNA manipulation allowed us to perform site-directed mutagenesis by PCR on the glyA gene . To overcome the problem of the presence of wild-type protein in the various mutant enzyme preparations, the E . coli strain GS245 used to express recombinant serine hydroxymethyltransferase was made recA deficient through generalized transduction mediated by phage P1 . The new strain was used for the production of a mutant form of the enzyme, in which the pyridoxal 5'-phosphate binding lysine was substituted by a glutamine . The preparation of this mutant form was completely devoid of wild-type enzyme contamination and measurements of its catalytic activity in the transamination reactions of L- and D-alanine confirmed the suggestion that the active site lysine is not the base that removes the alpha-proton from the substrate.

Protein Expr Purif, 1996 May, 7(3), 289 - 93
Specific replacement of consecutive AGG codons results in high-level expression of human cardiac troponin T in Escherichia coli; Hu X et al.; The adult isoform of human cardiac troponin T (TnT) contains 288 amino acids, 14 of which (4.9%) are encoded by the rarely used arginine codons (12 AGG, 2 AGA) in Escherichia coli genes . To generate sufficient quantity of TnT protein for antibody production, we cloned the corresponding cDNA and expressed it in E . coli . A low-level expression of TnT that comprised only about 1% of total cell protein was initially observed with the use of the native cDNA . The existence of two pairs of consecutive AGG codons AGG(165) AGG(166) and AGG(215) AGG(216) in the cDNA was suspected to be the main cause for this low-level expression . These two pairs of consecutive AGG codons were successively replaced with the major synonymous codon CGT by site-directed mutagenesis . As suspected, a 10-fold increase in TnT expression was obtained when one pair of the rare arginine codons was replaced and a 40-fold increase was achieved when both pairs of the rare codons were replaced . Our finding demonstrates the importance of consecutive rare codons in the suppression of high-level expression of heterologous proteins in E . coli and suggests that in order to maximize protein expression, a similar approach may be taken with other genes which contain consecutive rare codons.

Protein Expr Purif, 1996 May, 7(3), 269 - 74
Vector-derived expression of recombinant rat interleukin-6; Braciak TA et al.; Rat interleukin-6 (IL-6) cDNA, coding for an important inflammation- and immune-regulatory polypeptide cytokine, was cloned into the novel expression vector pH6EX3 which directs the synthesis of inserted genes as a fusion protein with histidine hexapeptide (HH) . The resultant vector (pRIL6.992) was shown to produce significant amounts of recombinant rat IL-6 fusion protein with HH at its N-terminus in various strains of Escherichia coli . The expression of the HH-IL-6 fusion protein was demonstrated to be under the control of the tac promoter and could be induced by IPTG . This protein was isolated from bacterial inclusion bodies and purified to homogeneity in a one-step procedure by affinity chromatography using a nickel-chelating column . The HH-IL-6 fusion protein isolated in this manner was biologically active as determined by hepatocyte stimulation and B9 hybridoma growth assays . Further, this activity was neutralized with a polyclonal antiserum raised against rat IL-6 protein generated in a novel fashion from rabbits infected with a recombinant human type-5 adenovirus vector expressing rat IL-6 protein (Ad5E3rIL6) . The recombinant HH-IL-6 protein was then used to boost Ad5E3rIL6-immunized rabbits . This resulting antiserum was shown to neutralize recombinant and natural rat and murine IL-6 bioactivity in vitro and was useful in Western blot analysis and immunohistochemistry of rat IL-6.

Protein Expr Purif, 1996 May, 7(3), 237 - 46
Expression, isolation, and characterization of a signal sequence-appended chimeric precursor protein; Kaderbhai N et al.; This report describes the properties and the functional utility of an unprocessed precursor protein overproduced in Escherichia coli . The precursor protein is from a fusion between DNA sequences coding for the alkaline phosphatase signal sequence and the full-length of rat liver cytochrome b(5) . The intact precursor protein accumulated in the membranes represented to over 5% of the total bacterial protein . A procedure involving disruption of the bacterial cells by sonication, isolation of the membranes by differential centrifugation, solubilization with a polar solvent, and ion-exchange chromatography provided milligram quantities of the undegraded precursor in a homogeneous and soluble form . The chimeric precursor protein displayed a characteristic b-type hemoprotein spectrum, identical to that of the native cytochrome b(5) . The properties of the precursor protein have been examined by a range of biophysical and biochemical methods . Molecular modeling suggests an amphipathic structure in which a fully preserved soluble core of cytochrome b(5) is terminally bonded by hydrophobic interactions between the amino-terminal signal sequence and the carboxy-terminal membrane anchoring hemoprotein sequence . The precursor substrate was recognized and efficiently cleaved by signal peptidase.

Pain, 1996 May-Jun, 65(2-3), 211 - 9
Hyperalgesia in rats following intracerebroventricular administration of endotoxin: effect of bradykinin B1 and B2 receptor antagonist treatment; Walker K et al.; The present study investigated the development of thermal and mechanical hyperalgesia following intracerebroventricular (i.c.v.) injections of E . coli lipopolysaccharide (LPS) . Hind paw withdrawal to von Frey filament stimulation and thermal withdrawal latencies were measured before and up to 24 or 48 h following an i.c.v . injection of LPS (dose range: 0.02--200 micrograms) . Thermal and mechanical hyperalgesia were evident by 6 h after LPS injection . LPS-induced hyperalgesia was reversed by the B2 receptor antagonist, HOE 140 (10--30 pmol), when administered i.c.v . but not systemically (0.01--1 mmol/kg, i.v.) . Central co-administration of the B1 receptor antagonists, des-Arg9-Leu8 Bk (0.1--1 nmol) or des-Arg10 HOE 140 (0.1--1 nmol) had no effect on thermal or mechanical hyperalgesia . LPS-induced hyperalgesia was also inhibited by indomethacin administered either i.c.v . (10 nmol) or i.v . (1 mumol/kg) . These results indicate that administration of endotoxin to the CNS induces the development of hyperalgesia and that this response involves the activity of kinins, via the stimulation of centrally located B2 receptors, and the formation of prostanoids.

J Protein Chem, 1996 May, 15(4), 351 - 8
Human neurotrophin-3: a one-step peptide mapping method and complete disulfide characterization of the recombinant protein; Hui JO et al.; Human neurotrophin-3 (NT-3) is a member of the nerve growth factor (NGF) family of neurotrophic factors, and the recombinant protein is being developed as a therapeutic for neurodegenerative diseases . The final product purity and lot-to-lot variation are monitored routinely by peptide mapping . However, only the N-terminal region of NT-3 was susceptible to proteolysis under native conditions . Complete digestion required that the protein be chemically modified by reduction and S-alkylation prior to proteolysis . Complete proteolytic degradation of the protein was achieved simply by an initial denaturation of NT-3 in 6 M guanidinium chloride (pH6) for 2 hr at 37 degrees C, followed by a tenfold dilution with the digestion buffer (0.1 M Tris-HCl, 1 mM CaCl2 at pH 7.0) and immediate addition of chymotrypsin at 1% by weight . Direct comparison of the peptide map with an identical aliquot that had been reduced and alkylated also allowed the establishment of the cystine linkages present in NT-3: Cys14 to Cys79, Cys57 to Cys108, and Cys67 to Cys110 . This disulfide structure is homologous to the NGF family of neurotrophic factors.

Bioconjug Chem, 1996 May-Jun, 7(3), 380 - 4
S-{2-(4-azidosalicylamido)ethylthio}-2-thiopyridine: radioiodinatable, cleavable, photoactivatible cross-linking agent; Ebright YW et al.; S-{2-(4-Azidosalicylamido)ethylthio}-2-thiopyridine (AET) contains a 2-thiopyridyl moiety, which permits cysteine-specific incorporation into protein through a cleavable disulfide bond, and a 4-azidosalicylamido moiety, which permits radioiodination and photoactivatible cross-linking . In contrast to the related compound S-{2-{N-{4-(4-azidosalicylamido)butyl}carbomoyl}ethylthio}-2 -thiopyridine {APDP; Zecherle, G., Oleinikov, A., and Traut, R . (1992) Biochemistry 31, 9526}, AET contains a relatively short linker arm between the 2-thiopyridyl moiety and the 4-azidosalicylamido moiety . In a previous paper, it was shown that AET could be used in site-specific protein-protein photocross-linking to identify nearest-neighbor protein domains within a multiprotein complex {Chen, Y., Ebright, Y., and Ebright, R . (1994) Science 265, 90} . In this paper, the synthesis, radioiodination, and incorporation into protein of AET are described.

Anal Chem, 1996 May 1, 68(9), 1550 - 5
Affinity chromatography of recombinant peptides/proteins based on a calmodulin fusion tail; Hentz NG et al.; An affinity chromatography system has been developed for the separation of recombinant fusion proteins based on the Ca(2+)-dependent binding of calmodulin (CaM) to the drug phenothiazine . Specifically, in the presence of Ca2+, a recognition site for phenothiazine is exposed on calmodulin, allowing the binding of this drug to CaM . Upon removal of Ca2+ with EGTA, the conformation of calmodulin changes, and the phenothiazine--CaM complex dissociates . This Ca(2+)-dependent binding has been exploited in the development of a fusion tail approach for the affinity purification of recombinant proteins and peptides . Protein A (ProtA) was employed as a model protein to demonstrate the advantages of this approach . In particular, the developed affinity chromatography system was used to isolate several ProtA--CaM fusion proteins . These recombinant fusion proteins were expressed in Escherichia coli and Saccharomyces cerevisiae from appropriately designed plasmids . Four different plasmids (two each for the bacteria and yeast) were used that encoded the fusion of CaM to the immunoglobulin-binding portion of protein A . After expression of the fusion protein, the crude cell lysates were loaded onto the phenothiazine affinity column in the presence of a Ca(2+)-containing buffer . Upon elution with an EGTA buffer, the ProtA--CaM fusion protein was purified, as confirmed by SDS-PAGE electrophoresis and Western blot analysis.

Vet Microbiol, 1996 May, 50(1-2), 105 - 15
Role of Adhesive Factor/Rabbit 2 in experimental enteropathogenic Escherichia coli O103 diarrhea of weaned rabbit; Pillien F et al.; The Adhesive Factor/Rabbit 2 (AF/R2) is found in Escherichia coli strains of serovar O103:K-:H2 and rhamnose-negative biovars isolated from weaned rabbits with diarrhea . This adhesin allows the colonization of the distal parts of the digestive tract, a first step leading to severe inflammatory diarrhea and death of the animals . In vitro, AF/R2 expression mediates diffuse adhesion of E . coli on HeLa cells, adhesion to ileal villi of newborn and weaned rabbits and the presence of a major 32 kDa subunit in bacterial surface extracts . In this work, we constructed TnphoA mutants of the prototype strain B10 and selected an isogenic clone that did not express the AF/R2 32 kDa subunit when grown in permissive conditions in vitro . The pathogenicity of the wild type strain and of the isogenic mutant was compared by oral inoculation to 35-day-old weaned rabbits . The mutant showed impaired colonization and a highly significant loss of pathogenicity . However, the occurrence of residual weight losses, and of diarrheas and mortalities in some inoculated rabbits suggest that pathogenicity of rabbit O103 enteropathogeniclike E . coli (EPEC-like) strains is due to multiple virulence factors and that other virulence traits remain to be found.

Arch Oral Biol, 1996 May, 41(5), 439 - 44
Lipopolysaccharides from Porphyromonas gingivalis, Prevotella intermedia and Actinobacillus actinomycetemcomitans promote osteoclastic differentiation in vitro; Ito HO et al.; Bacterial lipopolysaccharides possess bone-resorbing activity . Here, lipopolysaccharides from three putative periodontopathic bacteria were examined for effects on osteoclast-like cell formation of bone marrow cells from lipopolysaccharide-responsive C3H-HeN and non-responsive C3H/HeJ mice . The bone marrow cells were cultured with or without various doses of lipopolysaccharide in the presence of 1,25-dihydroxyvitamin D3 and dexamethasone . These lipopolysaccharide preparations significantly increased the number of osteoclast-like cells formed in the culture of C3H/HeN marrow cells; the same as lipopolysaccharides from Escherichia coli and a synthetic lipid A with E . coli-type structure (LA-15-PP), at doses from 0.1 to 1 microgram/ml . This stimulating effect of each lipopolysaccharides was uniformly abrogated by the addition of polymyxin B at 5 micrograms/ml . All the lipopolysaccharide and the synthetic lipid A had no effect on osteoclast formation of the C3H/HeJ marrow cells, whereas lipopolysaccharide from Porphyromonas gingivalis and Prevotella intermedia showed significant mitogenic activity on C3H/HeJ spleen cells . It seems likely that the activity of lipopolysaccharides to augment osteoclast-like cell formation in the bone marrow cell cultures is derived from a common structure of the lipid A portion.

FEMS Immunol Med Microbiol, 1996 May, 14(1), 1 - 13
Differential induction of IL-1 beta and IL-6 production by the nontoxic lipid A from Porphyromonas gingivalis in comparison with synthetic Escherichia coli lipid A in human peripheral blood mononuclear cells; Ogawa T et al.; Porphyromonas gingivalis 381 lipid A possesses 1-phospho beta(1-6)-linked glucosamine disaccharide with 3-hydroxy-15-methylhexadecanoyl and 3-hexadecanoyloxy-15-methylhexadecanoyl groups at the 2- and 2'-positions, respectively . P . gingivalis lipid A indicated lower activities in inducing interleukin-1 beta (IL-1 beta) mRNA expression, pro-IL-1 beta protein synthesis and IL-1 beta production than those of synthetic Escherichia coli lipid A (compound 506) in human peripheral blood mononuclear cells (PBMC) . The induction of IL-6 mRNA and IL-6 synthesis by P . gingivalis lipid A were comparable to those of compound 506 . Herbimycin A, H-7 and H-8, inhibitors of tyrosine kinase, protein kinase C and cyclic nucleotide-dependent protein kinase, inhibited P . gingivalis lipid A- and compound 506-induced IL-1 beta and IL-6 synthesis . W-7, an inhibitor of calmodulin (CaM) kinase, inhibited only P . gingivalis lipid A-induced IL-1 beta production . The result suggests that the CaM kinase-dependent cascade is involved in the down-regulation of IL-1 beta production by P . gingivalis lipid A . P . gingivalis lipid A and compound 506 also functioned in the induction of tyrosine and serine/threonine phosphorylation of several proteins in PBMC . P . gingivalis lipid A inhibited specific binding of fluorescein-labelled E . coli LPS to the PBMC . The nontoxic lipid A of P . gingivalis, having a chemical structure different from toxic compound 506, appears to induce the up- and down-regulation of the differential cytokine-producing activities following the activation of various intracellular enzymes including the CaM kinase through the common receptor sites of LPS.

Med Microbiol Immunol (Berl), 1996 May, 185(1), 1 - 10
The peptidolytic capacity of the spirochete system; Makinen KK et al.; Relatively scant chemical information has been available on the proteinases and peptidases of spirochetes in spite of the association of spirochetes with several serious infections known to plague humans and other animal species . This situation has partly resulted from difficulties in growing some spirochetes under laboratory conditions . The cells of Treponema denticola, a spirochete suggested to be associated with periodontal infections, have turned out to be a good source of new chemical information on those enzymes . Latest studies suggest that the outer cell envelope or the periplasmic space of T . denticola contains several novel proteinases and peptidases (hence called "ectoenzymes") which may contribute to the chronicity of periodontal infections . Some of the oligopeptidases discovered are specific for proline-containing host tissue peptides such as substance P, bradykinin, neurotensin, etc., and possibly small collagen fragments . The only spirochetal peptidases purified to give a single band on sodium dodecyl sulfate-polyacrylamide gel electrophoresis have been obtained from T . denticola . One particular peptidase, suggested to be similar to the oligopeptidase B (EC 3.4.21.83) of Escherichia coli seems to be present in the cell envelope or in the periplasmic space at quite large concentrations . The presence of this and several other peptidases in the outer cell structures of the treponemes suggests that such enzymes are important for the nutrition of these highly motile and invasive organisms . The biological role of these enzymes can thus be envisaged in the peptidolytic processing of host tissue proteins and peptides to gradually smaller molecules to fulfill the nutritional requirements of these organisms . Although the genetic similarity between T . denticola and some other treponemes and spirochetes can be hotly debated, it is nevertheless now possible to use T . denticula enzymes as suitable objects for comparison when the chemistry of other spirochetes is studied.

J Biochem (Tokyo), 1996 May, 119(5), 970 - 8
Pyridoxal 5'-phosphate binding of a recombinant rat serine: pyruvate/alanine:glyoxylate aminotransferase; Ishikawa K et al.; Serine: pyruvate/alanine: glyoxylate aminotransferase in the liver is a class IV amino-transferase . The present study was undertaken to characterize the pyridoxal 5'-phosphate (PLP) binding to a recombinant rat serine: pyruvate/alanine: glyoxylate aminotransferase (SPT10), which is a homodimer of 44.4 kDa subunits . Purified SPT10 exhibited absorption maxima at approximately 330 nm in addition to a 278 nm protein peak and a approximately 420 nm peak of PLP bound via Schiff base, and contained 0.56-0.69 mol of PLP per mol of subunit . Apo-SPT10 without measurable bound PLP did not exhibit the absorbance at approximately 420 nm, but still showed the approximately 330 nm peak . Upon reconstitution, 0.73-0.79 mol of PLP per mol of subunit was bound to apo-SPT10 with an apparent Kd of approximately 0.1 microM, resulting in a holo-SPT10 preparation whose specific activity and A approximately 420/A approximately 330 absorbance ratio were higher than those of the original SPT10 . On SDS/PAGE of BrCN-cleavage peptides of NaBH4-reduced SPT10, 22-23 kDa fragments migrated as a pair of bands . On amino acid sequencing, the approximately 22 and approximately 23 kDa pair gave the same sequence, except that Lys was released only from the approximately 22 kDa band material in the cycle corresponding to Lys209 . NaB3H4-treated SPT10 also migrated as a pair of 44-45 kDa bands and 3H was incorporated only into the approximately 45 kDa band . It appears that SPT10 has the capacity to bind 1 mol of PLP to Lys209 of every subunit, but usually binds less PLP in a Schiff base structure, probably due to the presence of a 330 nm-absorbing chromophore.

J Biochem (Tokyo), 1996 May, 119(5), 914 - 9
Immunofluorescence localization of a 23-kDa Tetrahymena calcium-binding protein, TCBP-23, in the cell cortex; Hanyu K et al.; Previously, we succeeded in cloning a cDNA of the 23-kDa Tetrahymena Ca(2+)-binding protein, designated TCBP-23 . Analysis of the deduced amino acid sequences showed that TCBP-23 is a member of the EF-hand family of Ca(2+)-binding proteins . However, its physiological function was not elucidated . In the studies reported here, recombinant TCBP-23 was expressed in Escherichia coli and purified . Since recombinant TCBP-23 binds Ca2+ in vitro, Ca(2+)-binding domains of the protein are likely to be functional in vivo . Rabbit antibodies against TCBP-23 were raised and used to determine the intracellular localization of the protein in Tetrahymena cells by indirect immunofluorescence . The antibodies strongly stained the whole cell cortex except for the oral apparatus and around the basal bodies . TCBP-23 remained in detergent-extracted cells, suggesting that it is associated with the epiplasm, the membrane skeleton of Tetrahymena . These results suggest that TCBP-23 may mediate Ca(2+)-regulated processes in the cell cortex.

Protein Eng, 1996 May, 9(5), 433 - 8
Study of cysteine residues in the alpha subunit of Escherichia coli tryptophan synthase . 2 . Role in enzymatic function; Hiraga K et al.; To understand the functional roles of Cys residues in the alpha subunit of tryptophan synthase from Escherichia coli, single mutants of the alpha subunit, in which each of the three Cys residues was substituted with Ser, Gly, Ala or Val, were constructed by site-directed mutagenesis . The effects of the substitutions on the function of tryptophan synthase were investigated by activity measurements, calorimetric measurements of association with the beta subunit and steady-state kinetic analysis of catalysis . Although the three Cys residues are located away from the apparently important parts for enzymatic activity, substitutions at position 81 by Ser, Ala or Val caused decreases in the intrinsic activity of the alpha subunit . Furthermore, Cys81Ser and Cys81Val reduced stimulation activities in the alpha and beta reactions due to formation of a complex with the beta subunit . The lower stimulation activities of the mutant proteins were not correlated with their abilities to associate with the beta 2 subunit but were correlated with decreases in kcat . The present results suggest that position 81 plays an indirectly important role in the activity of the alpha subunit itself and the mutual activation mechanism of the complex.

Protein Eng, 1996 May, 9(5), 425 - 31
Study of cysteine residues in the alpha subunit of Escherichia coli tryptophan synthase . 1 . Role in conformational stability; Hiraga K et al.; To eludicate the role in conformational stability of Cys residues buried in the interior of a protein, the thermodynamic properties of denaturation of mutant alpha subunit of Escherichia coli tryptophan synthase, in which Ser, Ala, Val or Gly was substituted for each of the three Cys residues, were analyzed using calorimetry . The mutants were less stable than the wild type, indicating that Cys residues contribute greatly to the stability of the alpha subunit . In most cases, a large decrease in denaturation enthalpy was observed, compensated for by the denaturation entropy to a major extent . The extent of changes in the denaturation Gibbs energy and denaturation enthalpy varied greatly depending on both substituting residues and positions . Of all the mutant proteins, the Cys154Ser mutant showed the greatest decrease in denaturation enthalpy; its denaturation enthalpy was half that of the wild type, and was considerably repaired by adding a ligand of the alpha subunit . Because the enthalpy of ligand binding to Cys154Ser in the native state did not change, it seems that the decrease in the denaturation enthalpy of Cys154Ser and its recovery by ligand binding are caused by conformational changes in the denatured state due to the mutation.

Mol Microbiol, 1996 May, 20(4), 875 - 84
Involvement of the narJ and mob gene products in distinct steps in the biosynthesis of the molybdoenzyme nitrate reductase in Escherichia coli; Palmer T et al.; The Escherichia coli mob locus is required for synthesis of active molybdenum cofactor, molybdopterin guanine dinucleotide . The mobB gene is not essential for molybdenum cofactor biosynthesis because a deletion of both mob genes can be fully complemented by just mobA . Inactive nitrate reductase, purified from a mob strain, can be activated in vitro by incubation with protein FA (the mobA gene product), GTP, MgCl2, and a further protein fraction, factor X . Factor X activity is present in strains that lack MobB, indicating that it is not an essential component of factor X, but over-expression of MobB increases the level of factor X . MobB, therefore, can participate in nitrate reductase activation . The narJ protein is not a component of mature nitrate reductase but narJ mutants cannot express active nitrate reductase A . Extracts from narJ strains are unable to support the in vitro activation of purified mob nitrate reductase: they lack factor X activity . Although the mob gene products are necessary for the biosynthesis of all E . coli molybdoenzymes as a result of their requirement for molybdopterin guanine dinucleotide, NarJ action is specific for nitrate reductase A . The inactive nitrate reductase A derivative in a narJ strain can be activated in vitro following incubation with cell extracts containing the narJ protein . NarJ acts to activate nitrate reductase after molybdenum cofactor biosynthesis is complete.

Mol Microbiol, 1996 May, 20(4), 853 - 65
Characterization of an Escherichia coli mutant, feeA, displaying resistance to the calmodulin inhibitor 48/80 and reduced expression of the rare tRNA3Leu; Bouquin N et al.; We previously described a mutation feeB1 conferring a temperature-sensitive filamentation phenotype and resistance to the calmodulin inhibitor 48/80 in Escherichia coli, which constitutes a single base change in the acceptor stem of the rare tRNA3Leu recognizing CUA codons . We now describe a second mutant, feeA1, unlinked to feeB, but displaying a similar phenotype, 48/80 resistance and a reduced growth rate at the permissive temperature, 30 degrees C, and temperature-sensitive, forming short filaments at 42 degrees C . In the feeA mutant, tRNA3Leu expression (but not that of tRNA1Leu) was reduced approximately fivefold relative to the wild type . We previously showed that the synthesis of beta-galactosidase, which unusually requires the translation of 6-CUA codons, was substantially reduced, particularly at 42 degrees C, in feeB mutants . The feeA mutant also shows drastically reduced synthesis of beta-galactosidase at the non-permissive temperature and reduced levels even at the permissive temperature . We also show that increased copy numbers of the abundant tRNA1Leu, which can also read CUA codons at low efficiency, suppressed the effects of feeA1 under some conditions, providing further evidence that the mutant was deficient in CUA translation . This, and the previous study, demonstrates that mutations which either reduce the activity of tRNA3Leu or the cellular amount of tRNA3Leu confer resistance to the drug 48/80, with concomitant inhibition of cell division at 42 degrees C.

Mol Microbiol, 1996 May, 20(4), 823 - 32
Protein engineering studies of A-chain loop 47-56 of Escherichia coli heat-labile enterotoxin point to a prominent role of this loop for cytotoxicity; Feil IK et al.; Heat-labile enterotoxin (LT), produced by enterotoxigenic Escherichia coli, is a close relative of cholera toxin (CT) . These two toxins share approximately 80% sequence identity, and consists of one 240-residue A chain and five 103-residue B subunits . The B pentamer is responsible for GM1 receptor recognition, whereas the A subunit carries out an ADP-ribosylation of an arginine residue in the G protein, Gs alpha, in the epithelial target cell . This paper explores the importance of specific amino acids in loop 47-56 of the A subunit . This loop was observed to be highly mobile in the inactive R7K mutant of the A subunit . The position of the loop in wild-type protein is such that it might require considerable reorganization during substrate binding and is likely to have a crucial role in substrate binding . Five single-site substitutions have been made in the LT-A subunit 47-56 loop to investigate its possible role in the enzymatic activity and toxicity of LT and CT . The wild-type residues Thr-50 and Val-53 were replaced either by a glycine or by a proline . The glycine substitutions were intended to increase the mobility of this active-site loop, and the proline substitutions were intended to decrease the mobility of this same loop by restricting the accessible conformational space . Under the hypothesis that mobility of the loop is important for catalysis, the glycine-substitution mutants T50G and V53G would be expected to exhibit activity equal to or greater than that of the wild-type A subunit, while the proline substitution mutants T50P and T53P would be less active . Cytotoxicity assays showed, however, that all four of these mutants were considerably less active than wild-type LT . These results lend support for assignment of a prominent role to loop 47-56 in catalysis by LT and CT.

Mol Microbiol, 1996 May, 20(4), 813 - 22
Independent interaction of the acyltransferase HlyC with two maturation domains of the Escherichia coli toxin HlyA; Stanley P et al.; The apparently unique fatty acylation mechanism that underlies activation (maturation) of Escherichia coli haemolysin and related toxins is further clarified by investigation of the interaction of protoxin with the specific acyltransferase HlyC . Using deleted protoxin variants and protoxin peptides as substrates in an in vitro maturation reaction dependent upon HlyC and acyl-acyl carrier protein, two independent HlyC recognition domains were identified on the 1024-residue protoxin, proA, and they were shown to span the two target lysine residues K564 (KI) and K690 (KII) that are fatty acylated . Each domain required 15-30 amino acids for basal recognition and 50-80 amino acids for wild-type acylation . The two domains (FAI and FAII) competed with each other in cis and in trans for HlyC . The affinity of FAI for HlyC is approximately four times greater than that of FAII resulting in an overall 80% acylation at KI and 20% acylation at KII in both whole toxin and peptide derivatives . No other proA sequences were required for toxin maturation, and excess Ca2+ prevented acylation of both lysines . The lack of primary sequence identity between FAI and FAII domains in proA and among corresponding sites on related protoxins currently precludes an explanation of the basis of HlyC recognition by proA.

J Virol Methods, 1996 May, 59(1-2), 161 - 72
A major antigenic domain for the human humoral response to Puumala virus nucleocapsid protein is located at the amino-terminus; Elgh F et al.; Nephropathia epidemica (NE), the major form of hemorrhagic fever with renal syndrome in Europe, is caused by the hantavirus serotype Puumala (PUU) . The PUU virus nucleocapsid protein (N) has been shown to be highly immunogenic both in laboratory animals and in man . We aimed to locate domains important in humoral immune reactivity and to use this information to develop a specific and sensitive enzyme-linked immunosorbent assay (ELISA) for serological diagnosis of NE . Escherichia coli poly-histidine fusion protein expression vectors containing over-lapping gene segments encoding the PUU virus N (PUU rN) were constructed . The resulting gene products were examined by immunoblots and ELISA with polyclonal and monoclonal antibodies . The dominating antigenic region of PUU rN was located between amino acids (aa) 7 and 94 . A recombinant fusion protein containing aa 7-137 of PUU virus N (PUU rN delta 5) was used for the detection of specific IgG and IgM responses in NE . ELISA based on PUU rN delta 5 was found to have equal sensitivity and specificity as compared to the full length recombinant PUU rN by ELISA, for both acute serological diagnosis of NE and for seroepidemiological screening purposes . Furthermore, this protein is easier to handle than full length PUU rN due to its higher solubility in aqueous solutions.

J Virol Methods, 1996 May, 59(1-2), 91 - 8
Immunoreactive core peptides of hepatitis C virus produced in Escherichia coli and in vitro DNA amplification-restricted transcription-translation system; Esumi M et al.; Three kinds of hepatitis C virus (HCV) core peptides were produced directly and efficiently in E . coli: 1-120 aa of the C region as NCC, 1-157 aa as NCCT and 1-190 aa as NCCL . These peptides were estimated to be 16, 22 and 24 kDa, respectively, by SDS-polyacrylamide gel electrophoresis . The processing to produce p22 core protein observed in insect cells and mammalian systems did not occur in E . coli . These peptides were similarly reactive with serum antibody from patients with hepatitis C . A mutant clone of NCC recombinant plasmid pKNCC4 was obtained, whose product, NCC4, was more stable in the E . coli lysate and was highly immunoreactive with sera of hepatitis C patients . This stable immunoreactive core peptide produced by pKNCC4 is useful for the detection of anti-HCV core antibody . Immunoreactive core peptides were also produced by DNA amplification-restricted transcription-translation . Five kinds of cDNA from C to E1 region were amplified and transcribed in vitro, and these five transcripts were then translated in vitro using rabbit reticulocyte lysate: 1-120 aa as 17 kDa of C1, 1-155 aa as 21 kDa of C2, 1-174 aa as 22 kDa of C3, 1-192 aa as 24 kDa of C4, and 1-213 aa as 26 kDa of C5 . Cotranslational processing using microsomal membranes occurred in peptides C4 and C5 to produce p22 the same size as C3 . These results indicate that the C-terminus of the mature core protein p22 may be generated at around aa 174 by cleavage with the signal peptidase.

J Virol Methods, 1996 May, 59(1-2), 13 - 21
Overexpression and simple purification of a truncated, immunologically reactive GST-HCV core (1-123) fusion protein; Seong YR et al.; A full-length and a truncated gene for the core protein of hepatitis C virus (HCV) were linked to the gene for glutathione S-transferase (GST), and the expression of each GST-HCV core fusion protein was analyzed . The truncated GST-HCV core (1-123) fusion protein was expressed as a mostly soluble and partly insoluble form comprising more than 50% of the total protein in Escherichia coli after induction by isopropylthio-beta-D-galactoside (IPTG), while the full length GST-HCV core (1-191) fusion protein was not expressed, suggesting that the hydrophobic carboxy terminal region in the core protein affects its expression . In addition, the GST-HCV core (1-123) fusion protein purified by GST-agarose chromatography reacted specifically with an anti-HCV serum from a patient.

J Infect, 1996 May, 32(3), 187 - 96
In vitro induction of nitric oxide by an extract of Plasmodium falciparum; Rockett KA et al.; Malarial illness and pathology is generally accepted to be caused by material released when the infected red cells burst at schizogony . The released material has been partially purified and shown to stimulate macrophages to make TNF . We have extended this work to show that these same preparations, isolated from parasitized erythrocytes, induce the mouse macrophage cell line RAW 264.7 to produce inducible nitric oxide synthase and release nitric oxide . By using cytokine-specific antisera we have found that this induction is independent of TNF and IL-1 alpha and partly independent of IL-1 beta.

Eur J Clin Microbiol Infect Dis, 1996 May, 15(5), 398 - 402
Detection of genes coding for extended-spectrum SHV beta-lactamases in clinical isolates by a molecular genetic method, and comparison with the E test; Nuesch-Inderbinen MT et al.; A highly sensitive and specific method, termed PCR/NheI, for the detection of genes coding for SHV extended-spectrum beta-lactamases (ESBL) in clinical isolates is presented . It is based on polymerase chain reaction (PCR) amplification of the blaSHV genes, followed by restriction with NheI . Due to the glycine (positive 238) (SHV-non-ESBL)-->serine (position 238) (SHV-ESBL) mutation, only PCR fragments from the genes coding for SHV-ESBLs were cleaved . A commercially available test for ESBLs, the E test ESBL, identified 52% of our 29 clinical isolates carrying blaSHV-ESBL genes as ESBL producers.

J Dairy Sci, 1996 May, 79(5), 886 - 94
Effect of a whey protein concentrate used as a colostrum substitute or supplement on calf immunity, weight gain, and health; Mee JF et al.; The efficacy of a whey protein concentrate was evaluated as a colostrum substitute or supplement in two experiments using four groups of 29 calves . In Experiment 1, calves were fed either 2 L of pooled colostrum (group 1) or 500 g of whey protein concentrate (group 2) . A mean total of 123.6 and 17.7 g of Ig was fed to calves in groups 1 and 2, respectively . Mean serum IgG, total protein, and globulin concentrations and Ig antibody activities to Escherichia coli K99 and rotavirus were significantly higher for calves in group 1 at 24 to 36 h and at 3 wk of age . Weight gain from birth to 3 wk of age was significantly lower for calves in group 2 . The incidence of diarrhea was high but not different between treatments . The mortality rate (0 to 3 wk) was significantly higher for calves in group 2 (27.6%) than for calves in group 1 (3.4%) . In Experiment 2, calves were fed either 2 L of pooled colostrum (group 3) or a solution of 1 L of pooled colostrum plus 500 g of whey protein concentrate (group 4) . A mean total of 117.2 and 69.1 g of Ig was fed to calves in groups 3 and 4, respectively . Absorption rate of IgG was significantly lower for calves in group 4 . Mean serum IgG, total protein, and globulin concentrations and Ig antibody activities to E . coli K99 and rotavirus were significantly higher for calves in group 3 at 24 to 36 h and at 3 wk of age . Mortality rate, BW gain, and incidence of diarrhea did not differ significantly between groups.

Mol Med, 1996 May, 2(3), 334 - 48
Eotaxin triggers eosinophil-selective chemotaxis and calcium flux via a distinct receptor and induces pulmonary eosinophilia in the presence of interleukin 5 in mice; Rothenberg ME et al.; BACKGROUND: Understanding the processes that control selective eosinophilia is of fundamental importance in a variety of human diseases (e.g., allergies, parasitic infections, malignancy) . Interleukin 5, an eosinophil-specific growth and activating factor, and eotaxin appear to collaborate in this process . Eotaxin is a recently described chemotactic factor that belongs to the C-C (or beta) chemokine family and has been implicated in animal and human eosinophilic inflammatory states . We have recently reported the molecular characterization of murine eotaxin and now report the biological properties of purified recombinant murine eotaxin in vitro and in vivo in the presence or absence of interleukin 5 (IL-5) in mice . MATERIALS AND METHODS: Murine eotaxin was expressed in bacteria and purified by affinity chromatography and HPLC . Activity was tested in vitro by examining chemotactic and calcium flux responses of purified murine leukocytes . Additionally, desensitization of calcium flux responses to other chemokines, eosinophil survival assays, and basophil histamine release were examined . Finally, eotaxin was delivered to wild-type or IL-5 transgenic mice and the host response was examined . RESULTS: Eotaxin had activity only when the recombinant molecule had the native mature amino terminus and contained the first 25 amino acids of the mature protein . It was active in vitro at an effective concentration between 10 and 100 ng/ml in both chemotaxis and calcium flux assays toward eosinophils, but not macrophages or neutrophils . Furthermore, intranasal or subcutaneous application of eotaxin selectively recruited large numbers of eosinophils into the mouse lung and skin, respectively, only in the presence of interleukin 5 . Macrophage inflammatory protein-1 alpha, a related C-C chemokine active on eosinophils, and eotaxin were not able to cross-desensitize . Eotaxin had no affect on the in vitro survival of eosinophils and did not induce basophil histamine release . CONCLUSIONS: Mouse eotaxin is an eosinophil specific chemoattractant that has a markedly enhanced effect in vivo in the presence of another eosinophil selective cytokine IL-5, and utilizes a signal transduction receptor pathway that is distinct from that utilized by macrophage inflammatory protein-1 alpha . This data suggests that the development of tissue eosinophilia in vivo involves a two-step mechanism elicited by interleukin 5 and eotaxin.

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