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