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| J Dairy Sci, 1994 Feb, 77(2), 619 - 27 Factors affecting milk somatic cells and their role in health of the bovine mammary gland; Kehrli ME Jr et al.; Milk somatic cells play a protective role against infectious disease in the bovine mammary gland . Many genetic and environmental factors affect the number and kinds of leukocytes that account for the vast majority of somatic cells in milk . Neutrophils constitute the vast majority of somatic cells in mammary glands that are infected with mastitis pathogens . The recruitment of neutrophils into the infected mammary gland is a normal part of the cow's defense mechanisms that is very effective for eradicating the majority of infections that occur . For many reasons, milk production and milk quality are negatively impacted by the presence of inflammation in infected glands . Because of the negative effects of high SCC in milk, various approaches are needed to reduce milk SCC . In the future, genetic gains for milk quality and mastitis resistance may be made by removing bulls from breeding programs when their daughters are predisposed to high SCC. Microbiology, 1994 Feb, 140 ( Pt 2), 369 - 78 Stability and function of the signal peptide of the pCloDF13-derived bacteriocin release protein; van der Wal FJ et al.; The pCloDF13-derived bacteriocin release protein (BRP) is synthesized as a prelipoprotein with a signal peptide which remains stable after processing . This signal peptide accumulates in the cytoplasmic membrane and is, together with the mature BRP, required for efficient release of cloacin DF13 . We investigated the structural requirements for stability of the BRP signal peptide by constructing hybrid signal peptides consisting of parts of the BRP and Lpp signal peptides . Signal peptide stability was investigated by pulse-labelling and pulse-chase experiments . To study the functioning of the BRP signal peptide, the hybrid constructs were tested for their ability to promote BRP-mediated cloacin DF13-release and their ability to affect the viability of the host cells . The results obtained suggest that the N-terminal part of the BRP signal peptide together with the C-terminal alanine residue are important for stability . When expressed as a separate entity, all mutant signal peptides that contain a part of the BRP signal peptide are capable of affecting cell viability . The results indicated a possible correlation between stability of the BRP signal peptide and cloacin DF13-release. Mol Reprod Dev, 1994 Feb, 37(2), 167 - 71 Introduction of exogenous DNA into somatic and germ cells of chickens by microinjection into the germinal disc of fertilized ova; Naito M et al.; The plasmid DNA, pAcZ, containing Escherichia coli beta-galactosidase (lacZ) gene under the control of chicken beta-actin gene promoter was injected in a linearized form into the germinal disc of fertilized chick ova at the single-cell stage . The manipulated embryos were cultured by the method of Naito et al . (1990) until hatching . The rate of hatching was 11.8% (31/263), and 19 males and 6 females were matured . DNA from blood and semen samples of the 25 matured chickens was analyzed for the presence of the injected DNA by Southern blot hybridization . The injected DNA was detected in the blood DNA of one male and in the sperm DNA of another male up to 13 months after hatching, indicating that the injected DNA was stably maintained in these chickens . Restriction digestion analysis of the injected DNA suggested that it was not rearranged and was organized as head-to-tail multimers . The copy numbers of the DNA were 0.07-0.02 in the blood DNA of one male per diploid genome, and 0.02-0.015 in the sperm DNA of another male, indicating that the exogenous DNA was present in limited populations of blood and sperm cells. DNA Cell Biol, 1994 Feb, 13(2), 183 - 92 Recombinant and cellular expression of the murine chemotactic protein, CP-10; Iismaa SE et al.; The S100 protein CP-10 (chemotactic protein, 10 kD), a potent chemotactic factor for murine and human polymorphonuclear cells (PMN) and murine monocytes, has been purified in small amounts from supernatants of activated murine spleen cells (Lackmann et al., 1992) . To obtain a more abundant source of the protein, CP-10 was expressed in Escherichia coli as a fusion protein with glutathione S-transferase (GST) . The property of S100 proteins to undergo calcium-dependent conformational changes was used in a novel approach to optimize the release of recombinant (r) CP-10 by thrombin cleavage . Purified rCP-10 was characterized by amino-terminal sequence analysis and bioassays . Optimal chemotactic activity of rCP-10 for murine PMN and WEHI-265 monocytoid cells was 10(-11) M (native protein has optimal chemotactic activity between 10(-11) and 10(-13) M) . Immunization of rabbits with the GST/CP-10 fusion protein bound to glutathione-agarose beads resulted in high titer, specific antibodies that neutralized CP-10-initiated chemotaxis and were suitable for immunoblotting . A combination of Western and Northern analyses identified CP-10 in murine peritoneal exudate PMN and macrophages, splenocytes, bone marrow cells, and WEHI-265 cells (all of myeloid origin), but not in thymus, liver, lung, 3T3 fibroblasts, EL4 lymphoma cells, or bEND 3 brain endothelial cells, indicating cell-specific regulation of CP-10 expression. Anal Biochem, 1994 Feb 1, 216(2), 427 - 30 Measurement of the uptake of radioactive para-aminobenzoic acid monitors folate biosynthesis in Escherichia coli K-12; Herrington MB; This study was undertaken to develop a method for rapidly and easily estimating the folate content of different strains of Escherichia coli . Cells were grown to stationary phase in medium containing radioactive para-aminobenzoic acid . The amount of label incorporated into cells was measured by collecting the cells on a filter, washing, and then determining the radioactivity retained on the filter . The addition of unlabeled para-aminobenzoic acid or the antifolate drugs sulfathiazole or trimethoprim reduced the uptake of the radioactive compound . These results indicate that uptake measurements monitored folate biosynthesis . The assay is well suited for the analysis of large numbers of samples and does not require specialized equipment. Anal Biochem, 1994 Feb 1, 216(2), 413 - 7 Release of proteins and peptides from fusion proteins using a recombinant plant virus proteinase; Parks TD et al.; An improved method for the production, cleavage, and purification of fusion proteins and peptides is described . The unique aspect of this method is dependent on the use of a proteinase from tobacco etch virus (TEV) . The proteinase used is a recombinant TEV proteinase produced with a polyhistidine tract positioned at the amino terminus . The proteinase recognizes a specific, extended cleavage site sequence . The peptide or protein of interest is purified as a fusion protein with a TEV proteinase cleavage site sequence located between it and an affinity carrier portion of the fusion . Incubation with the recombinant TEV proteinase mediates release of the peptide or protein of interest . Use of the recombinant TEV proteinase to cleave fusion proteins is an improvement over use of other proteinases for several reasons, including its high degree of specificity, its insensitivity to many proteinase inhibitors generally used in protein purification, and the ready separation of both the affinity tag and the proteinase from the cleaved product of interest. J Ethnopharmacol, 1994 Feb, 41(3), 185 - 92 Immunostimulatory activity of Picrorhiza kurroa leaf extract; Sharma ML et al.; A 50% ethanolic extract of Picrorhiza kurroa Royle ex Benth . (Scrophulariaceae) leaves (PKLE) was found to stimulate the cell-mediated and humoral components of the immune system as well as phagocytosis in experimental animals . PKLE elicited a dose-related increase in SRBC, induced 4 h (early) and 24 h (delayed) hypersensitivity reactions in mice and rats, and horse serum induced Arthus reaction in guinea pigs . It also enhanced the humoral immune responses in mice and rats and phagocytic function of the cells of the reticuloendothelial system in mice . PKLE exhibited no mitogenic activity but augmented the responsiveness of murine splenocytes to T cell mitogens phytohaemagglutinin (PHA) and concanavalin A (Con A) and B cell mitogen lipopolysaccharide (LPS E . coli). J Photochem Photobiol B, 1994 Feb, 22(2), 131 - 8 Are enzymatically produced single-strand breaks involved in UV-induced inactivation of plasmid DNA? Gurzadyan GG, Schulte-Frohlinde D. pBR322 plasmid DNA was exposed to 254 nm UV radiation and examined for enzymatically produced single-strand break (sbb) and double-strand break (dsb) formation by treatment with an extract containing the proteins of Escherichia coli (AB1157 (uvrA+ recA+) and AB2480 (uvrA- recA-)) . Enzymatic conversion of the 254 nm-induced lesions into ssbs on treatment with an extract from AB1157 was observed, but not conversion into dsbs . The rate of enzymatic ssb formation in the AB1157 extract is initially higher than in the AB2480 extract, the sbb formation levels off leading to plateau values with increasing incubation time . The rate of ssb formation in the AB2480 extract is initially lower, but does not level off, and the ssb yield becomes larger at longer incubation times than that with the AB1157 extract . The biological inactivation of the plasmids was measured as a function of 254 nm fluence by transformation of E . coli AB1157 and AB2480 . Inactivation with AB2480 is mainly due to a single photoproduct, a cyclobutane-type pyrimidine dimer, per DNA molecule . Inactivation with AB1157 occurs with a quantum yield which is virtually identical with that of the plateau values of enzymatic ssb formation, as measured by incubation in the AB1157 extract . A possible interpretation is that the formation of irreparable ssbs is the lethal step in the sequence of events leading to inactivation of plasmid DNA in the repair wild-type strain . The quantum yield of inactivation is 10-20 times smaller for transformation of AB1157 than for AB2480, indicating that enzymatic repair of photolesions of the plasmid occurs in AB1157. J Cell Biochem, 1994 Feb, 54(2), 247 - 55 Recombinant GST-human osteopontin fusion protein is functional in RGD-dependent cell adhesion; Xuan JW et al.; Osteopontin (OPN) is a secreted phosphoprotein expressed by many tumor cells, as well as a limited set of normal cells . Native OPN has been shown to support cell adhesion in an RGD-peptide-inhibitable fashion . Here we expressed human OPN in E . coli as a recombinant fusion protein with glutathione-S-transferase (GST) . We report that the GST-OPN fusion protein has functional activity . PAP2 (ras-transformed, metastatic murine NIH 3T3) and MDA-MB-435 human mammary carcinoma cells bound to GST-OPN in an in vitro cell adhesion assay nearly as well as to native bovine OPN . Adhesion to the recombinant fusion protein was blocked by addition of GRGDS peptide, suggesting that the cells adhere to the recombinant and native OPN proteins by similar, integrin-mediated mechanisms . Adhesion to both sources of OPN also was inhibited by thrombin treatment of the protein . Thrombin cleaves GST from OPN in the fusion protein, and also cleaves internally in OPN, adjacent to the RGD sequence of the protein . Our results suggest that (a) thrombin cleavage of native OPN may be a natural regulator of OPN function, and (b) the majority of OPN cell binding activity is mediated by the RGD sequence in the protein backbone, with little or no requirement for post-translational modifications that occur in native OPN for adhesive function as measured here. J Appl Physiol, 1994 Feb, 76(2), 793 - 800 Gut and muscle tissue PO2 in endotoxemic dogs during shock and resuscitation; Vallet B et al.; There is indirect evidence that tissue hypoxia occurs in human sepsis and surface measures of muscle tissue PO2 (PtiO2) in hypodynamic endotoxic animals are decreased . This study assessed systemic and regional tissue oxygenation in a more relevant model of hyperdynamic endotoxicosis . We isolated venous outflow from the left hindlimb and a segment of ileum in six anesthetized dogs to measure muscle and gut O2 delivery and uptake (VO2) and lactate flux, gut intramucosal pH (pHi) by tonometry, and PtiO2 by multi-point surface electrodes placed on mucosal and serosal surfaces of gut and on muscle . We then infused Escherichia coli lipopolysaccharide (LPS; 2 mg/kg) over 1 h followed by a 2-h infusion of dextran (0.5 ml.kg-1.min-1) . LPS infusion significantly decreased systemic and gut VO2, cardiac output (Q), and blood pressure and increased arterial lactate and gut lactate flux . Resuscitation increased Q to above baseline and restored systemic VO2 . In response to LPS and then resuscitation, muscle PtiO2 distribution did not change, suggesting little microcirculatory disturbance, although mean PtiO2 first decreased and then increased . In contrast, gut VO2 and pHi remained low and lactate output remained high, despite restoration of gut blood flow . Gut VO2, lactate flux, pHi, and PtiO2 histograms were consistent with a marked redistribution of blood flow within the gut wall, away from the mucosa and toward the muscularis . These data show that, in hyperdynamic acute endotoxemia, skeletal muscle PtiO2 and VO2 are well maintained, but blood flow within the gut is significantly disturbed with mucosal hypoxia. J Appl Physiol, 1994 Feb, 76(2), 516 - 22 Pulmonary inflammatory cell response to sustained endotoxin administration; Wang CZ et al.; We have developed a model of human sepsis in sheep . Twenty-four hours after continuous infusion of Escherichia coli endotoxin (lipopolysaccharide) (10 ng.kg-1.min-1) was begun, pulmonary transvascular fluid flux was almost five times the baseline values, cardiac output was nearly doubled, and mean arterial pressure was reduced by approximately 20 mmHg . At this time, the animals were killed and their lungs were fixed by endotracheal installation of 2.5% glutaraldehyde at 25 cmH2O pressure . Morphometry was performed by point counting, and data were expressed as relative volume density . Pulmonary edema and congestion were observed in sheep receiving lipopolysaccharide, whereas sham controls appeared normal . There was an increase in interstitial volume density . There was a significant increase (P < 0.01) in volume density of the pulmonary intravasculature (180%), interstitial macrophages (270%), and mast cells (240%) . The volume densities of intravascular and interstitial polymorphonuclear neutrophils also showed a small insignificant increase. Ophthalmologe, 1994 Feb, 91(1), 98 - 102 {Effect of platelet-derived growth factor PDGF on replication of cultivated bovine lens epithelial cells}; Wunderlich K et al.; It has previously been reported that PDGF isoforms AB and BB induce an increase of cytosolic free calcium in cultured bovine lens epithelial cells in a dose-dependent manner . To evaluate the biological capacity of PDGF, we investigated the proliferative response of bovine lens epithelial cells to stimulation with PDGF-AA, -AB and -BB . Since various unspecific components of serum-containing media act as mitogenes and mask the effect of PDGF, serum-free culture conditions were a prerequisite for growth-factor-induced effects . Therefore, a basic medium (Waymouth's MB 752/1 with Ham F12 Nutrient Mixture 1:2, v/v; Gibco BRL) was supplemented with only 2 mM CaCl2, 10 micrograms/ml Transferrin, 10 micrograms/ml Thyroglobulin (both Sigma Chemie) and standard amounts of antibiotics . PDGF isoforms were obtained by separate expression of cloned genes in Escherichia coli, which has been previously described . Under these conditions the isoforms PDGF-AB and -BB caused an increase in proliferation in a dose-dependent manner . The maximum increase of the cell number was 21% for PDGF-AB with an EC5 of 5 ng/ml . PDGF-BB revealed a maximum increase of 33% with an EC5 of 1.5 ng/ml . PDGF-AA, when used in similar concentration was ineffective . These data show the involvement of PDGF isoforms AB and BB in the replicative action of BLEC. Am J Vet Res, 1994 Feb, 55(2), 221 - 6 Effect of in vitro and in vivo migration of bovine neutrophils on binding and expression of Fc receptors for IgG2 and IgM; Worku M et al.; Binding of endogenous and exogenous homologous IgG2 and IgM to bovine neutrophils before and after in vitro migration through micropore filters, and in vivo migration through mammary tissues after intramammary injection of endotoxin was evaluated by use of flow cytometry . Immunoglobulin binding to neutrophils at 4 and 37 C was also evaluated . Before and after in vitro migration, neutrophils with endogenously bound IgG2 and IgM averaged 1 and 2% and 23 and 7%, respectively . Before and after in vivo migration, IgG2 and IgM binding averaged 1 and 7% and 26 and 15%, respectively . Before and after in vitro migration, binding of purified IgG2 and IgM averaged 75 and 67% and 8 and 24%, respectively . Before and after in vivo migration, percentage of neutrophils binding purified IgG2 and IgM averaged 92 and 98% and 54 and 70%, respectively . When serum was used as a source of exogenous immunoglobulins, binding of total IgG after in vitro migration increased from 5% to 28% and of IgM from 4% to 20% . After in vivo migration, binding increased from 21% to 47% and from 24% to 56%, respectively . Exogenous binding of IgG2 at 4 and 37 C averaged 75 and 84%, and binding of IgM averaged 8% at either temperature . Endogenous IgG2 was unaffected by temperature, however, binding of IgM decreased from 23% at 4 C to 2% at 37 C . These data indicate that endogenous binding was higher for IgM before migration than after migration, in vitro and in vivo . Furthermore, migration in vivo through cellular matrices induced receptor upregulation for IgG and IgM.(ABSTRACT TRUNCATED AT 250 WORDS) Ultramicroscopy, 1994 Feb, 53(2), 121 - 36 Three-dimensional reconstruction from random projections: orientational alignment via Radon transforms; Radermacher M; A method for alignment of projections with unknown projecting directions towards a three-dimensional reference has been developed . The technique has been applied to the three-dimensional reconstruction from images of a frozen-hydrated preparation of 50S ribosomal subunits from Escherichia coli recorded in the electron microscope . The algorithm as used here combines the Single Exposure Conical Reconstruction Technique (SECReT) with a three-dimensional orientation search . The algorithm allows for the refinement of a reconstruction obtained with SECReT by refinement of the true projection angles, and by the inclusion of projections with a priori unknown random orientation . With model data it is demonstrated that the algorithm works reliably even for signal-to-noise ratios lower than 1. Protein Eng, 1994 Feb, 7(2), 271 - 80 Bifunctional hybrids between the variable domains of an immunoglobulin and the maltose-binding protein of Escherichia coli: production, purification and antigen binding; Bregegere F et al.; Hybrids were constructed between the maltose-binding protein of Escherichia coli (MalE) and the variable domains (V-domains) of D1.3, a mouse antibody directed against hen lysozyme . Each V-domain was fused with the C- or N-terminus of MalE and expressed in E . coli, either alone or associated with the other V-domain, as a heterodimer (Fv) or as a single-chain fragment (scFv) . The hybrids were exported into the bacterial periplasm, purified by affinity chromatography on cross-linked amylose and separated from incomplete products by ion-exchange chromatography . Hybrids between MalE and Fv bound the antigen specifically, with affinities increased up to 10-fold when compared to native D1.3 . This strongly suggests that MalE contributed to the binding . The affinities and specificities of the different hybrids, as well as their levels of contamination by incomplete products, depended on their fusion pattern with MalE . Hybrids between MalE and either single V-domain also bound hen lysozyme specifically, which shows that each V-domain can recognize the antigen when fused with MalE . The high affinity of VH-MalE (KD = 3 nM) could be due to both participation of MalE in the binding and a conformational adaptation of the lone V-domain. Protein Eng, 1994 Feb, 7(2), 263 - 70 Thermostable variants of bovine beta-lactoglobulin; Cho Y et al.; The thermal stability of bovine beta-lactoglobulin (BLG) has been enhanced by the introduction of an additional disulfide bond . Wild-type BLG has two disulfide bonds, C106-C119 and C66-C160, with a free cysteine at position 121 . We have designed, with the aid of molecular modeling calculations, two mutants of a recombinant BLG (rBLG), L104C and A132C . Molecular dynamics simulations were performed at 300K to study the effect of these alterations on the conformation of the protein . These mutants were then created by site-directed mutagenesis and purified from Escherichia coli carrying a tac expression vector using a two-step renaturation method . Formation of disulfide linkages in the correct arrangement, as designed, was confirmed by peptide mapping . In contrast to wild-type rBLG, which polymerizes at temperatures > 65 degrees C, neither of the mutant proteins polymerized . The conformational stability of the L104C and A132C mutant proteins against thermal denaturation has been substantially increased (8-10 degrees C) as compared with wild-type rBLG . Furthermore, the A132C rBLG exhibits an enhanced stability against denaturation by guanidine hydrochloride as compared with the wild-type or L104C rBLG. J Microsc, 1994 Feb, 173 ( Pt 2), 143 - 7 A cryoglue to mount vitreous biological specimens for cryoultramicrotomy at 110K; Richter K; Mixtures of ethanol, 2-propanol and 2-butanol can be used as a cryoglue to mount vitrified biological specimens for ultrathin cryosectioning . Brought directly from room temperature to a cutting support at 140 K in the cold chamber of a cryoultramicrotome, these alcohols stiffen to a viscous and gluey consistency allowing the attachment or embedding of a vitreous biological sample . The mass hardens at lower temperatures fixing the sample well for microtomy . With ethanol: 2-propanol (2:3), samples are applied at 140 K and ultrathin cutting can be done at 115 K. Surg Laparosc Endosc, 1994 Feb, 4(1), 65 - 7 Laparoscopic cholecystectomy for empyema of gallbladder during pregnancy; Shaked G et al.; Laparoscopic cholecystectomy has gained great popularity during the last few years . This procedure has advantages over the traditional open operation, thus making it the standard method for removal of the gallbladder at present . Only a few cases of laparoscopic cholecystectomy during pregnancy have been reported . On the other hand, some authors classify pregnancy as one of the contraindications for this procedure. Protein Expr Purif, 1994 Feb, 5(1), 89 - 95 An expression system for secretion and purification of a genetically engineered thermostable chimera of protein A and alkaline phosphatase; Chowdhury PS et al.; A chimera between gene segments of Protein A and a mutated alkaline phosphatase (lysine328 mutated to alanine) of Escherichia coli has been constructed . This chimeric gene was cloned in a T7 promoter-based IPTG-inducible expression vector . The chimeric protein was expressed in E . coli and was efficiently secreted into the periplasm, from which it could be easily purified by a combination of ion-exchange and gel permeation chromatography . The purified chimera was found to be thermostable and exhibited both IgG binding and high alkaline phosphatase activity . It was used as a probe in enzyme-linked immunosorbent assays and results indicate that it is a promising substitute for secondary antibodies in enzyme-linked immunoassays. Protein Expr Purif, 1994 Feb, 5(1), 57 - 64 Overexpression and reactivation of binding activity of the recombinant CTF/NF-1 transcription factor; Amemiya K et al.; The transcription factor CTF/NF-1 is a multifunctional cellular protein which participates in the expression of host and viral genes and in the replication of viral DNA . A procedure was developed to obtain recombinant CTF/NF-1 protein in order to help identify the binding sites of CTF/NF-1 protein in the regulatory region of viral sequences . Cloning and expression of the CTF/NF-1 gene downstream from a T7 RNA polymerase promoter resulted in an insoluble protein produced in Escherichia coli . Specific binding by the recombinant CTF/NF-1 protein to its cognate recognition site was obtained only after a denaturation and renaturation procedure . No special chromatography steps were required to obtain a rCTF/NF-1 preparation which could be used for our binding studies . Specific binding was detected with an NF-1 oligonucleotide and with viral DNA templates . Binding to the viral DNA sequences was identical to that previously published with biochemically purified cellular CTF/NF-1 protein. Protein Expr Purif, 1994 Feb, 5(1), 44 - 9 Overexpression in Escherichia coli and purification of the 6-phosphogluconate dehydrogenase of Trypanosoma brucei; Barrett MP et al.; The gene encoding 6-phosphogluconate dehydrogenase (EC 1.1.1.44) from Trypanosoma brucei was cloned into the overexpression vector pET3a which utilizes the T7 polymerase gene expression system . Up to 40% of total cell protein consisted of the trypanosome enzyme when expression was induced in Escherichia coli host strains at 28 degrees C . The enzyme was rapidly degraded at temperatures higher than 30 degrees C . The T . brucei enzyme was purified to near homogeneity (as judged by SDS-polyacrylamide gel electrophoresis) using a two-step purification method, involving a DE-52 cellulose batch preparation followed by 2' AMP-agarose affinity chromatography . The purified protein crystallized readily . A molecular model of the trypanosome enzyme based on its mammalian counterpart revealed differences between the two enzymes in residues involved in cofactor binding. Protein Expr Purif, 1994 Feb, 5(1), 22 - 6 Purification and biochemical characterization of a recombinant mouse seminal vesicle trypsin inhibitor produced in Escherichia coli; Lai ML et al.; Escherichia coli cells were transformed with an expression vector constructed by inserting a DNA fragment encoding a Kazal-type trypsin inhibitor from mouse seminal vesicle into pGEX-2 . The cloned cells were able to produce a high yield of a chimeric polypeptide made by fusing the trypsin inhibitor to glutathione S-transferase . The chimeric polypeptide could be purified through an affinity column of glutathione agarose beads . The purified protein could be digested with thrombin to release the recombinant trypsin inhibitor which could be further purified by HPLC of the thrombin digests on a reverse-phase C4 column . The recombinant trypsin inhibitor was homogeneous and showed trypsin inhibitor activity as strong as that of the naturally occurring trypsin inhibitor. Protein Expr Purif, 1994 Feb, 5(1), 14 - 21 Refolding and characterization of human recombinant heparin-binding neurite-promoting factor; Seddon AP et al.; Heparin-binding neurite-promoting factor (HBNF) is a highly basic, cysteine-rich 136-residue protein, and a member of a new class of heparin-binding proteins . It exhibits a neurite-outgrowth promoting activity and its expression is both temporally and spacially regulated during fetal and postnatal development . A high interspecies sequence conservation suggests important, presently unknown, biological functions . HBNF is structurally and most likely functionally related to the product of a developmentally regulated gene, MK (midkine) . To elucidate biological roles of these proteins, recombinant forms of the proteins were produced . Expression of human recombinant HBNF and MK in Escherichia coli lead to the formation of insoluble aggregated protein that accounted for about 25% of the total cellular protein . Homogeneous, monomeric forms of each protein were recovered from inclusion bodies by reduction with dithiothreitol and solubilization in 8 M urea . Refolding of the reduced and denatured protein occurred upon dialysis at pH 7.4 . Human recombinant (hr) HBNF and hrMK prepared in this manner were further purified by heparin affinity chromatography . Chromatographic evidence demonstrates that refolding and concomitant disulfide bond formation in hrHBNF proceeds in high yield with minimal formation of stable nonnative disulfides . Studies on the redox status of the 10 cysteine residues of bovine brain HBNF and the refolded recombinant protein indicate that all cysteines are engaged in disulfide bond formation . The disulfide arrangements for the recombinant protein were found to be identical to those in the native protein isolated from bovine brain.(ABSTRACT TRUNCATED AT 250 WORDS) Biophys J, 1994 Feb, 66(2 Pt 1), 335 - 44 The rational design of amino acid sequences by artificial neural networks and simulated molecular evolution: de novo design of an idealized leader peptidase cleavage site; Schneider G et al.; A method for the rational design of locally encoded amino acid sequence features using artificial neural networks and a technique for simulating molecular evolution has been developed . De novo in machine design of Escherichia coli leader peptidase (SP1) cleavage sites serves as an example application . A modular neural network system that employs sequence descriptions in terms of physicochemical properties has been trained on the recognition of characteristic cleavage site features . It is used for sequence qualification in the design cycle, representing the sequence fitness function . Starting from a random sequence several cleavage site sequences were generated by a simulated molecular evolution technique . It is based on a simple genetic algorithm that takes the quality values calculated by the artificial neural network as a heuristic for inductive sequence optimization . Simulated in vivo mutation and selection allows the identification of predominant sequence positions in Escherichia coli signal peptide cleavage site regions (positions -2 and -6) . Various amino acid distance maps are used to define metrics for the step size of mutations . Position-specific mutability values indicate sequence positions exposed to high or low selection pressure in the simulations . The use of several distance maps leads to different courses of optimization and to various idealized sequences . It is concluded that amino acid distances are context dependent . Furthermore, a method for identification of local optima during sequence optimization is presented. Trends Biochem Sci, 1994 Feb, 19(2), 78 - 82 Phosphoryl transfer in Flp recombination: a template for strand transfer mechanisms; Jayaram M; The basic chemistry involved in DNA recombination, RNA splicing and DNA transposition is a phosphoryl transfer reaction . This review is an attempt to provoke a unified thinking on the reaction mechanisms in these nucleic acid transactions . Some of the recent results with the Flp site-specific recombinase that reveal how the chemical reactivity for recombination is derived from cooperative protein-subunit interactions on the DNA substrate are discussed . At least some of the features of Flp reaction are likely to have global implications in other DNA and RNA strand-transfer systems. Plant Physiol, 1994 Feb, 104(2), 591 - 6 A small GTP-binding protein from Arabidopsis thaliana functionally complements the yeast YPT6 null mutant; Bednarek SY et al.; A clone designated A.t.RAB6 encoding a small GTP-binding protein was isolated from a cDNA library of Arabidopsis thaliana leaf tissue . The predicted amino acid sequence was highly homologous to the mammalian and yeast counterparts, H.Rab6 and Ryh1/Ypt6, respectively . Lesser homology was found between the predicted Arabidopsis protein sequence and two small GTP-binding proteins isolated from plant species (44% homology to Zea mays Ypt1 and 43% homology to Nicotiana tabacum Rab5) . Conserved stretches in the deduced amino acid sequence of A.t.Rab6 include four regions involved in GTP-binding, an effector region, and C-terminal cysteine residues required for prenylation and subsequent membrane attachment . Northern blot analysis demonstrated that A.t.Rab6 mRNA was expressed in root, leaf, stem, and flower tissues from A . thaliana with the highest levels present in roots . Escherichia coli produced histidine-tagged A.t.Rab6 protein-bound GTP, whereas a mutation in one of the guanine nucleotide-binding sites (asparagine122 to isoleucine) rendered it incapable of binding GTP . Functionally, the A.t.RAB6 gene was able to complement the temperature-sensitive phenotype of the YPT6 null mutant in yeast . The isolation of this gene will aid in the dissection of the machinery involved in soluble protein sorting at the trans-Golgi network of plants. Plant Physiol, 1994 Feb, 104(2), 417 - 23 Cloning and characterization of a cDNA encoding aspartate aminotransferase-P1 from Lupinus angustifolius root tips; Winefield CS et al.; A root tip cDNA library, constructed in the lambda Zap II expression vector, was immunoscreened with a monoclonal antibody raised against aspartate aminotransferase-P1 from Lupinus angustifolius L . var Uniharvest . One 1452-base pair clone was isolated . The encoded protein sequence had high homology to both plant and animal aspartate aminotransferase sequences . The clone was converted to the phagemid form and expressed in Escherichia coli . The expressed protein was enzymically active and could be immunocomplexed with aspartate aminotransferase-P1-specific antibodies . The complete aspartate aminotransferase-P1 cDNA was cloned into the yeast expression vector pEMBL-yex4 and transformed into Saccharomyces cerevisiae strain BRSCS6, which possesses a mutated aspartate aminotransferase gene (the asp5 mutation) . Complementation of the mutation was achieved using this construct. Proteins, 1994 Feb, 18(2), 161 - 73 The structure of Trypanosoma cruzi trypanothione reductase in the oxidized and NADPH reduced state; Lantwin CB et al.; The three-dimensional structure of trypanothione reductase (TR) (EC 1.6.4.8) from Trypanosoma cruzi has been solved at 0.33 nm resolution by molecular replacement using the structure of C . fasciculata TR as a starting model . Elucidation of the T . cruzi TR structure represents the first step in the rational design of a drug against Chagas' disease . The structure of T . cruzi TR is compared with those of C . fasciculata TR as well as human and E . coli glutathione reductase (GR) . In the FAD-binding domain, TR has two insertions, each about 10 residues long, which do not occur in GR . The first one is a rigid loop stabilizing the position of helix 91-117 which is responsible for the wider active site of TR as compared to GR . The second insertion does not occur where it is predicted by sequence alignment; rather the residues extend three strands of the 4-stranded beta-sheet by one or two residues each . This increases the number of hydrogen bonds within the sheet structure . The structure of the NADPH.TR complex has been solved at 0.33 nm resolution . The nicotinamide ring is sandwiched between the flavin ring and the side chain of Phe-198 which undergoes the same conformational change upon coenzyme binding as Tyr-197 in GR . In addition to Arg-222 and Arg-228, which are conserved in TR and GR, Tyr-221--the last residue of the second beta-sheet strand of the beta alpha beta dinucleotide binding fold--is in hydrogen bonding distance to the 2' phosphate group of NADPH. Proteins, 1994 Feb, 18(2), 119 - 32 Evaluation of the conformational free energies of loops in proteins; Smith KC et al.; In this paper we discuss the problem of including solvation free energies in evaluating the relative stabilities of loops in proteins . A conformational search based on a gas-phase potential function is used to generate a large number of trial conformations . As has been found previously, the energy minimization step in this process tends to pack charged and polar side chains against the protein surface, resulting in conformations which are unstable in the aqueous phase . Various solvation models can easily identify such structures . In order to provide a more severe test of solvation models, gas-phase conformations were generated in which side chains were kept extended so as to maximize their interaction with the solvent . The free energies of these conformations were compared to that calculated for the crystal structure in three loops of the protein E . coli RNase H, with lengths of 7, 8, and 9 residues . Free energies were evaluated with a finite difference Poisson-Boltzmann (FDPB) calculation for electrostatics and a surface area-based term for nonpolar contributions . These were added to a gas-phase potential function . A free energy function based on atomic solvation parameters was also tested . Both functions were quite successful in selecting, based on a free energy criterion, conformations quite close to the crystal structure for two of the three loops . For one loop, which is involved in crystal contacts, conformations that are quite different from the crystal structure were also selected . A method to avoid precision problems associated with using the FDPB method to evaluate conformational free energies in proteins is described. Proteins, 1994 Feb, 18(2), 107 - 18 Crystallization and structural analysis of bullfrog red cell L-subunit ferritins; Trikha J et al.; Ferritin is a 24 subunit protein that controls biomineralization of iron in animals, bacteria, and plants . Rates of mineralization vary among members of the ferritin family, particularly between L and H type subunits of animal ferritins which are differentially expressed in various cell types . To examine ferritin from a highly differentiated cell type and to clarify the relationship between ferritin structure and function, bullfrog red cell L ferritin has been cloned, overexpressed in E . coli, and crystallized under two conditions . Crystals were obtained at high ionic strength in the presence of MnCl2 at a concentration comparable to that of the protein and in the presence of MgCl2 at a concentration much higher than that of the protein . Under both crystallization conditions, the crystals are tetragonal bipyramids in the space group F432 with unit cell dimensions a = b = c = 182 +/- 0.5 A . Crystals obtained in the presence of manganese and ammonium sulfate diffract to 1.9 A, while those obtained in the presence of magnesium and sodium tartrate diffract to 1.6 A . Isomorphous crystals have been obtained under similar conditions for a site-directed mutant with a reduced mineralization rate in which Glu-57, -58, -59, and -61 are all replaced by Ala . The structure of wild type L-subunit with magnesium has been solved by molecular replacement using the calcium salt of human liver H subunit (Lawson et al., Nature (London) 349:541-544, 1991) as the model . The crystallographic R factor for the 6-2.2 A shell is 0.21 . The overall fold of human H and bullfrog L ferritins is similar with an rms difference in backbone atomic positions of 0.97 A . The largest structural differences occur in the D helix and the loop connecting the D and E helices of the four helix bundle . Because red cell L ferritin and liver H ferritin show differences in both rates of mineralization and three-dimensional structure, more detailed comparisons of these structures are likely to shed new light on the relationship between conformation and function. J Periodontol, 1994 Feb, 65(2), 139 - 46 The secretion of PGE2, IL-1 beta, IL-6, and TNF alpha by adherent mononuclear cells from early onset periodontitis patients; Shapira L et al.; The secretion of prostaglandin E2 (PGE2), tumor necrosis factor alpha (TNF alpha), interleukin 1 beta (IL-1 beta), and interleukin 6 (IL-6) by adherent mononuclear cells (AMNC) from 28 patients with early-onset periodontitis was studied . The early onset-periodontitis patients consisted of 12 patients with localized juvenile periodontitis (LJP) and 16 patients with severe generalized periodontitis (SGP) . The AMNC responses to different concentrations of lipopolysaccharide (LPS) (E . coli) were determined in these 28 patients and compared to 14 healthy controls . Mediator levels in the supernatant were measured using radioimmunoassays for PGE2, IL-1 beta, and IL-6 determination and an enzyme linked immunosorbent assay for TNF alpha levels . The mean age of the patients was 19.9 years for the LJP group, 30.4 years for SGP, and 28.0 years for the controls . The mean number of teeth per patient with attachment loss of > 6 mm was 4.75 in the LJP patients and 17.3 in the SGP group . In the absence of LPS, LJP AMNC secreted significantly more PGE2 than unstimulated control or SGP AMNC, while similar baseline amounts of IL-1 beta, IL-6, and TNF alpha were secreted by AMNC from the 3 patient groups . LPS stimulation resulted in the dose-dependent secretion of significantly higher levels of PGE2 by LJP AMNC compared to SGP AMNC which in turn secreted significantly more than controls . TNF alpha secretion by LJP monocytes was significantly greater than the SGP and the control groups while IL-1 beta secretion by the SGP AMNC was depressed compared to the other two patient groups.(ABSTRACT TRUNCATED AT 250 WORDS) J Anim Sci, 1994 Feb, 72(2), 309 - 14 Lipopolysaccharide-induced sickness behavior in pigs is inhibited by pretreatment with indomethacin; Johnson RW et al.; Many of the behavioral responses following acute bacterial or viral infection are now considered important for maintaining homeostasis during inflammation . In the present study, we extend this concept to pigs (16 crossbred barrows) by demonstrating that lipopolysaccharide (LPS, .5, 5, or 50 micrograms/kg BW) from Escherichia coli injected i.p . reduces feed intake, decreases activity, and elevates body temperature . To determine whether any of these effects could be mediated via a prostaglandin (PG)-dependent mechanism, a second experiment with 16 crossbred barrows was conducted . Barrows were pretreated with indomethacin (IND, 5 mg/kg BW {a cyclooxygenase inhibitor}), and their behavior and body temperature following a challenge i.p . injection of LPS (5 micrograms/kg BW) were assessed . Pretreatment with IND inhibited the anorexia and inactivity caused by LPS, suggesting that the behavioral effects of LPS are dependent on activation of a PG system . Lipopolysaccharide alone, however, did not elevate body temperature in this case; thus, the involvement of PGs in this response was not determined . Collectively, these data indicate that pigs respond to LPS by reducing feed intake, decreasing activity, and becoming febrile . The ability of IND to inhibit behavioral effects of LPS is consistent with the hypothesis that a PG system is involved in mediating sickness behavior . Perhaps, by altering the activity of cyclooxygenase it is possible to enhance or inhibit the behavioral symptoms of sickness in pigs. Hear Res, 1994 Feb, 73(1), 65 - 6 Construction of a cDNA library from microdissected guinea pig crista ampullaris; Wilcox ER et al.; Poly(A) RNA was isolated from microdissected guinea pig crista ampullaris epithelium and converted into cDNA with RNase H- murine leukemia virus reverse transcriptase . After size fractionation, the cDNA was directionally ligated into the vector pSPORT 1 and the plasmids electroporated into E . coli . The library was found to have 1.6 x 10(7) independent colonies with 5% of the colonies lacking an insert . Thirty randomly selected colonies were checked for inserts and the average insert size was 833 base pairs with a range of 400 to 2300 base pairs . The library was screened with a beta-actin guinea pig cDNA probe and 0.16% of the colonies contained an insert hybridizing to the probe. Int J Sports Med, 1994 Feb, 15(2), 96 - 9 Effect of long-distance running on polymorphonuclear neutrophil phagocytic function of the upper airways; Muns G; A high incidence of infections predominantly of the upper and lower respiratory tract have been observed for years after strenuous exercise . The mucosal surfaces represent a first-line-of-defense for airborne pathogens, but little is known about their function during exercise . The purpose of this study was thus to assess the influence of long-distance running on polymorphonuclear neutrophils (PMNs) phagocytic function of the upper respiratory tract . The number of PMNs recovered by nasal lavage (NAL) was determined, and the percentage of phagocytizing PMNs and number of phagocytized E . coli per PMN was measured utilizing a fluorescence activated cell sorter . A 20 km race resulted in a 2.0-fold higher count of PMNs in the nasal lavage fluid of 12 male amateur runners (aged 34 +/- 11.3 years) immediately after the competition and a 1.6-fold higher count 1 day after the race in comparison to the pre-race value . During training (7 and 3 days before the race) and after 3 days of recreation (3 days after the race) the runners' counts were lower than immediately after the race . The percentage of phagocytizing PMNs was significantly reduced during the pre-race period, but the reduction was most striking immediately and 1 day after the race (48.7 +/- 6.4% and 54.5 +/- 6.2%) . The number of bacteria ingested by the athletes' phagozytizing PMNs was 3.2 +/- 0.3 E . coli/PMN immediately after the race and 5.2 +/- 0.3 1 day after the competition . The findings suggest chronic upper airway inflammation and impaired phagocyte function after strenuous exercise . This might contribute to the observed high incidence of respiratory tract infections among participants in sports. Plant Mol Biol, 1994 Feb, 24(4), 585 - 602 Conserved gene clusters in the highly rearranged chloroplast genomes of Chlamydomonas moewusii and Chlamydomonas reinhardtii; Boudreau E et al.; We have extended to about 75 the number of genes mapped on the Chlamydomonas moewusii and Chlamydomonas reinhardtii chloroplast DNAs (cpDNAs) by partial sequencing of the very closely related C . eugametos and C . moewusii cpDNAs and by hybridizations with Chlamydomonas chloroplast gene-specific sequences . Only four of these genes (tscA and three reading frames) have not been identified in any other algal cpDNAs and thus may be specific to Chlamydomonas . Although the C . moewusii and C . reinhardtii cpDNAs differ by complex sequence rearrangements, 38 genes scattered throughout the genome define 12 conserved clusters of closely linked loci . Aside from the rRNA operon, four of these gene clusters share similarity to evolutionarily primitive operons found in other cpDNAs, representing in fact remnants of these operons . Our results thus indicate that most of the ancestral bacterial operons that characterize the chloroplast genome organization of land plants and early-diverging photosynthetic eukaryotes have been disrupted before the emergence of the polyphyletic genus Chlamydomonas . All gene rearrangements between the C . moewusii and C . reinhardtii cpDNAs, with the exception of those accounting for the relocations of atpA, psbI and rbcL, occurred within corresponding regions of the genome . One of these rearrangements seems to have led to disruption of the ancestral region containing rpl23, rpl2, rps19, rpl16, rpl14, rpl5, rps8 and the psaA exon 1 . This gene cluster, which bears striking similarity to the Escherichia coli S10 and spc operons, spans a continuous DNA segment in C . reinhardtii, while it maps to two separate fragments in C . moewusii. Bioorg Khim, 1994 Feb, 20(2), 114 - 25 {ATP-dependent proteinase La from Escherichia coli}; Romanova TV et al.; Homogeneous preparations of the ATP-dependent La proteinase from E . coli and two its mutant forms, containing an alanine residue instead of Ser679 or Ser368, were isolated . Ser679 was shown to be catalytically active rather than Ser368 as suggested in the literature . To choose between the alternative structures of the gene lon La proteinase fragments within the controversial regions were analysed and the gene structure established at the Laboratory of Proteolytic Enzymes (Institute of Bioorganic Chemistry) was confirmed . Inactivity of La proteinase in some in vitro systems suggests its functioning in vivo to be not autonomous, requiring additional factors. Mol Microbiol, 1994 Feb, 11(3), 537 - 44 Expression and role of the universal stress protein, UspA, of Escherichia coli during growth arrest; Nystrom T et al.; The synthesis of the small, cytoplasmic protein UspA universal stress protein A) of Escherichia coli is induced as soon as the cell growth rate falls below the maximal growth rate supported by the medium, regardless of the condition inhibiting growth . The increase in UspA synthesis appears to be the result of induction of the monocistronic uspA gene . Induction of this gene during a heat-shock treatment is demonstrated to be the result of transcriptional activation of a sigma 70-dependent promoter which has previously been shown to be activated also during carbon starvation-induced growth arrest . Mutant cells lacking UspA grow at rates indistinguishable from the isogenic parent at different temperatures and in the presence of different growth inhibitors but are impaired in their ability to survive prolonged periods of complete growth inhibition caused by a variety of diverse stresses, including CdCl2, H2O2, DNP, CCCP exposure, and osmotic shock . Moreover, the uspA mutation results in an increased sensitivity of cells to carbon-source starvation (i.e . glucose, glycerol or succinate depletion) . Also, the mutation causes a marked alteration in the timing of starvation protein expression but protein expression during steady-state growth appears to be normal . The results presented have prompted us to postulate that UspA may have a general protective function related to the growth arrest state. Mol Microbiol, 1994 Feb, 11(3), 525 - 36 Leucine-responsive regulatory protein, IS1 insertions, and the negative regulator FaeA control the expression of the fae (K88) operon in Escherichia coli; Huisman TT et al.; Nucleotide sequence analysis of the fae operon encoding the biosynthesis of K88 fimbriae revealed the presence of two divergently transcribed regulatory genes, faeA and faeB, separated by two inverted IS1 insertions . The amino acid sequences of the regulatory proteins FaeA and FaeB show similarity to the primary structure of corresponding regulatory proteins involved in the biosynthesis of Pap and S fimbriae . Expression of faeA is positively controlled by the FaeA protein, whereas K88 fimbriae production is negatively controlled by the co-operative activity of FaeA and the leucine-responsive regulatory protein (Lrp) . Exchange of FaeA for Papl, a positive regulator of Pap fimbriae expression, also represses K88 production indicating that the combination Papl/Lrp has opposite effects on fae and pap expression . Mutations in faeB had no effect on the biosynthesis of K88 fimbriae . The presence of the two IS1 insertions is hypothesized to neutralize part of the repression of K88 biosynthesis by FaeA/Lrp . Like pap, the fae operon does not respond to exogenous leucine. Mol Microbiol, 1994 Feb, 11(3), 459 - 69 Mutational and physical analysis of F plasmid traY protein binding to oriT; Luo Y et al.; F plasmid traY protein binding to wild-type or deleted regions containing the TraY-binding site, sbyA, was studied in vitro . The principal DNA-protein complex was formed with DNA segments including the sbyA site defined by footprinting and (with lesser affinity) with truncated segments that retained the leftward two-thirds of sbyA . This located the major sequence determinants for TraY binding between bp 204 and 227 on the oriT map . For all sequences tested, bound TraY induced bending of approximately 50 to 55 degrees, and centred between bp 214 and 221 . Thermodynamic and mobility analyses indicated that two TraY protomers bind to sbyA . At higher TraY concentrations, additional TraY bound to the left of the sbyA in a region previously shown to bind IHF (site IHF A) . TraY binding to this additional site (sbyC) was inhibited by IHF . Sequence similarities shared by sbyA, sbyB, and sbyC may include the critical base pairs for TraY binding. Vaccine, 1994 Feb, 12(2), 109 - 16 Epidemiological model of diarrhoeal diseases and its application in prevention and control; Jose MV et al.; In this paper, a prototype epidemiological model of acute bacterial and viral diarrhoeal diseases occurring in young children is formulated . The model is able to mimic the observed epidemiological patterns of infantile diarrhoeal diseases associated mainly with enterotoxigenic Escherichia coli or with rotavirus . The proposed mathematical model predicts a plausible pattern of the serological profile of an enteric infection . According to computer simulation experiments (CSE) with this model, it is not necessary to develop an enteric vaccine conferring total and long-lasting immunity in order to achieve protection from diarrhoeal diseases in young children . Given a protective efficacy and a finite duration of vaccine-induced protection, the optimal immunization policy must be sought . Oral rehydration therapy (ORT) intervention has a clear effect in diminishing the number of individuals dying from diarrhoeal illness . The CSE also predict an apparent reduction in age-prevalence of diarrhoeal diseases by use of ORT. Am J Physiol, 1994 Feb, 266(2 Pt 2), R614 - 21 Antipyresis caused by stimulation of vasopressinergic neurons and intraseptal or systemic infusions of gamma-MSH; Bock M et al.; Antipyretic properties have been ascribed to arginine vasopressin (AVP), and the site where its antipyretic effects are mediated in the brain was identified as the ventrolateral septum of the limbic system . In guinea pigs, the majority of AVP projections to the septum originate from parvocellular neurons of the hypothalamic paraventricular nucleus (PVN) . Electrical stimulation of the PVN with 10-s trains of current pulses (duration 1 ms, frequency 20 Hz, amplitude 8 V, current 0.205 +/- 0.017 mA) reduced the febrile response to an intramuscular injection of 20 micrograms/kg lipopolysaccharide (LPS from Escherichia coli, 0111: B4) by 54% compared with unstimulated animals . This reduction in fever by electrical PVN stimulation was partly reversed by a simultaneous intraseptal microinfusion of the vasopressinergic V1-receptor antagonist d(CH2)5{Tyr(Met)2}AVP at a concentration of 10(-5) mol for 6 h with an infusion speed of 0.1 microliter/min . We further investigated the effects of intraseptal microinfusions or systemic infusions of the gamma-melanocyte-stimulating hormone (gamma-MSH), a derivative of the proopiomelanocortin, on LPS-induced fever . Intraseptal microinfusions of gamma-MSH at a concentration of 10(-5) mol/l for 6 h with an infusion speed of 0.1 microliter/min caused a 38% reduction in fever . A significantly greater 57% reduction in fever was observed when the intraseptal microinfusion of gamma-MSH was combined with electrical stimulation of the PVN (for parameters see above) . A systemic infusion of 0.261 mumol gamma-MSH for 6 h reduced LPS fever to approximately 50% compared with animals infused with vehicle (0.9% saline).(ABSTRACT TRUNCATED AT 250 WORDS) Biotechnol Appl Biochem, 1994 Feb, 19 ( Pt 1), 3 - 15 Towards an understanding of structure-function relationships of elongation factor Tu; Wiborg O et al.; In light of the recently determined structure of elongation factor Tu, and taking into account chemical studies mapping functional sites, a number of residues have been selected for site-directed mutagenesis studies . Gly94, Gly126, His66, His118, Lys89 and Asp90 have each been point-mutated . Preliminary in vitro characterization data are presented. FEMS Microbiol Lett, 1994 Feb 1, 116(1), 61 - 6 Characterization of translucent segments observed in an smbA mutant of Escherichia coli; Yamanaka K et al.; The smbA gene of Escherichia coli is essential for cell proliferation . The smbA2 mutant shows cold-sensitive colony formation at 22 degrees C . A novel morphological phenotype, formation of a translucent segment at midcell or at a cell pole, was observed by phase-contrast microscopy at a high frequency in the smbA2 mutant cells incubated in L medium lacking NaCl at 22 degrees C, but not observed in L medium containing 1% NaCl or 20% sucrose at the same temperature . No translucent segment was observed in the wild-type cells in any of the media used . Electron microscopic observation revealed that the translucent segments resulted from the enlargement of a periplasmic space by separation of the inner membrane from the peptidoglycan layer and the outer membrane. FEMS Microbiol Lett, 1994 Feb 1, 116(1), 37 - 42 Rescue by vitamin B12 of Escherichia coli cells treated with colicins A and E allows measurement of the kinetics of colicin binding on BtuB; Cavard D; Sensitivity of Escherichia coli bacteria to colicins A and E1 was significantly increased by overproduction of the BtuB receptor protein . The amount of vitamin B12 needed before colicins A and E1 treatment to protect cells against killing was found to be a function of the number of BtuB molecules present at the cell surface . Cells treated by colicins A and E were rescued from killing by addition of vitamin B12 shortly after colicin treatment . The rate of reversal by vitamin B12 may correspond to the kinetics of irreversible binding to BtuB of the various colicins. Plant Mol Biol, 1994 Feb, 24(3), 495 - 503 In vitro characterization of a cassette to accumulate multiple proteins through synthesis of a self-processing polypeptide; Marcos JF et al.; The strategy for processing the polyprotein encoded by plant potyviruses has been mimicked by constructing an expression cassette based on the nuclear inclusion (Nla) proteinase from tobacco etch virus (TEV) . This cassette (pPR01), includes the TEV Nla coding region flanked on each side by its heptapeptide cleavage sequence and cloning sites for the in frame insertion of two different open reading frames . pPR01 allows the synthesis, under the control of a single transcriptional promoter, of two proteins in equimolar amounts as part of a polyprotein which is cleaved into individual mature products by the TEV protease . In in vitro reactions the cassette functioned as expected when several different protein-coding sequences were used . The potential uses of pPR01 are discussed. Mol Gen Genet, 1994 Feb, 242(4), 484 - 9 Regulation of the expression of the isocitrate lyase gene (acuD) of Aspergillus nidulans; Bowyer P et al.; Analysis of the promoter region of the acetate-induced isocitrate lyase gene (acuD) of Aspergillus nidulans is described . Transcription start sites were detected at positions -163, -170 and approximately -281 upstream of the ATG . Transcription analysis showed that the acuD gene is transcribed during growth on acetate but not on hexoses or glycerol . Expression of the acuD gene was studied under inducing and repressing conditions in cre+, creA, creB and creC mutant strains, showing that the creA(d)-1 mutation led to slight derepression of isocitrate lyase . Regulation of expression of the acuD gene was also studied using an in-frame fusion with the lacZ gene of Escherichia coli . Several deletions were made in order to identify the regions responsible for acetate induction and repression . A deletion of the -412 to -200 bp upstream region resulted in loss of all promoter activity and a smaller deletion within this region abolished most of the acetate inducibility. Mol Gen Genet, 1994 Feb, 242(4), 467 - 71 An 'instant gene bank' method for gene cloning by mutant complementation; Gems D et al.; We describe a new method of gene cloning by complementation of mutant alleles which obviates the need for construction of a gene library in a plasmid vector in vitro and its amplification in Escherichia coli . The method involves simultaneous transformation of mutant strains of the fungus Aspergillus nidulans with (i) fragmented chromosomal DNA from a donor species and (ii) DNA of a plasmid without a selectable marker gene, but with a fungal origin of DNA replication ('helper plasmid') . Transformant colonies appear as the result of the joining of chromosomal DNA fragments carrying the wild-type copies of the mutant allele with the helper plasmid . Joining may occur either by ligation (if the helper plasmid is in linear form) or recombination (if it is cccDNA) . This event occurs with high efficiency in vivo, and generates an autonomously replicating plasmid cointegrate . Transformants containing Penicillium chrysogenum genomic DNA complementing A . nidulans niaD, nirA and argB mutations have been obtained . While some of these cointegrates were evidently rearranged or consisted only of unaltered replicating plasmid, in other cases plasmids could be recovered into E . coli and were subsequently shown to contain the selected gene . The utility of this "instant gene bank" technique is demonstrated here by the molecular cloning of the P . canescens trpC gene. Mol Gen Genet, 1994 Feb, 242(4), 455 - 60 A luxAB transcriptional fusion to the cryptic celF gene of Escherichia coli displays increased luminescence in the presence of nickel; Guzzo A et al.; From a library of 3000 Escherichia coli clones, each containing a single, chromosomally located luxAB transcriptional gene fusion, one clone was found in which luminescence increased in the presence of 1 to 50 ppm of NiSO4 . A molecular analysis revealed that the insertion occurred within the celF gene of E . coli . This gene encodes the phospho-beta-glucosidase involved in cleavage of the sugars cellobiose, salicin and arbutin . Cloning and sequencing of DNA downstream of the celF gene revealed three open reading frames (potentially encoding polypeptides of 9.9, 14.1 and 28.5 kDa) that could be co-expressed with the celF gene and that may underlie the observed induction of the celF gene by nickel . A polypeptide of 26 kDa was produced when this region was placed under the control of the Ptac promoter . Moreover, this region was found to be directly adjacent to, and transcribed in the opposite orientation from, the katE gene of E . coli. Mol Gen Genet, 1994 Feb, 242(4), 448 - 54 An "instant gene bank" method for heterologous gene cloning: complementation of two Aspergillus nidulans mutants with Gaeumannomyces graminis DNA; Bowyer P et al.; We present a novel technique for gene cloning by complementation of mutations in Aspergillus nidulans with DNA from a heterologous organism, Gaeumannomyces graminis . This technique bypasses the time-consuming and difficult construction of gene libraries, making it both rapid and simple . The method relies on recombination between a fungal replicating vector pHELP1 and linear G . graminis genomic DNA during co-transformation . We were able to complement two out of seven A . nidulans mutants tested and to rescue transforming DNA from both in Escherichia coli . Complementation of the A . nidulans argB mutation resulted from integration of 8-10 kb segments of G . graminis DNA into pHELP1 . The complementation of the A . nidulans pyrG mutation resulted from a complex rearrangement . Complementing DNA was shown to originate from G . graminis, and was capable of retransforming the original mutants to give the expected phenotype. Mol Gen Genet, 1994 Feb, 242(4), 440 - 7 Inducible transcription of the dnaA gene from Streptomyces lividans 66; Zakrzewska-Czerwinska J et al.; The dnaA gene of Streptomyces lividans was cloned using the Escherichia coli medium-copy-number vector pSU18 and E . coli strain TC1963, which can by-pass the requirement for the DnaA protein . Its regulatory region was subcloned in the Streptomyces probe vector pIJ4083 . Primer extension and S1 mapping studies allowed the identification of a class I Streptomyces promotor (P2) . An additional, previously unknown promoter type (P1) was found by S1 mapping . The presence of two DnaA box motifs between P1 and P2 suggests that the transcriptions of the S . lividans dnaA gene is autoregulated by its gene product . It was shown that the transcription of the dnaA gene is significantly induced by mitomycin C, an agent known to inhibit DNA replication . The data suggest that, as in E . coli, one of the regulatory mechanisms governing the transcription of the dnaA gene in S . lividans is probably related to the SOS response network. Mol Gen Genet, 1994 Feb, 242(4), 374 - 82 The linear Streptomyces plasmid pBL1: analyses of transfer functions; Zotchev SB et al.; pBL1 is a conjugative linear extrachromosomal element of 43 kb previously isolated after interspecific mating between Streptomyces bambergiensis and S . lividans . Cloning experiments using the non-conjugative, circular Streptomyces vector pIJ702 allowed the identification of a 5.74 kb region from pBL1 which facilitates plasmid transfer . Insertion and deletion mutagenesis, gene disruptions, and sequence data suggest that at least five previously unknown genes of pBL1 are required for efficient plasmid transfer and its regulation. J Surg Res, 1994 Feb, 56(2), 117 - 22 Interleukin-2-induced lymphocyte infiltration of multiple organs is differentially suppressed by soluble tumor necrosis factor receptor; Quinn TD et al.; Interleukin-2 (IL-2) mediates the regression of metastatic cancer, but clinical application is restricted by associated toxicities . Previous studies implicate tumor necrosis factor (TNF) as an important mediator of certain IL-2-induced toxicities . We hypothesized that soluble TNF receptor (sTNFr), a TNF antagonist, would alter lymphocyte trafficking into normal tissues and ameliorate IL-2-induced toxicity . Four groups of C57BL/6 mice were treated for 4 days with intraperitoneal injections of 100,000 IU IL-2 alone, 100,000 IU IL-2 and 30 micrograms sTNFr combined, 30 micrograms sTNFr alone, or equal volumes of saline . Animal activity was graded and blood obtained for SGPT and SGOT . At necropsy, organs were harvested for wet:dry ratios as a measurement of organ edema . The lung, liver, and thymus were examined histologically for lymphocytic infiltration and graded on a scale of 1 to 5 . IL-2-treated groups had a statistically significant increase in organ edema, lymphocytic infiltration into the lung and liver, liver enzyme elevation, and pancytopenia when compared with controls . Soluble TNFr significantly suppressed IL-2-induced pulmonary lymphocytic infiltration and associated serum lymphopenia without significant alteration of other IL-2-induced effects . These data implicate TNF as a mediator of the pulmonary lymphocytic infiltration and of lymphopenia that accompanies IL-2 therapy and further suggest that alternative mechanisms are involved in other IL-2-induced deleterious effects. Epidemiol Infect, 1994 Feb, 112(1), 63 - 7 Prospective study of verocytotoxin-producing, enteroaggregative and diffusely adherent Escherichia coli in different diarrhoeal states; Brook MG et al.; One hundred and eighty-one stool specimens from patients with various types of diarrhoea (135 patients) or from non-diarrhoeal controls (23 acute medical patients, 23 inflammatory bowel disease in remission) were investigated using a colony-blot DNA hybridization assay for the presence of Verocytotoxin-producing (VTEC), enteroaggregative (EAggEC) and diffusely adherent (DAEC) Escherichia coli . Twelve patients had probe-positive EAggEC in the stool and 8 of these had diarrhoea, 6 following recent travel . Eight patients had DAEC, 7 of whom had travellers' diarrhoea . Six of 10 (60%) travellers with gastroenteritis, but without a recognized enteric pathogen, were positive for EAggEC (4) or DAEC (2) . Five of 10 (50%) travellers with gastroenteritis related to a recognized enteric pathogen also had DAEC identified in their stool . Of the 23 acute medical control patients 11 had been abroad, 4 of these were immigrants and had EAggEC . VTEC were not found and, with one exception, immunoassays for antibodies to E . coli O 157 and O 2 lipopolysaccharides were negative. Mol Immunol, 1994 Feb, 31(3), 219 - 26 Spontaneous assembly of bivalent single chain antibody fragments in Escherichia coli; McGregor DP et al.; The ability of immunoglobulin Fab and single chain (ScFv) fragments to penetrate effectively into tissue from the vascular system has made these molecules excellent candidates as drug delivery systems and imaging tools . This study investigates the use of single chain antibody fragment bacterial expression vectors as a possible strategy for the production of these molecules . We have modified the pSW1-VHD1.3-VKD1.3-TAG1 vector {Ward et al . (1989) Nature 341, 544-546} which originally, when expressed in E . coli, produced an Fab fragment . In an effort to improve the affinity of the parent vector product a novel single chain antibody construct which encodes a protein with anti-P . aeruginosa activity was generated using a 14 amino acid linker {Chaudhary et al . (1990) Proc . natn . Acad . Sci . U.S.A . 87, 1066-1070} . In addition to the heavy and light chain variable domain genes, our construct also contained the light chain kappa constant domain gene to aid purification of the fragments . To underline this difference from the conventional ScFv fragment we have described this protein as a ScAb . The ScAb generated had an antigen binding capacity similar to the parent anti-P . aeruginosa antibody but was superior to the recombinant anti-P . aeruginosa Fab fragment . On HPLC and non-denaturing gel electrophoresis analysis, the ScAb was found to exist in multimeric forms while the Fab fragment existed only as a single unit . Dimeric ScAb had a similar antigen binding profile to the parent antibody. Mol Pharmacol, 1994 Feb, 45(2), 201 - 11 In vitro reconstitution of a functional peripheral-type benzodiazepine receptor from mouse Leydig tumor cells; Garnier M et al.; The peripheral-type benzodiazepine receptor (PBR) was identified and characterized by its high affinity for two distinct classes of compounds, the benzodiazepines (BZs) and the isoquinolines (IQs) . An M(r) 18,000 IQ-binding protein has been identified as the PBR . In this report we isolated and sequenced a 626-base pair cDNA, specifying an open reading frame of 169 amino acid residues with a predicted molecular weight of 18,843, from MA-10 mouse tumor Leydig cells {i.e., mouse peripheral-type benzodiazepine receptor (mPBR)} . Expression of mPBR cDNA in simian virus 40-transformed 3T3 fibroblasts resulted in an increase in the density of both BZ and IQ binding sites . To examine whether the increased drug binding was due to the M(r) 18,000 PBR protein alone or to other constitutively expressed components of the receptor, an in vitro system was developed using recombinant mPBR protein . The mPBR cDNA was inserted in the pMAL-c2 vector downstream from the malE gene, which encodes maltose-binding protein (MBP) . Transfection of the recombinant pMAL-c2 in Escherichia coli provided high levels of expression of the MBP-mPBR fusion protein . Purified MBP-mPBR recombinant fusion protein incorporated into liposomes, but not MBP alone, was able to bind IQs but not BZs . Addition of MA-10 mitochondrial extracts to the liposomes resulted in the restoration of BZ binding . The protein responsible for this effect was then purified and identified as the M(r) 34,000 voltage-dependent anion channel protein, which by itself does not express any BZ and IQ binding . These results provide strong evidence that PBR is not a single protein receptor but a multimeric complex in which the IQ binding site is on the M(r) 18,000 subunit and expression of the BZ binding site requires both the M(r) 18,000 and 34,000 voltage-dependent anion channel subunits. J Gen Virol, 1994 Feb, 75 ( Pt 2), 283 - 93 Ribosomal protein S2/Sa kinase purified from HeLa cells infected with vaccinia virus corresponds to the B1R protein kinase and phosphorylates in vitro the viral ssDNA-binding protein; Beaud G et al.; A ribosomal protein S2 kinase was purified 6000-fold from cytoplasmic extracts of HeLa cells infected with vaccinia virus, using 80S ribosomes or 40S ribosomal subunits as a substrate . Although the preparation was not homogeneous, a 34K component was identified, the chromatographic behaviour of which correlated with enzyme activity . During its purification the ribosomal protein S2 kinase was resolved from a less abundant ribosomal protein S13 kinase, demonstrating the two to be different entities . A second protein kinase activity against a 43K ribosomal protein comigrated with the ribosomal protein S2 kinase activity during all five chromatographic procedures employed, and we conclude that the two activities are properties of a single species . Two-dimensional gel electrophoresis demonstrated that this second substrate was the acidic ribosomal protein Sa, of isoelectric point approximately 5.2, previously shown to be phosphorylated during infection with vaccinia virus . Another substrate for the ribosomal protein S2/Sa kinase in vitro was the 36K viral ssDNA-binding protein, of isoelectric point approximately 5.0, which is also known to be phosphorylated in vivo . The 34K protein correlating with the catalytic activity in the most purified preparations of the ribosomal protein S2/Sa kinase was recognized by an antibody specific for a protein expressed in Escherichia coli from vaccinia virus gene B1R . This and other evidence suggest strongly that the ribosomal protein S2/Sa kinase is the product of this gene. J Gen Virol, 1994 Feb, 75 ( Pt 2), 277 - 81 Functional oligomerization of purified human papillomavirus types 16 and 6b E7 proteins expressed in Escherichia coli; Chinami M et al.; Purified non-fused soluble human papillomavirus type 16 and 6b E7 proteins expressed in Escherichia coli were found to form oligomers . For both proteins, several degrees of oligomerization were demonstrated by gel filtration, dynamic laser light scattering and scanning electron microscopy . Oligomerization was dependent on the concentration of E7 protein . Oligomerized E7 proteins were able to bind the retinoblastoma gene product pRB and stimulated DNA synthesis when introduced into cells. Eur J Biochem, 1994 Feb 1, 219(3), 945 - 52 The glucose transporter of Escherichia coli . Assignment of the 1H, 13C and 15N resonances and identification of the secondary structure of the soluble IIB domain; Golic Grdadolnik S et al.; The IICBGlc subunit of the Escherichia coli glucose transporter consists of two domains, the membrane-spanning IIC domain, and the hydrophilic IIB domain which contains the phosphorylation site (Cys421) . A functional form of the IIB domain was over-expressed separately and isotopically labelled with 13C and 15N . A variety of 15N-edited and 13C, 15N triple-resonance NMR experiments yielded a nearly complete assignment of the 1H, 13C and 15N resonances . Based on the evaluation of conformationally sensitive parameters including NOE effects, scalar couplings and chemical shifts, the secondary structure of the IIB domain is presented . The protein is comprised of four beta-strands forming an antiparallel beta-sheet, two larger alpha-helices at the N- and C-termini and a smaller helical structure of residues 52-58. Eur J Biochem, 1994 Feb 1, 219(3), 877 - 86 Cloning, expression, and spectroscopic studies of the Jun leucine zipper domain; Riley LG et al.; Association of the human c-Jun and c-Fos proteins depends upon interactions involving their leucine zipper domains . We are interested in elucidating the tertiary structure of the Jun and Fos leucine zipper domains with a view to understanding the precise intermolecular interactions which govern the affinity and specificity of interaction in these proteins, which have the unusual capacity to form either homodimeric or heterodimeric zipper pairs . With this goal in mind, we have developed a bacterial expression system for the efficient production of both unlabelled and isotopically labelled c-Jun leucine zipper domain . A synthetic junLZ gene was created by annealing, ligation, and polymerase-chain-reaction amplification of overlapping synthetic oligonucleotides which comprised 132 bp of coding sequence encompassing residues Arg276-Asn314 of c-Jun plus a total of five engineered non-native residues at the N- and C-termini . The junLZ gene was cloned into the pGEX-2T vector from which recombinant c-Jun leucine zipper domain (rJunLZ; 46 residues, 5.1 kDa) was overexpressed (approximately 15% total cell protein) in Escherichia coli as a fusion protein of 31.4 kDa, consisting of rJunLZ fused to the carboxy-terminal portion of Schistosoma japonicum glutathione S-transferase . Two markedly different expression strategies have been devised which allow purification of rJunLZ from the soluble or inclusion-body fraction of induced cells . We have used these strategies to produce unlabelled and uniformly 15N-labelled rJunLZ for NMR studies which, in combination with circular dichroic measurements, reveal that rJunLZ most likely forms a symmetric coiled-coil of parallel alpha-helices . We also present 15N-NMR chemical shift assignments for the backbone and sidechain amide nitrogens of rJunLZ, which should assist in determination of a high-resolution structure of the homodimeric Jun leucine zipper using heteronuclear three-dimensional NMR spectroscopy. Eur J Biochem, 1994 Feb 1, 219(3), 855 - 63 The bilin-binding protein of Pieris brassicae . cDNA sequence and regulation of expression reveal distinct features of this insect pigment protein; Schmidt FS et al.; The bilin-binding protein (BBP) is a blue pigment protein which is abundant in the butterfly Pieris brassicae . In an attempt to clarify the physiological role of this member of the lipocalin family of proteins, its complete cDNA was cloned and expression of the BBP gene in P . brassicae was investigated . It was found that synthesis of the BBP mRNA is highly regulated during the insect's ontogenesis . In larvae after the third ecdysis as well as in pupae and adults, large amounts of the mRNA are present . Each of these stages itself displays a distinct time course of mRNA synthesis . In addition, BBP is expressed tissue specifically, with the fat body being the major source of this secretory protein in the larvae . Hence, the expression pattern of BBP in this organism is markedly different from the closely related pigment protein insecticyanin in Manduca sexta . Finally, the bacterial expression of BBP in a functional state was established as a basis for the future analysis of its ligand-binding functions by protein engineering. Eur J Biochem, 1994 Feb 1, 219(3), 1041 - 51 Kinetic resolution of the reaction catalysed by proton-translocating transhydrogenase from Escherichia coli as revealed by experiments with analogues of the nucleotide substrates; Hutton M et al.; The mechanism, by which transhydrogenase couples transfer of H- equivalents between NAD(H) and NADP(H) to the translocation of protons across a membrane, has been investigated in the solubilised, purified enzyme from Escherichia coli using analogues of the nucleotide substrates . The key observation was that, at low pH and ionic strength, solubilised transhydrogenase catalysed the very rapid reduction of acetylpyridine adenine dinucleotide (an analogue of NAD+) by NADH, but only in the presence of either NADP+ or NADPH . This indicates that the rates of release of NADP+ and NADPH from their binary complexes with the enzyme are slow . The dependences on pH and salt concentration suggest that (a) release of both NADP+ and NADPH are accompanied by the release of H+ from the enzyme and (b) increased ionic strength decreases the value of the pKa of the group responsible for H+ release . Modification of the enzyme with N,N1-dicyclohexylcarbodiimide led to inhibition of the rate of release of NADP+ and NADPH from the enzyme, but had a much smaller effect on the binding and release of NAD+, NADH and their analogues and on the interconversion of the ternary complexes of the enzyme with its substrates . It is considered that the binding and release of H+, which accompany the binding and release of NADP+/NADPH, might be central to the mechanism of proton translocation by the enzyme in its membrane-bound state. Mol Gen Genet, 1994 Feb, 242(3), 363 - 4 Location of the ompT gene relative to that of appY on the physical map of the Escherichia coli chromosome; Holmes Z et al.; Results concerning the precise location of the ompT gene (encoding the outer membrane protease OmpT) on the Escherichia coli chromosome were obtained which disagree with published restriction sites in the gene . It is shown that the gene, together with appY, is present on a 3.075 PstI fragment, encompassing positions 596-598 of the E . coli physical map. Mol Gen Genet, 1994 Feb, 242(3), 280 - 8 A reproducible method for identification of human genomic DNA autonomously replicating sequences; Nielsen T et al.; We demonstrate a method for the isolation of autonomously replicating sequences from pools of clones obtained from genomic DNA libraries constructed using affinity purification of cruciform DNA . The selection of autonomously replicating sequences was based on their differential ability to replicate as episomes after transfection of pools of plasmid clones into human HeLa cells . Two separate libraries containing affinity-purified cruciform DNA were used, one prepared from DNA of log phase primary human genital fibroblasts and the other prepared from DNA of log phase SW48 colon adenocarcinoma cells . Representative samples of the entire phage libraries were converted to phagemid clones by filamentous helper phage-mediated mass excision to produce pBluescript libraries in Escherichia coli . Clones were grown up individually and the bacteria pooled into groups of 48 for recovery of plasmid DNA . Plasmid pools of 48 independent clones (120 micrograms total) were then transfected by calcium phosphate coprecipitation onto log phase HeLa cells, which were allowed to grow for 3 days before recovery of plasmid by Hirt lysis . The recovery of plasmid from each transfection was estimated to range from 10 to 60 ng . DpnI digestion was then used to digest plasmids which had not been replicated and therefore retained a bacterial methylation pattern which was sensitive to digestion . We estimated from agarose electrophoresis gels that 40-200 pg of recovered plasmid DNA per transfected pool of DNA was resistant to DpnI and therefore was capable of transforming competent E . coli cells . The DpnI-resistant fraction yielded from one to seven independent clones from each pool, with genomic DNA inserts ranging in size from 0.35 to 3.4 kb.(ABSTRACT TRUNCATED AT 250 WORDS) Mol Gen Genet, 1994 Feb, 242(3), 241 - 9 The fadD gene of Escherichia coli K12 is located close to rnd at 39.6 min of the chromosomal map and is a new member of the AMP-binding protein family; Fulda M et al.; The fadD gene of Escherichia coli K12 was cloned and sequenced . The gene was identified by its ability to complement the corresponding mutant and by measuring the enzymatic activity after its expression in this mutant . The deduced polypeptide sequence exhibits similarity to other long chain acyl-CoA (coenzyme A) synthetases and a variety of other proteins, which together form a family of AMP-binding proteins . This family is extended by several new members and subdivided into four groups . fadD is assigned to a subgroup that does not include long chain acyl-CoA synthetases from eukaryotic organisms. J Med Microbiol, 1994 Feb, 40(2), 95 - 7 Adherence of non-enteropathogenic Escherichia coli to HeLa cells; Bouzari S et al.; Three hundred and nine strains of Escherichia coli isolated from infants and children with diarrhoea but not belonging to any recognised classes of diarrhoeagenic E . coli were investigated for their ability to adhere to HeLa cells in the presence of D-mannose . An enteroadherent-aggregative pattern (EAgg) was observed in 32.03%, localised adherence (LA) in 4.5%, diffuse adherence (DA) in 5.8%, and LA/DA and EAgg/LA in 1.9% and 1.2% of the isolates respectively . The results obtained with 100 control isolates were: EAgg 17%, LA 2%, DA 2%, LA/DA 2%, EAgg/LA 6% and DA/EAgg 1% . No adherence was manifested by 168 (54.36%) of 309 diarrhoeal isolates and 70% of the 100 control isolates . The results of this study showed that amongst non-enteropathogenic E . coli, strains exhibiting the EAgg pattern are significantly associated with diarrhoea (p < 0.005) . Most of these strains showed a pattern of multiple drug resistance. J Bacteriol, 1994 Feb, 176(4), 992 - 8 Influence of translational efficiency on the stability of the mRNA for ribosomal protein S20 in Escherichia coli; Rapaport LR et al.; A set of plasmids was constructed so as to contain point mutations which limit the efficiency and/or extent of translation of the gene for ribosomal protein S20 . These plasmids were transformed into strains carrying mutations in the genes for polynucleotide phosphorylase (pnp-7), RNase E (rne-1), or both . Subsequently, the effect of translational efficiency on mRNA abundance and chemical half-life was determined . The data indicated that mutations altering translational efficiency also affected mRNA levels over a 10-fold range . This variation in mRNA abundance occurred independently of mutations in either RNase E or polynucleotide phosphorylase, both of which determine the stability of the S20 mRNAs . Moreover, a mutation at codon 15 which caused premature termination of translation of the S20 mRNA did not significantly reduce its stability in different genetic backgrounds . We propose a model in which initiation of translation competes for early steps in mRNA turnover, which could be the binding of RNase E itself or as a complex to one or more sites near the 5' terminus of the S20 mRNA. J Bacteriol, 1994 Feb, 176(4), 948 - 52 Translocation of an outer membrane protein into prey cytoplasmic membranes by bdellovibrios; Tudor JJ et al.; Within minutes of Bdellovibrio bacteriovorus attack on prey cells, such as Escherichia coli, the cytoplasmic membrane of the prey is altered . Sodium dodecyl sulfate-polyacrylamide gel electrophoresis of purified invaded prey cell (bdelloplast) membranes revealed the appearance of a noncytoplasmic membrane protein . This protein is not observed in preparations of noninvaded E . coli membranes and migrates in a manner similar to that of E . coli OmpF . Isoelectric focusing and two-dimensional gel electrophoresis of bdelloplast cytoplasmic membrane preparations also revealed the presence of a protein with electrophoretic properties similar to those of OmpF and the major Bdellovibrio outer membrane proteins . The protein appears in cytoplasmic membrane preparations within minutes of attack and persists throughout most of the intraperiplasmic developmental cycle . The appearance of this protein is consistent with our hypothesis that bdellovibrios translocate a pore protein into the bdelloplast cytoplasmic membrane to kill their prey and to gain access to the cytoplasmic contents for growth. J Bacteriol, 1994 Feb, 176(4), 1184 - 7 Cloning, sequencing, and expression in Escherichia coli of the gene encoding a 45-kilodalton protein, elongation factor Tu, from Chlamydia trachomatis serovar F; Zhang YX et al.; The gene encoding a 45-kDa protein (45K) of Chlamydia trachomatis serovar F was cloned, sequenced, and overexpressed in Escherichia coli . Alignment of the deduced peptide sequence with E . coli elongation factor Tu (EF-Tu) demonstrated 69% identity . The 45K was recognized by a Chlamydia genus-specific monoclonal antibody GP-45 and cross-reacted with a monospecific polyclonal antibody to E . coli EF-Tu . Purified recombinant 45K has the capability to bind GDP, and the binding was enhanced in the presence of E . coli elongation factor Ts (EF-Ts) . The GDP binding was specifically inhibited by the monoclonal antibody GP-45 . These data suggest that the 45K is a chlamydial EF-Tu, and it forms a functional complex with E . coli EF-Ts protein. J Bacteriol, 1994 Feb, 176(4), 1157 - 63 Transmembrane signalling by a hybrid protein: communication from the domain of chemoreceptor Trg that recognizes sugar-binding proteins to the kinase/phosphatase domain of osmosensor EnvZ; Baumgartner JW et al.; Chemoreceptor Trg and osmosensor EnvZ of Escherichia coli share a common transmembrane organization but have essentially unrelated primary structures . We created a hybrid gene coding for a protein in which Trg contributed its periplasmic and transmembrane domains as well as a short cytoplasmic segment and EnvZ contributed its cytoplasmic kinase/phosphatase domain . Trz1 transduced recognition of sugar-occupied, ribose-binding protein by its periplasmic domain into activation of its cytoplasmic kinase/phosphatase domain as assessed in vivo by using an ompC-lacZ fusion gene . Functional coupling of sugar-binding protein recognition to kinase/phosphatase activity indicates shared features of intramolecular signalling in the two parent proteins . In combination with previous documentation of transduction of aspartate recognition by an analogous fusion protein created from chemoreceptor Tar and EnvZ, the data indicate a common mechanism of transmembrane signal transduction by chemoreceptors and EnvZ . Signalling through the fusion proteins implies functional interaction between heterologous domains, but the minimal sequence identity among relevant segments of EnvZ, Tar, and Trg indicates that the link does not require extensive, specific interactions among side chains . The few positions of identity in those three sequences cluster in transmembrane segment 1 and the short chemoreceptor sequence in the cytoplasmic part of the hybrid proteins . These regions may be particularly important in physical and functional coupling . The specific cellular conditions necessary to observe ligand-dependent activation of Trz1 can be understood in the context of the importance of phosphatase control in EnvZ signalling and limitations on maximal receptor occupancy in binding protein-mediated recognition. J Bacteriol, 1994 Feb, 176(4), 1121 - 7 Cloning of a gene involved in rRNA precursor processing and 23S rRNA cleavage in Rhodobacter capsulatus; Kordes E et al.; In Rhodobacter capsulatus wild-type strains, the 23S rRNA is cleaved into {16S} and {14S} rRNA molecules . Our data show that a region predicted to form a hairpin-loop structure is removed from the 23S rRNA during this processing step . We have analyzed the processing of rRNA in the wild type and in the mutant strain Fm65, which does not cleave the 23S rRNA . In addition to the lack of 23S rRNA processing, strain Fm65 shows impeded processing of a larger 5.6-kb rRNA precursor and slow maturation of 23S and 16S rRNAs from pre-23S and pre-16S rRNA species . Similar effects have also been described previously for Escherichia coli RNase III mutants . Processing of the 5.6-kb precursor was independent of protein synthesis, while the cleavage of 23S rRNA to generate 16S and 14S rRNA required protein synthesis . We identified a DNA fragment of the wild-type R . capsulatus chromosome that conferred normal processing of 5.6-kb rRNA and 23S rRNA when it was expressed in strain Fm65. J Bacteriol, 1994 Feb, 176(4), 1037 - 46 Characterization of the genome region encoding an fdxH-type ferredoxin and a new 2{4Fe-4S} ferredoxin from the nonheterocystous, nitrogen-fixing cyanobacterium Plectonema boryanum PCC 73110; Schrautemeier B et al.; A genomic DNA region with four consecutive open reading frames, including an fdxH-type gene, has been sequenced and initially characterized for the nonheterocystous nitrogen-fixing cyanobacterium Plectonema boryanum PCC 73110 . The fdxH gene encodes a {2Fe-2S}-type ferredoxin, 98 amino acids in length, with a deduced molecular mass of 10.9 kDa . Conserved residues include two characteristic lysines at positions 10 and 11, shown recently to be important for interaction with nitrogenase reductase (S . Schmitz, B . Schrautermeier, and H . Bohme, Mol . Gen . Genet . 240:455-460, 1993) . The gene is transcribed only under anaerobic nitrogenase-inducing conditions, whereas the Plectonema petF gene, encoding a different (type 1) {2Fe-2S} ferredoxin, is only transcribed in cultures growing with combined nitrogen . The fdxH gene was expressed in Escherichia coli as a holoprotein . The purified protein was able to effectively donate electrons to cyanobacterial nitrogenase, whereas PetF from the same organism was not . The occurrence of FdxH in the nonheterocystous genus Plectonema demonstrates for the first time that FdxH-type ferredoxins are not exclusively expressed within heterocysts, as is true for cyanobacteria differentiating these cells for nitrogen fixation under aerobic growth conditions . Two open reading frames that precede fdxH have high similarity to those found at a corresponding location in Anabaena sp . strain PCC 7120 . In the latter organism, they are transcribed only under nitrogen-fixing conditions, but the functions of their gene products remain unclear (D . Borthakur, M . Basche, W . J . Buikema, P . B . Borthakur, and R . Haselkorn, Mol . Gen . Genet . 221:227-234, 1990) . An fdxB-type gene encoding a 2{4Fe-4S} ferredoxin not previously identified in cyanobacteria is located immediately downstream of fdxH in P . boryanum. J Bacteriol, 1994 Feb, 176(4), 1009 - 13 Regulation of Escherichia coli purA by purine repressor, one component of a dual control mechanism; He B et al.; Escherichia coli purA encodes adenylosuccinate synthetase, one of two enzymes required for synthesis of AMP from IMP . purA is subject to two- to threefold regulation by purR and about twofold regulation by a purR-independent mechanism . The 5'-flanking region of purA confers purR-dependent transcriptional regulation of purA but not the purR-independent regulation . Two operator sites in the 5'-flanking region which bind purine repressor in vitro and are required for in vivo regulation were identified . The purR-independent regulation may be posttranscriptional . It is now established that all transcription units involved in de novo synthesis of purine nucleotides, nine pur operons, as well as purR itself and guaBA, are subject to purR control. Res Microbiol, 1994 Feb, 145(2), 99 - 109 Synthesis of DnaK protein during the division cycle of Escherichia coli; Hupp TR et al.; DnaK protein is involved in the initiation of DNA synthesis from the Escherichia coli chromosome as well as from the replication origins of phage lambda and P1 . The synthesis of dnaK mRNA and protein has been reported to vary during the cell cycle of Caulobacter crescentus (Gomes et al., 1990) . We have measured the expression of DnaK protein during the E . coli division cycle using the membrane-elution method . Cells labelled with a radioactive amino acid at different times during the division cycle were analysed for radiolabelled DnaK protein by quantitative immunoprecipitation, gel electrophoresis and autoradiography . In contrast to reports of cell-cycle-specific synthesis of DnaK protein in C . crescentus, we find the synthesis of DnaK protein to be invariant during the E . coli division cycle . Its synthesis occurs exponentially, as does the synthesis of total cell protein. Res Microbiol, 1994 Feb, 145(2), 141 - 50 Differentiation of Escherichia coli strains using randomly amplified polymorphic DNA analysis; Cave H et al.; Fifty-nine Escherichia coli strains isolated from 54 unrelated patients over a six-year period, as well as the reference strain of the species, were studied by analysis of randomly amplified polymorphic DNA (RAPD) to assess the usefulness of this genotyping approach in molecular epidemiology . Using a 10-mer oligonucleotide primer, 28 different RAPD fingerprints were distinguished among the 60 strains previously delineated in 36 ribotypes by EcoRI and HindIII digests . The patterns were reproducible and stable after in vitro and in vivo studies . Some strains harbouring an identical ribotype exhibited distinct RAPD fingerprints . Thus, these data illustrate the usefulness of the association of two genetic markers in assessing the relationship between strains . Interestingly, among the RAPD patterns, several bands were present only in the highly virulent carboxylesterase type B2 strains, which are more homogeneous than the carboxylesterase type B1 strains . Because of its simplicity and rapidity, RAPD analysis appears to be a highly valuable tool for studying E . coli molecular epidemiology. Res Microbiol, 1994 Feb, 145(2), 111 - 20 Growth and division of Escherichia coli under microgravity conditions; Gasset G et al.; The growth rate in glucose minimal medium and time of entry into the stationary phase in pepton cultures were determined during the STS 42 mission of the space shuttle Discovery . Cells were cultured in plastic bags and growth was stopped at six different time points by lowering the temperature to 5 degrees C, and at a single time point, by formaldehyde fixation . Based on cell number determination, the doubling time calculated for the flight samples of glucose cells was shorter (46 min) than for the ground samples (59 min) . However, a larger cell size expected for more rapidly growing cells was not observed by volume measurements with the electronic particle counter, nor by electron microscopic measurement of cell dimensions . Only for cells fixed in flight was a larger cell length and percentage of constricted cells found . An optical density increase in the peptone cultures showed an earlier entry into the stationary phase in flight samples, but this could not be confirmed by viability counts . The single sample with cells fixed in flight showed properties indicative of growth stimulation . However, taking all observations together, we conclude that microgravity has no effect on the growth rate of exponentially growing Escherichia coli cells. Mol Cell Neurosci, 1994 Feb, 5(1), 87 - 93 Characterization of recombinant mouse tryptophan hydroxylase expressed in Escherichia coli; Park DH et al.; Recombinant mouse tryptophan hydroxylase (TPH) was expressed in large quantities in Escherichia coli strain MC 1061, using a bacterial expression vector, pKS, containing the full coding region of mouse TPH . Specific polyclonal antiserum to the subunit of the recombinant mouse TPH was produced in rabbit by injecting the TPH band cut from SDS-polyacrylamide slab gels . The resultant antiserum recognized a single identical protein band (MW = 54,000) from rat dorsal raphe area, pineal gland, and brain stem by Western blot analysis . The specific activity of recombinant mouse TPH obtained was equivalent to that of TPH purified from rat brain . The recombinant mouse TPH was stable for 3 days at 4 degrees C but lost 25% of the original activity for the same period at -20 degrees C . A serotonin concentration greater than 1 mM inhibited TPH activity under our assay conditions in a concentration-dependent fashion . The recombinant mouse TPH exhibited a charge isozyme corresponding to that of pineal gland TPH as applied to chromatofocusing column chromatography . Taken together, our results show that recombinant mouse TPH, expressed in large quantities in E . coli is not only enzymatically highly active but also shares many biochemical and immunochemical properties with native TPH. Immunobiology, 1994 Feb, 190(1-2), 53 - 66 Lipopeptide-polyoxyethylene conjugates as mitogens and adjuvants; Kleine B et al.; Two lipopeptide analogues of the Escherichia coli lipoprotein rendered water-soluble by polyoxyethylene were tested for mitogenicity in vitro in murine and human B lymphocytes and for adjuvant activity in vivo in mice . These highly amphiphilic lipopeptides retained the biological activity other lipopeptides usually exerted which supports the hypothesis of specific interactions of lipopeptides with membranes of reactive cells . The activation of human B lymphocytes by these lipopeptides was much less pronounced compared to that of murine cells . However, given in combination with anti-CD40 antibodies plus interleukin-4, human B lymphocytes could synergistically be stimulated to proliferate . As an adjuvant, the polyoxyethylene linked lipopeptides were almost as potent as Freund's adjuvants and other basic lipopeptides . Being water-soluble, these novel analogues are easy to apply and they are suitable for field studies as adjuvants when sonication can not usually be provided. J Protein Chem, 1994 Feb, 13(2), 217 - 25 Reconstitution of heterologous and chimeric casein kinase II with recombinant subunits from human and Drosophila: identification of species-specific differences in the beta subunit; Lin WJ et al.; Casein kinase II is composed of two catalytic (alpha) and two regulatory (beta) subunits, the amino acid sequences of the alpha and beta subunits are highly conserved between species . To examine whether heterologous casein kinase II could be formed, recombinant alpha and beta subunits from human and Drosophila were reconstituted from inclusion bodies . Casein kinase II containing either human alpha and Drosophila beta or Drosophila alpha and human beta subunits exhibited enzymatic properties similar to those of the homologous holoenzymes with regard to specific activity, salt optima, and autophosphorylation . However, renaturation and reconstitution of casein kinase II was dependent on the type of beta subunits and the redox conditions, with the Drosophila beta subunits requiring more reduced conditions . Chimeric beta subunits prepared from human and Drosophila cDNA revealed that the N-terminal region was responsible for the requirement for the reduced redox state during renaturation . The N-terminal region also affected solubility and electrophoretic mobility of the beta subunit. Microb Pathog, 1994 Feb, 16(2), 131 - 9 Colonization factor antigens (CFAs) of enterotoxigenic Escherichia coli can prime and boost immune responses against heterologous CFAs; Rudin A et al.; The fimbrial colonization factor antigen I (CFA/I) and coli surface antigen 4 (CS4) of enterotoxigenic Escherichia coli (ETEC) are antigenically different, but have closely related N-terminal amino acid sequences . We have studied the capacity of purified CFA/I and CS4 fimbriae respectively to prime and boost immune responses against the homologous and heterologous CFAs in parenterally immunized mice . Based on initial characterization of the kinetics of the primary and secondary CFA/l immune responses in serum as well as antibody-secreting cell (ASC) responses in spleen, two doses with different combinations of the purified CFAs were given 7 weeks apart . It was shown, using either assay, that CFA/l could both prime and boost immune responses against CS4, and, conversely, that CS4 could prime and boost immune responses against CFA/l . These findings suggest the presence of immunorecessive epitopes, or epitopes that are not surface-exposed on whole fimbriae, that are shared between CFA/l and CS4 and which can expand B-cell clones with specificity for heterologous fimbriae. S Afr Med J, 1994 Feb, 84(2), 85 - 7 Incidence of heat-labile enterotoxin-producing Escherichia coli detected by means of polymerase chain reaction amplification; Winterbach R et al.; Diarrhoea can be caused by many different organisms, some of which are notoriously difficult to identify . One of these is enterotoxin-producing Escherichia coli . Recently a new diagnostic technique that uses polymerase chain reaction DNA amplification was developed for detection of the 'A' subunit of the labile enterotoxin-producing E . coli gene . This technique was used to evaluate the incidence of heat-labile (LT+) enterotoxin-producing E . coli in the causation of diarrhoea . The results from this study showed that LT+ E . coli is a cause of diarrhoea in the western Cape and that 5.3% of non-diagnosed diarrhoea patients in Tygerberg Hospital were infected with this pathogen . This represented less than 1% of the total number of cases of diarrhoea investigated in this hospital . The peak coincides with the wetter months in this locality and the infection rate is lower than that reported in most other countries . Given the low incidence of occurrence of this organism we do not recommend routine implementation of the diagnostic procedure . However, this test may be useful at times, e.g . to ascertain the source of a diarrhoea epidemic. J Comp Pathol, 1994 Feb, 110(2), 103 - 15 Gross and histopathological study of endotoxin-induced hoof lesions in cattle; Singh SS et al.; In young cattle infusion of Escherichia coli endotoxin resulted in sole and wall haemorrhages that were more severe in the hind than in the fore feet . Histopathological examination of biopsies taken 5 days after infusion showed separation at the tips of the laminae, changes in the vascularity of the dermal papillae and laminae, lightly stained material in the sole horn and degeneration of the epidermal cells of the laminae and papillae . Later, densely stained material in the tubules of the sole, arteriosclerosis in all parts of the corium and proliferative changes in the laminae were seen . It is concluded that endotoxin can induce diffuse aseptic pododermatitis in cattle, characterized by initial degenerative changes in the papillae and laminae followed by proliferative changes in the laminae and arteriosclerosis in all parts of the corium. J Interferon Res, 1994 Feb, 14(1), 41 - 6 Construction and activity of phosphorylatable human interferon-alpha B2 and interferon-alpha A/D; Wang P et al.; The polymerase chain reaction (PCR) was used to introduce a phosphorylation site into human interferon-alpha B2 (Hu-IFN-alpha B2) and the chimeric human interferon-alpha A/D (Hu-IFN-alpha A/D) . The phosphorylation sites were created by adding an amino acid consensus sequence for phosphorylation by the cAMP-dependent protein kinase to the carboxyl termini of the IFNs . The resultant modified IFNs (Hu-IFN-alpha B2-P and Hu-IFN-alpha A/D-P) were expressed in Escherichia coli and purified . The purified proteins exhibited antiviral activities similar to that of unmodified Hu-IFN-alpha B2 and Hu-IFN-alpha A/D . The Hu-IFN-alpha B2-P and Hu-IFN-alpha A/D-P can be phosphorylated by the catalytic subunit of the cAMP-dependent protein kinase and {gamma-32P}ATP with retention of biological activities . The introduction of phosphorylation sites into Hu-IFN-alpha B2 and Hu-IFN-alpha A/D provides new reagents for studies of receptor binding, pharmacokinetics, and other studies where labeled IFNs are useful. J Bioenerg Biomembr, 1994 Feb, 26(1), 67 - 88 Structure-function studies of {2Fe-2S} ferredoxins; Holden HM et al.; The ability to overexpress {2Fe-2S} ferredoxins in Escherichia coli has opened up exciting research opportunities . High-resolution x-ray structures have been determined for the wild-type ferredoxins produced by the vegatative and heterocyst forms of Anabaena strain 7120 (in their oxidized states), and these have been compared to structural information derived from multidimensional, multinuclear NMR spectroscopy . The electron delocalization in in these proteins in their oxidized and reduced states has been studied by 1H, 2H, 13C, and 15N NMR spectroscopy . Site-directed mutagenesis has been used to prepare variants of these ferredoxins . Mutants (over 50) of the vegetative ferredoxin have been designed to explore questions about cluster assembly and stabilization and to determine which residues are important for recognition and electron transfer to the redox partner Anabaena ferredoxin reductase . The results have shown that serine can replace cysteine at each of the four cluster attachment sites and still support cluster assembly . Electron transfer has been demonstrated with three of the four mutants . Although these mutants are less stable than the wild-type ferredoxin, it has been possible to determine the x-ray structure of one (C49S) and to characterize all four by EPR and NMR . Mutagenesis has identified residues 65 and 94 of the vegetative ferredoxin as crucial to interaction with the reductase . Three-dimensional models have been obtained by x-ray diffraction analysis for several additional mutants: T48S, A50V, E94K (four orders of magnitude less active than wild type in functional assays), and A43S/A45S/T48S/A50N (quadruple mutant). Arzneimittelforschung, 1994 Feb, 44(2A), 251 - 3 Mutagenicity study of the new cognition-enhancing agent nefiracetam; Shimada H et al.; A new cognition-enhancing agent, nefiracetam (N-(2,6-dimethylphenyl)-2- (2-oxo-1-pyrrolidinyl) acetamide, DM-9384, CAS 77191-36-7) was studied for mutagenicity by using the following short-term in vitro and in vivo tests: 1 . reverse mutation test (Ames method) on S . typhimurium and E . coli, 2 . cytogenetic test on Chinese hamster cells, and 3 . mouse micronucleus test . In the cytogenetic study, nefiracetam caused a slight but significant increase of chromosomal aberration at the highest dose in the 48 h treatment group, but no mutagenicity was observed with the same indicator in the in vivo micronucleus test . Furthermore, nefiracetam did not show any positive response in the reverse mutation test . These results suggest that nefiracetam has no biologically significant respectively relevant mutagenic potential. Parasite Immunol, 1994 Feb, 16(2), 63 - 7 Immunization against malaria with a recombinant protein; Ling IT et al.; We have expressed in bacteria the C-terminal part of Plasmodium yoelii merozoite surface protein-1 (MSP1) containing the two epidermal growth factor-like domains . The protein, either alone or fused to glutathione S-transferase, was highly effective as a vaccine and protected mice against challenge infection . Reduction and alkylation abolished the protection obtained with the protein . This shows for the first time the absolute requirement of the disulphide-bonded conformation for immunogenicity . In a short term experiment, mice were protected against a massive challenge . The immunity was effective at the time of merozoite release/reinvasion . Recombinant protein based on this part of MSP1 may be suitable as a vaccine against malaria. Mol Biochem Parasitol, 1994 Feb, 63(2), 265 - 73 Purification and characterization of dihydrofolate reductase of Plasmodium falciparum expressed by a synthetic gene in Escherichia coli; Sano G et al.; We have expressed the dihydrofolate reductase (DHFR) part of the DHFR-thymidylate synthetase complex of P . falciparum in Escherichia coli, by constructing a gene with synthetic oligonucleotides that changed the gene's codon usages . The induced expression in an E . coli cell of the synthetic gene yielded a product that constituted about 30% of the total bacterial protein . The product was precipitated in an inclusion body in a cell . Its enzymatic activity was restored after denaturation and renaturation procedures with guanidine-HCl . Recombinant DHFRs with Ser or Thr at position 108 were prepared . Kinetic characterization showed that the DHFRSer108 has less of an affinity for NADPH and dihydrofolate than the DHFRThr108. Mol Biochem Parasitol, 1994 Feb, 63(2), 221 - 9 Isolation, sequencing and expression of the gene encoding hypoxanthine-guanine-xanthine phosphoribosyltransferase of Tritrichomonas foetus; Chin MS et al.; We have cloned and expressed the full-length gene encoding the hypoxanthine-guanine-xanthine phosphoribosyltransferase (HGXPRTase) from the anaerobic protozoan parasite Tritrichomonas foetus . This enzyme is essential in nucleic acid metabolism of T . foetus because the parasite is unable to synthesize purine nucleotides de novo and relies on the HGXPRTase activities for its purine requirements . Initially, a cDNA clone encoding part of the HGXPRTase was isolated by complementation of an Escherichia coli mutant, SO609, with a cDNA library of T . foetus . Northern blot analysis identified a single mRNA band of approximately 700-800 bases . The full-length genomic clone was then isolated and identified to have an open reading frame of 549 bp encoding an 183-amino acid sequence with an estimated size of 21.1 kDa . The sequence is only 27.3% identical to that of the human HGPRTase . The T . foetus HGXPRTase gene was subsequently cloned into the pBAce vector for expression in E . coli . This construct yields completely soluble and enzymatically active recombinant T . foetus HGXPRTase, which constitutes approximately 20% of the total cellular protein of the transformed E . coli . It has the same molecular weight as the authentic native enzyme, and the N-terminal amino acid sequence of the recombinant enzyme is identical to that predicted from the open reading frame . The high expression of this apparently native T . foetus HGXPRTase will provide large quantities of purified protein, necessary for detailed kinetic and structural analysis of this enzyme for its potential value as a target for antitrichomonial chemotherapy . To our knowledge, this is also the first time a gene from T . foetus was cloned and expressed. Free Radic Biol Med, 1994 Feb, 16(2), 177 - 85 New roles for quin2: powerful transition-metal ion chelator that inhibits copper-, but potentiates iron-driven, Fenton-type reactions; Sandstrom BE et al.; The objective of this study was to investigate whether quin2, through its metal chelating properties, could affect copper- or iron-driven Fenton reactions . Chelation of ferric ion with quin2 uniformly strongly enhanced the formation of oxidizing species, detected with the DMSO and deoxyribose assays, both by H2O2 and a mixture of superoxide/hydrogen peroxide produced by hypoxanthine/xanthine oxidase . Fe(3+)-EDTA gave the same effects, but lacked reactivity with bolus H2O2 as detected with the DMSO assay . Whereas the formation of oxidizing species with Fe(3+)-EDTA and ferric ions alone were strongly inhibited by superoxide dismutase both in the bolus H2O2 and hypoxanthine/xanthine oxidase systems, such formation in the presence of Fe(3+)-quin2 either did not decrease or decreased only moderately . Fe(3+)-quin2 also strongly enhanced plasmid DNA strand breakage in the presence of H2O2 . Our findings suggest that quin2 as chelator of ferric ion may be a more powerful enhancer of oxidant formation than other chelators so far tested . The formation of oxidizing species from copper ions and bolus H2O2 was found to be fundamentally dependent on the choice of buffer system . We could only detect significant amounts of oxidants in both assays in Hepes buffer, but not in the phosphate, cacodylate or unbuffered systems, which all gave low reactivity in the DMSO assay compared to the deoxyribose assay . Quin2 chelation of cupric ion effectively inhibited the formation of oxidants as well as plasmid DNA strand breakage.(ABSTRACT TRUNCATED AT 250 WORDS) Protein Sci, 1994 Feb, 3(2), 282 - 90 Enzyme IIBcellobiose of the phosphoenol-pyruvate-dependent phosphotransferase system of Escherichia coli: backbone assignment and secondary structure determined by three-dimensional NMR spectroscopy; Ab E et al.; The assignment of backbone resonances and the secondary structure determination of the Cys 10 Ser mutant of enzyme IIBcellobiose of the Escherichia coli cellobiose-specific phosphoenol-pyruvate-dependent phosphotransferase system are presented . The backbone resonances were assigned using 4 triple resonance experiments, the HNCA and HN(CO)CA experiments, correlating backbone 1H, 15N, and 13C alpha resonances, and the HN(CA)CO and HNCO experiments, correlating backbone 1H,15N and 13CO resonances . Heteronuclear 1H-NOE 1H-15N single quantum coherence (15N-NOESY-HSQC) spectroscopy and heteronuclear 1H total correlation 1H-15N single quantum coherence (15N-TOCSY-HSQC) spectroscopy were used to resolve ambiguities arising from overlapping 13C alpha and 13CO frequencies and to check the assignments from the triple resonance experiments . This procedure, together with a 3-dimensional 1H alpha-13C alpha-13CO experiment (COCAH), yielded the assignment for all observed backbone resonances . The secondary structure was determined using information both from the deviation of observed 1H alpha and 13C alpha chemical shifts from their random coil values and 1H-NOE information from the 15N-NOESY-HSQC . These data show that enzyme IIBcellobiose consists of a 4-stranded parallel beta-sheet and 5 alpha-helices . In the wild-type enzyme IIBcellobiose, the catalytic residue appears to be located at the end of a beta-strand. Protein Sci, 1994 Feb, 3(2), 240 - 7 Cysteine scanning mutagenesis of the N-terminal 32 amino acid residues in the lactose permease of Escherichia coli; Sahin-Toth M et al.; Using a functional lactose permease mutant devoid of Cys residues (C-less permease), each amino acid residue in the hydrophilic N-terminus and the first putative transmembrane helix was systematically replaced with Cys (from Tyr-2 to Trp-33) . Twenty-three of 32 mutants exhibit high lactose accumulation (70-100% or more of C-less), and an additional 8 mutants accumulate to lower but highly significant levels . Surprisingly, Cys replacement for Gly-24 or Tyr-26 yields fully active permease molecules, and permease with Cys in place of Pro-28 also exhibits significant transport activity, although previous mutagenesis studies on these residues suggested that they may be required for lactose transport . As expected, Cys replacement for Pro-31 completely inactivates, in agreement with previous findings indicating that "helix-breaking" propensity at this position is necessary for full activity (Consler TG, Tsolas O, Kaback HR, 1991, Biochemistry 30:1291-1297) . Twenty-nine mutants are present in the membrane in amounts comparable to C-less permease, whereas membrane levels of mutants Tyr-3-->Cys and Phe-12-->Cys are slightly reduced, as judged by immunological techniques . Dramatically, mutant Phe-9-->Cys is hardly detectable when expressed from the lac promoter/operator at a relatively low rate, but is present in the membrane in a stable form when expressed at a high rate from the T7 promoter . Finally, studies with N-ethylmalemide show that 6 Cys-replacement mutants that cluster at the C-terminal end of putative helix I are inactivated significantly.(ABSTRACT TRUNCATED AT 250 WORDS) Bioorg Med Chem, 1994 Feb, 2(2), 79 - 84 Expression of mouse Gal beta 1,4GlcNAc alpha 2,6-sialyltransferase in an insoluble form in Escherichia coli and partial renaturation; Hamamoto T et al.; Mouse Gal beta 1,4GlcNAc alpha 2,6-sialyltransferase was produced in an insoluble form in Escherichia coli cells harboring expression plasmids . The insoluble protein was solubilized with 8 M urea and diluted for renaturation of the enzyme . The substrate specificity and kinetic parameters, except for the specific activity, of the renatured enzyme were similar to those of the enzyme obtained from rat liver . These results suggest that a bacterial expression system is a potentially powerful tool for the large scale production of sialyltransferases and for elucidating the molecular mechanisms of sialyltransferases. Mol Microbiol, 1994 Feb, 11(4), 629 - 39 Isolation and characterization of the aspartokinase and aspartate semialdehyde dehydrogenase operon from mycobacteria; Cirillo JD et al.; Diaminopimelic acid (DAP) is a major component of the peptidoglycan layer of the mycobacterial cell wall . The mycobacterial cell wall has been implicated as a potential virulence factor and is highly immunogenic . The pathway for biosynthesis of DAP may serve as a target in the design of antimycobacterial agents and construction of in vivo selection systems . Despite its significance, this biosynthetic pathway is poorly understood in mycobacteria . In order to develop a better understanding of mycobacterial DAP biosynthesis, the aspartate semialdehyde dehydrogenase (asd) genes of Mycobacterium smegmatis, bacille Calmette-Guerin (BCG), Mycobacterium avium, Mycobacterium leprae, and Mycobacterium tuberculosis were isolated . The M . smegmatis asd gene was isolated by complementation in Escherichia coli . This gene was then used to isolate the asd genes from other mycobacteria . The asd-complementing fragments from BCG and M . smegmatis were sequenced . An open reading frame upstream of the mycobacterial asd gene was identified as the mycobacterial aspartokinase gene (ask) . Primer extension analysis revealed that the only transcriptional start in this region is found 5' of the ask gene . This observation indicates that the mycobacterial ask and asd genes are in an operon. Mol Microbiol, 1994 Feb, 11(4), 605 - 18 Leucine-responsive regulatory protein and deoxyadenosine methylase control the phase variation and expression of the sfa and daa pili operons in Escherichia coli; van der Woude MW et al.; The Escherichia coli operons daa and sfa encode F1845 and S pili, respectively . In this paper we show that the expression of these operons is under phase variation control at a transcriptional level . The transcription of both operons is dependent on the global regulator leucine-responsive regulatory protein (Lrp) and deoxyadenosine methylase (Dam) . Lrp is required for methylation protection of two GATC sites located within conserved DNA sequences in the regulatory regions of these operons . These GATC sites are differentially methylated, establishing a methylation pattern which is characteristic of either the phase ON or phase OFF state . We also show that Lrp binds to the daa and sfa regulatory regions and that this binding is modulated by the methylation of the GATC sites . These results indicate that the phase variation of the daa and sfa operons is regulated by a mechanism involving differential binding of Lrp owing to methylation of GATC sites in the regulatory region, which is similar to the mechanism that controls phase variation of the pap operon. Protein Expr Purif, 1994 Feb, 5(1), 50 - 6 Purification of human big endothelin 1 derived through cleavage with collagenase and dipeptidylpeptidase IV from a fusion protein expressed in Escherichia coli; Becker A et al.; The cDNA coding for human big endothelin 1 (bigET-1), preceded by an optimized collagenase recognition sequence and followed by a stop codon, was fused in frame to the C-terminal region of alkaline phosphatase (AP) . The fusion protein (AP-bigET), expressed in Escherichia coli K12 upon the lowering of organic phosphate concentrations, consisted of alkaline phosphatase (1-447), the collagenase cleavage site (Gly-Pro-Ala)4, and glycylprolyl-bigET-1 . AP-bigET accumulated intracellularly in the form of inclusion bodies that were extensively washed and finally extracted by 8 M urea to yield highly enriched AP-bigET . Upon digestion of the fusion protein with collagenase, two disulfide conformeres of glycylprolyl-bigET-1 (bigET-1A and bigET-1B) could be purified by reverse-phase FPLC . Upon treatment with dipeptidylpeptidase IV to remove the N-terminal glycylprolyl-dipeptide, the later-eluting form of bigET-1 (bigET-1B) coeluted with authentic human bigET-1 on reverse-phase HPLC . BigET-1A and bigET-1B were formed at a ratio of 1:3 . After reduction and S-pyridylethylation, both conformers coeluted with authentic but reduced bigET-1 . Their amino acid sequences were identical . Both forms were converted by digestion with pepsin to the respective ET-1 conformeres (ET-1A and ET-1B) that were purified . In vasoconstriction assays, ET-1B but not ET-1A, at 10(-8) M, evoked a maximal response indistinguishable from that of authentic ET-1. Mol Microbiol, 1994 Feb, 11(3), 555 - 66 Adhesin regulatory genes within large, unstable DNA regions of pathogenic Escherichia coli: cross-talk between different adhesin gene clusters; Morschhauser J et al.; The uropathogenic Escherichia coli strain 536 possesses two large, unstable DNA regions on its chromosome, which were termed pathogenicity islands (pais) . Deletions of pais, which occur with relatively high frequency in vitro and in vivo, lead to avirulent mutants . The genetic determinants for production of haemolysin (Hly) and P-related fimbriae (Prf) are located in one of these islands . Deletion of this pathogenicity island (paill) not only removes the hly- and prf-specific genes, but also represses S fimbriae (Sfa), although the sfa genes of this virulence factor are not located on paill . We have identified two regulatory genes, prfB and prfl, of the prf gene cluster that are homologous to the sfa regulatory genes sfaB and sfaC, respectively . Mutations in sfaB and sfaC that inhibit transcription of the major fimbrial subunit gene sfaA were complemented by the homologous prf genes, suggesting communication between the two fimbrial gene clusters in the wild-type strain . Chromosomal mutagenesis of the two prf regulators in strain 536 repressed transcription of sfaA, detected by Northern hybridization and a chromosomal sfaA-lacZ fusion . In addition, haemagglutination assays measured a lower level of S fimbriae in these mutants . Expression of the cloned prf regulators in trans reversed the effect of the mutations; furthermore, constitutive expression of prfB or prfl could also over-come the repression of S fimbriae in a strain that had lost the pathogenicity islands . Virulence assays in mice established that the prf mutants were less virulent than the wild-type strain . The results demonstrate that cross-regulation of two unlinked virulence gene clusters together with the co-ordinate loss of large DNA regions significantly influences the virulence of an extraintestinal E . coli wild-type isolate. J Clin Microbiol, 1994 Feb, 32(2), 510 - 4 Hemagglutinating properties of enteroaggregative Escherichia coli; Qadri F et al.; Many intestinal bacterial pathogens possess hemagglutinating properties, which are indicative of their adhesive properties to the intestinal mucosal surface . To understand the bacteria-mucosa interaction, 41 strains of enteroaggregative Escherichia coli (EAggEC), a recently described category of diarrheagenic E . coli, isolated mostly from children with diarrhea in Bangladesh, India, Thailand, Central America, and South America were screened for mannose-sensitive hemagglutination and mannose-resistant hemagglutination of erythrocytes from humans, rats, mice, sheep, cattle, and rabbits . Some strains demonstrated mannose-sensitive hemagglutination of erythrocytes . Most isolates showed mannose-resistant hemagglutination of erythrocytes from all species except rabbits . The hemagglutination patterns could be classified into 18 groups . Studies with three selected isolates suggested that hemagglutinins are cell bound and are protein in nature . On the basis of the pattern of inhibition of hemagglutination by various chemicals, 39 isolates were classified into 19 groups . Hemagglutinations of many isolates were inhibited by sialic acid-containing compounds, suggesting that these compounds may be the receptors for these organisms on erythrocytes and possibly on the intestinal mucosa . These data indicate that strains of EAggEC are a heterogeneous group of organisms with different types of hemagglutinins or adhesins for the intestinal mucosal surface . Also, the adhesion characteristics of EAggEC strains may be too complex to be assessed by simple hemagglutination tests. Genetics, 1994 Feb, 136(2), 439 - 48 DNA polymerase II of Escherichia coli in the bypass of abasic sites in vivo; Tessman I et al.; The function of DNA polymerase II of Escherichia coli is an old question . Any phenotypic character that Pol II may confer upon the cell has escaped detection since the polymerase was discovered 24 yr ago . Although it has been shown that Pol II enables DNA synthesis to proceed past abasic sites in vitro, no role is known for it in the bypass of those lesions in vivo . From a study of phage S13 single-stranded DNA, we now report SOS conditions under which Pol II is needed for DNA synthesis to proceed past abasic sites with 100% efficiency in vivo . Overproduction of the GroES+L+ heat shock proteins, which are members of a ubiquitous family of molecular chaperones, eliminated this requirement for Pol II, which may explain why the role of Pol II in SOS repair had eluded discovery . Mutagenesis accompanied SOS bypass of abasic sites when the original occupant had been cytosine but not when it had been thymine; the quantitative difference is shown to imply that adenine was inserted opposite the abasic sites at least 99.7% of the time, which is an especially strict application of the A-rule . Most, but not all, spontaneous mutations from Rifs to Rifr, whether in a recA+ or a recA(Prtc) cell, require Pol II; while this suggests that cryptic abasic lesions are a likely source of spontaneous mutations, it also shows that such lesions cannot be the exclusive source. Eur J Biochem, 1994 Feb 1, 219(3), 727 - 36 Characterization and partial purification of the human receptor for the heat-stable enterotoxin; Visweswariah SS et al.; The receptor for the Escherichia coli heat-stable enterotoxin has been characterized and partially purified from the T84 human colonic cell line . Using a novel mutant heat-stable enterotoxin peptide as a radioligand (the C-terminal tyrosine residue is replaced by phenylalanine in the mutant), a single class of high-affinity receptor sites was detected in T84 cells, with a Kd of 0.1 nM, similar in affinity to the receptor described in human intestinal tissue . The receptor was solubilised from T84 cell membranes and affinity cross-linking of the solubilised preparation indicated that a single species of M(r) 160,000 served as the receptor . Freshly solubilised preparations of the receptor retained heat-stable enterotoxin-activable guanylyl cyclase activity . Purification of the receptor was achieved through sequential affinity chromatography on GTP--epoxy-Sepharose and wheat-germ-agglutinin columns resulting in purification of the receptor by 3000 fold . The heat-stable enterotoxin-binding characteristics of the receptor were unchanged during the purification and silver staining of the purified receptor preparation indicated a band of M(r) 160,000, which was specifically cross-linked to the 125I-labeled mutant peptide . The purified receptor retained guanylyl cyclase activity, but the activity was not stimulated on addition of human heat-stable enterotoxin, suggesting that accessory structural factors may be involved in the activation of the guanylyl cyclase/receptor. J Bacteriol, 1994 Feb, 176(4), 1099 - 110 Permissive linker insertion sites in the outer membrane protein of 987P fimbriae of Escherichia coli; Schifferli DM et al.; The FasD protein is essential for the biogenesis of 987P fimbriae of Escherichia coli . In this study, subcellular fractionation was used to demonstrate that FasD is an outer membrane protein . In addition, the accessibility of FasD to proteases established the presence of surface-exposed FasD domains on both sides of the outer membrane . The fasD gene was sequenced, and the deduced amino acid sequence was shown to share homologous domains with a family of outer membrane proteins from various fimbrial systems . Similar to porins, fimbrial outer membrane proteins are relatively polar, lack typical hydrophobic membrane-spanning domains, and posses secondary structures predicted to be rich in turns and amphipathic beta-sheets . On the basis of the experimental data and structural predictions, FasD is postulated to consist essentially of surface-exposed turns and loops and membrane-spanning interacting amphipathic beta-strands . In an attempt to test this prediction, the fasD gene was submitted to random in-frame linker insertion mutagenesis . Preliminary experiments demonstrated that it was possible to produce fasD mutants, whose products remain functional for fimbrial export and assembly . Subsequently, 11 fasD alleles, containing linker inserts encoding beta-turn-inducing residues, were shown to express functional proteins . The insertion sites were designated permissive sites . The inserts used are expected to be least detrimental to the function of FasD when they are inserted into surface-exposed domains not directly involved in fimbrial export . In contrast, FasD is not expected to accommodate such residues in its amphipathic beta-strands without being destabilized in the membrane and losing function . All permissive sites were sequenced and shown to be located in or one residue away from predicted turns . In contrast, 5 of 10 sequenced nonpermissive sites were mapped to predicted amphipathic beta-strands . These results are consistent with the structural predictions for FasD. Proc Natl Acad Sci U S A, 1994 Feb 1, 91(3), 883 - 7 Chimeric restriction endonuclease; Kim YG et al.; Fok I restriction endonuclease recognizes the nonpalindromic pentadeoxyribonucleotide 5'-GGATG-3'.5'-CATCC-3' in duplex DNA and cleaves 9 and 13 nt away from the recognition site . Recently, we reported the presence of two distinct and separable domains within this enzyme: one for the sequence-specific recognition of DNA (the DNA-binding domain) and the other for the endonuclease activity (the cleavage domain) . Here, we report the construction of a chimeric restriction endonuclease by linking the Drosophila Ultrabithorax homeodomain to the cleavage domain (FN) of Fok I restriction endonuclease . The hybrid enzyme, Ubx-FN, was purified, and its cleavage properties were characterized . The hybrid enzyme shows the same DNA sequence-binding preference as that of Ubx; as expected, it cleaves the DNA away from the recognition site . On the 5'-TTAATGGTT-3' strand the hybrid enzyme cleaves 3 nt away from the recognition site, whereas it cuts the complementary 5'-AACCATTAA-3' strand 8, 9, or 10 nt away from the binding site . Similarly engineered hybrid enzymes could be valuable tools in physical mapping and sequencing of large eukaryotic genomes. J Immunol, 1994 Feb 1, 152(3), 1171 - 81 Mechanism of differential regulation of IL-2 in murine Th1 and Th2 T cell subsets . 1 . Induction of IL-2 transcription in Th2 cells by up-regulation of transcription factors with the protein synthesis initiation factor 4E; Barve SS et al.; Regulation of IL-2 gene expression in response to receptor-mediated stimuli is known to be mediated primarily by the IL-2 transcriptional enhancer and multiple transcription factors . However, the mechanism that controls the differential expression of the IL-2 gene in both human and murine CD4+ Th cell subsets (Th1-IL-2+ and Th2-IL-2-) is not clearly understood . Differential IL-2 gene expression was assessed in murine Th1 and Th2 subsets by analyzing the expression of a Escherichia coli lacZ reporter gene under control of the human IL-2 enhancer (IL2ZH) transfected in both T cell subsets . Stimulation of transfected T cells with the mitogen Con A, anti-CD3 Ab, or PMA plus ionomycin activated the IL2ZH construct in Th1 but not Th2 cells . However, IL2ZH was activated in stimulated Th2 cells that were co-transfected with a vector that overexpressed the eukaryotic initiation factor 4E (eIF-4E) . It has been shown that eIF-4E is rate limiting for protein synthesis and its overexpression leads to increased rates of protein synthesis . Hence, eIF-4E overexpression could have overcome a deficiency in transcriptionally active levels of IL-2 regulatory factors in Th2 cells leading to IL-2 enhancer activation . This possibility was supported by demonstrating that transcriptionally active levels of the critical IL-2 transcription factor, nuclear factor of activated T cells (NF-AT), occurred only in Th2 cells overexpressing eIF-4E but not in normal Th2 cells, thus indicating that the inability of Th2 cells to express IL-2 was associated with inadequate levels of at least one transcription factor, NF-AT . Moreover, these results were confirmed by the observation that eIF-4E overexpression augmented NF-AT binding activity in Th2 cells . These data suggest that concentrations of inducible transcription factors are a major component of the regulatory mechanisms dictating IL-2 expression and may be under translational control in Th1/Th2 T cell subsets. Exp Parasitol, 1994 Feb, 78(1), 101 - 12 Cloning, sequencing, and structural analysis of the DNA encoding inosine monophosphate dehydrogenase (EC 1.1.1.205) from Tritrichomonas foetus; Beck JT et al.; The inosine monophosphate dehydrogenase (IMPDH) of the parasitic protozoan Tritrichomonas foetus is a purine salvage enzyme with a subunit molecular weight of 58,000 . The enzyme has been purified to homogeneity by Verham et al . (Molecular and Biochemical Parasitology 24, 1-12, 1987) and characterized in more detail by Hedstrom and Wang (Biochemistry 29, 849-854, 1990) . We used a polyclonal antibody directed against the purified enzyme to identify three cDNA clones from T . foetus . These clones were sequenced and found to contain an open reading frame encoding 497 amino acids . By complementation studies on an Escherichia coli mutant with its IMPDH gene deleted, the cDNA clones were able to transform the bacterial cells to grow on minimal medium without guanine . One of the cDNA clones, 2aa1, was used to identify two genomic clones, 2d1c and 3m4b, both containing a 4.1-kb HindIII fragment . The fragment was subcloned into the Bluescript KS+ plasmid, sequenced, and found to contain the same open reading frame as the cDNA clone except that it encodes six additional amino acid residues at the N-terminus . Its sequence has a 34% identity with that of the human IMPDH, 32% with that of E . coli IMPDH, and 31% with that of Leishmania donovani IMPDH . The molecular weight of the deduced protein is 55,534 . Two segments of polypeptide that are conserved in all other IMPDHs, containing the putative NAD+ and IMP binding sites, are also relatively conserved in T . foetus . Since the parasite enzyme differs from the bacterial and mammalian IMPDHs by a very high Km value for NAD+ and an even higher KI value for mycophenolic acid (MPA) (Verham et al . 1987; Hedstrom and Wang 1990), the sequence of the parasite enzyme may provide information on the mechanism of MPA binding and the chance for other specific inhibitor design. Mol Cell Biol, 1994 Feb, 14(2), 1510 - 9 Cloning and characterization of centromeric DNA from Neurospora crassa; Centola M et al.; The centromere locus from linkage group VII of Neurospora crassa has been cloned, characterized, and physically mapped . The centromeric DNA is contained within a 450-kb region that is recombination deficient, A+T-rich, and contains repetitive sequences . Repetitive sequences from within this region hybridize to a family of repeats located at or near centromeres in all seven linkage groups of N . crassa . Genomic Southern blots and sequence analysis of these repeats revealed a unique centromere structure containing a divergent family of centromere-specific repeats . The predominantly transitional differences between copies of the centromere-specific sequence repeats and their high A+T content suggest that their divergence was mediated by repeat-induced point (RIP) mutations. Bioseparation, 1994 Feb, 4(1), 39 - 61 Protein inactivations during novel bioseparation techniques; Sadana A; An analysis is presented for the quantitative and qualitative inactivation of proteins and other bioproducts during their separation utilizing the reverse micellar and the aqueous two-phase extraction techniques . Information on the influence of different parameters on the quantitative yields of the bioproducts separated is available, and more information is being gathered to provide further physical insights into improving the quantitative yields . However, very little information is available on the qualitative nature of the bioproducts separated utilizing either the reverse micelle or the aqueous two-phase extraction technique . More information is definitely required on the qualitative nature of the bioproducts separated by the above techniques to assist in their proper evaluation as effective bioseparation techniques. Biotechnology (N Y), 1994 Feb, 12(2), 178 - 80 Increasing the efficiency of protein export in Escherichia coli; Perez-Perez J et al.; Export of recombinant proteins to the periplasm of Escherichia coli is in many cases preferable to cytoplasmic production . However, when the protein is overexpressed, export efficiency decreases significantly and some advantages of the system are lost . This is what happens when attempting to produce recombinant human interleukin-6 (hIL-6) as a pre(OmpA) fusion in E . coli . Assuming that the host protein export machinery becomes overloaded, we have tested the effect of providing the host with additional copies of two key components of that machinery . Supplementation with a plasmid bearing prlA4 (secY allele) and secE genes increased the ratio of mature to precursor hIL-6 from 1.2 to 10.8 . The increase in processing ratio was associated with the accumulation of a larger amount of total (mature plus precursor forms) hIL-6 . Providing a plasmid-borne wild-type prlA was ineffective compared to prlA4 allele . This suggests that the PrlA protein, a component of the translocator, recognizes features at the mature portion of secretory substrates independently of those at the signal peptide portion. Shock, 1994 Feb, 1(2), 153 - 7 The role of histamine in the increased cardiac output in hyperdynamic endotoxemia; Tarnoky K et al.; The role of histamine in the hyperdynamic circulatory response to endotoxin (ETX) was investigated in 32 anesthetized dogs by means of histamine H1- and H2-receptor blockade . A hyperdynamic circulation was elicited with a prolonged, slow infusion of a low dose of ETX, and hemodynamic parameters were examined in control and histamine receptor-blocked groups . The following groups were studied: Group ETX received a 2 h infusion of Escherichia coli 055:B5 endotoxin in a total dose of 13.75 micrograms/kg at a rate of 10 micrograms/kg for 45 min and then 5 micrograms/kg for 75 min . In addition to the same dose of ETX, Groups ETX+TPA and ETX+RAN received 0.5 mg/kg of the H1-blocker tripelennamine (TPA) or 2 mg/kg of the H2-blocker ranitidine (RAN), respectively . Infusion of ETX caused a moderate decrease in arterial pressure in Group ETX, whereas TPA but not RAN inhibited this pressure fall . The cardiac output (CO) increased by 41% above the baseline level in Group ETX . Both TPA and RAN prevented this rise in CO . The total peripheral resistance was considerably lowered by ETX, but this decrease was significantly attenuated in the TPA or RAN-treated groups . The heart rate rose significantly after ETX infusion and was unaffected by TPA or RAN . The stroke volume remained unchanged following ETX but was decreased both by TPA and by RAN . TPA or RAN, when given alone, did not affect any of the measured hemodynamic parameters . These experiments provide evidence of the participation of histamine in the hyperdynamic circulatory response in endotoxemia. Shock, 1994 Feb, 1(2), 135 - 40 Dose-related pattern of sinusoidal leukocyte adhesion in sublobular regions of the liver after systemic endotoxin challenge in the rat; Bauer M et al.; The unique arrangement of large numbers of fixed tissue macrophages, endothelial, and parenchymal cells along hepatic sinusoids as well as their key role in the acute phase response makes the liver a primary target organ in endotoxemia . Pathogenesis of hepatic failure in endotoxemia is incompletely understood, but microcirculatory failure as well as leukocyte-endothelial interaction in response to inflammatory mediators may relate to it . Using intravital fluorescence microscopy, sinusoidal widths, leukocyte flow velocity, and sublobular leukocyte (white blood cell (WBC)) adhesion characteristics 1 h after randomized intravenous application of 0, 0.1, 1, or 5 mg/kg b.w . Escherichia coli endotoxin O 111 B4 (ETX) were evaluated in female Sprague-Dawley rats (n = 6-8/group) . Whereas the bolus injection of ETX caused only minor concurrent macrohemodynamic effects, a significant increase of permanent WBC adhesion especially in periportal areas (0 mg, 2.1 +/- 0.7%; 0.1 mg, 16.2 +/- 3.6%**; 1 mg, 15.5 +/- 1.0%**; 5 mg, 13.2 +/- 2.3%* (**p < .01, *p < .05, compared to vehicle)) was found in all groups 60 min after ETX challenge . In contrast, an increase of WBC margination in midzonal and pericentral regions was only seen with the higher doses of 1 or 5 mg/kg ETX, respectively . The sublobular pattern of WBC margination is consistent with the concept of regulation of WBC adhesion by inflammatory mediators released by lipopolysaccharide-stimulated Kupffer cells in vivo . We propose that overwhelming the detoxifying capacity of predominantly periportally located Kupffer cells with large amounts of ETX may lead to activation of pericentral-located resting macrophages paralleled by a rise of adhering leukocytes. Shock, 1994 Feb, 1(2), 115 - 22 The influence of sympathoadrenal activation on skeletal muscle oxygen extraction during endotoxemia; Hershey JC et al.; We have previously shown a direct relationship (r = .97) between the fall in arterial blood pressure and the increase in skeletal muscle oxygen extraction (MVO2) during canine endotoxemia . Since it is well known that hypotension activates the sympathetic system, the primary aim of these experiments was to determine if the increase in MVO2 during endotoxemia is a result of elevated levels of catecholamines due to increased sympathetic neural and/or humoral activity (sympathoadrenal system) . Canine gracilis muscles were vascularly isolated and perfused in situ at a constant flow (6-7 ml/min/100 g) . Endotoxemia was induced by a 30 min intravenous infusion of Escherichia coli endotoxin (2 mg/kg), which induced a 50% reduction in arterial pressure . Perfusion pressure, mean arterial pressure, and arteriovenous oxygen difference (a-v O2) were continuously measured . We found 1) no significant difference between the amount of O2 extracted by an innervated or a denervated muscle during endotoxemia; 2) the intra-arterial infusion of norepinephrine or epinephrine into a denervated gracilis muscle (plasma molar concentrations of; 10(-11), 10(-9), 10(-7), and 10(-5) failed to increase MVO2 to the level observed during endotoxemia; 3) pretreatment of a muscle with propranolol to block skeletal muscle beta-adrenergic receptors, did not suppress the endotoxin-induced rise in MVO2 . We concluded that the increase in MVO2 seen after the administration of endotoxin is not due to either increased sympathetic nerve activity or elevated levels of circulating catecholamines . We speculate that the increased MVO2 during endotoxemia is caused by nonadrenergic mediators released by endotoxin rather than the hypotensive stimulus. Nat Struct Biol, 1994 Feb, 1(2), 95 - 101 A conserved loop in the ATPase domain of the DnaK chaperone is essential for stable binding of GrpE; Buchberger A et al.; The activity of DnaK (Hsp70) chaperones in assisting protein folding relies on DnaK binding and ATP-controlled release of protein substrates . The ATPase activity of DnaK is tightly controlled by the nucleotide exchange factor GrpE . We find that GrpE interacts stably with the amino-terminal ATPase domain of DnaK . Analysis of the mutant DnaK756 protein, which has a lower affinity for GrpE, reveals a role for residue Gly 32 in GrpE binding . Gly 32 is located in an exposed loop near the nucleotide binding site of DnaK . Deletion of this loop prevents stable GrpE binding, ATPase stimulation by GrpE, and DnaK chaperone activity . Conservation of this loop within the Hsp70 family suggests that cooperation between Hsp70 and GrpE-like proteins may be a general feature of this class of chaperone. Cell Struct Funct, 1994 Feb, 19(1), 37 - 47 Angiogenic activity of a fusion protein of the cell-binding domain of fibronectin and basic fibroblast growth factor; Hashi H et al.; We constructed a fusion protein of the cell-binding domain of human fibronectin and human basic fibroblast growth factor, and prepared a polypeptide with both cell-adhesive activity and growth factor activity . A human gene fragment coding for basic fibroblast growth factor was amplified by the polymerase chain reaction, and introduced into the expression vector pTF7520, which encodes the cell-binding domain of human fibronectin . The resulting plasmid encoded a fusion protein in which basic fibroblast growth factor was added covalently to the C-terminal end of the fibronectin fragment . The fusion protein was expressed in Escherichia coli JM109 cells and purified from the extract by heparin affinity chromatography . The purified fusion protein had cell-adhesive activity toward BALB/c 3T3 cells, and stimulated their DNA synthesis in serum-depleted cultures . The fusion protein gave maximum mitogenic activity at the concentration of 10 nM . The fusion protein adsorbed to culture dishes, or added to collagen gels, stimulated the growth of human umbilical-vein endothelial cells . The fusion protein stimulated the angiogenesis in chorioallantoic membranes of developing chick embryos. J Biomol Struct Dyn, 1994 Feb, 11(4), 901 - 11 Mapping tRNA and 5S RNA tertiary structures by charge dependent Fe(II)-catalyzed cleavage; Zhong M et al.; Chemical and enzymatic footprinting experiments have made it possible to identify protein binding sites in DNA and RNA, and to localize structural differences within nucleic acids to a resolution of a single base pair . We show here that by combining three reagents, Fe(II).EDTA2-, Fe(II).EDDA and Fe2+, differential maps of sites in RNA that vary in their local conformation and/or charge can be constructed . Comparison of profiles with respect to controls in the absence of a counterion such as Mg2+ allows analysis of sites responsive to tertiary structure . A single site that is labile to metals such as Pb2+ exists in tRNA(Phe) and a number of other tRNA's; this site is hyper-reactive to Fe(II), but not to the other probes . Scission induced by the neutral complex, Fe(II).EDDA, offers the most general measure of surface accessibility, since its distribution about the target molecule is insensitive to charge . Enhanced cleavage by Fe(II) relative to the other agents is detected at several adjacent sites in 5S RNA, consistent with conformational mobility . Protection at a series of positions in the arm formed by loops E and D with helix IV suggests further that at low temperature this arm interacts with loop A and helix I. Mol Microbiol, 1994 Feb, 11(4), 655 - 70 mRNAs in the methanogenic archaeon Methanococcus vannielii: numbers, half-lives and processing; Hennigan AN et al.; Cells from the early exponential growth phase of cultures of the methanogenic archaeon Methanococcus vannielii have been shown to contain c . 180 transcripts of the mcrBDCGA (mcr) operon, c . 100 transcripts of the MvaL1,L10,L12 (Mva) operon, c . 8 transcripts of the argG gene and c . 1 transcript of the secY gene . These values decreased to c . 50 mcr transcripts, c . 30 Mva transcripts, c . 3 argG transcripts and < 1 secY transcript per cell as the cultures entered the stationary phase of growth . Addition of bromo-ethanesulphonate (BES) or removal of H2 inhibited growth and RNA synthesis in vivo and, at 37 degrees C in the presence of BES, the half-lives of the mcr, Mva, argG and secY transcripts were found to be 15 min, 30 min, 57 min and 7 min, respectively . Addition of puromycin, pseudomonic acid or virginiamycin also inhibited growth but did not inhibit transcription . In the presence of puromycin the half-lives of the mcr and Mva transcripts increased c . 4.6-fold and c . 3.5-fold, respectively, and there was a net accumulation of the Mva transcript . Addition of pseudomonic acid or virginiamycin also increased the half-life of the Mva transcript and also resulted in the accumulation of a second, shorter Mva transcript but did not increase the half-life of the mcr transcript . Transcription of the mcr operon was not stimulated by partial inhibition of methanogenesis. Comput Appl Biosci, 1994 Feb, 10(1), 13 - 7 Nucleotide sequence statistical analysis of pauses in RNA elongation by Escherichia coli RNA polymerase; Fedoseyeva VB et al.; A convenient motif-searching program has been developed, based on a double correlation algorithm, for analysis of the pulse character of Escherichia coli transcription . Activity in the zone of minimal pause formation (-1,2 bp) is precisely determined . Oligonucleotides (di-, tri- and tetranucleotides) are randomized by their pause-generating activity . 'CATG' and 'CATGC' are detected which coincide with the primary structure of RNA associated with distinctive delays in biologically meaningful situations. Antimicrob Agents Chemother, 1994 Feb, 38(2), 388 - 91 Expression of reverse transcriptase from feline immunodeficiency virus in Escherichia coli; North TW et al.; Reverse transcriptase from feline immunodeficiency virus (FIV) has been cloned and expressed in Escherichia coli . We have purified this recombinant enzyme and shown that it is a 66-kDa protein that is indistinguishable from virion-derived FIV reverse transcriptase in sensitivity to the 5'-triphosphates of 3'-azido-3'-deoxythymidine and the four 2',3'-dideoxynucleosides . The availability of large quantities of the FIV reverse transcriptase will allow more detailed physical and pharmacological studies. Blood Coagul Fibrinolysis, 1994 Feb, 5(1), 23 - 8 Antithrombin III infusion suppresses the hypercoagulable state in adult acute lymphoblastic leukaemia patients treated with a low dose of Escherichia coli L-asparaginase . A GIMEMA study; Mazzucconi MG et al.; Thrombotic events have been reported in acute lymphoblastic leukaemia patients, especially during or after L-asparaginase administration . A so-called L-asparaginase associated coagulopathy has been well recognized, being characterized by a hypercoagulable state (decrease of antithrombin III, plasminogen, protein C, protein S and increase of prothrombin fragment F1 + 2, thrombin-antithrombin complexes and fibrinopeptide A) . The aim of this study was to determine whether the supplementation of antithrombin III (AT-III) concentrates could improve the L-asparaginase associated coagulopathy, thereby blocking the activation of the haemostatic system . In 25 adult patients with acute lymphoblastic leukaemia (M 19, F6, mean age 34 years) antithrombin III (AT-III) concentrates were administered at daily doses of 50 U/kg for 10 consecutive days from the beginning of L-asparaginase therapy (6,000 U/m2/day s.c . for 7 days), given according to the GIMEMA ALL 0288 trial . A marked increase of antithrombin III was recorded on days IV-VIII-XI (P < 0.001) . No changes in protein C, protein S, plasminogen, alpha 2-antiplasmin, factor VII and platelet count were observed and there was no increase in markers of hypercoagulability . There was no evidence of disseminated intravascular coagulation . In conclusion, AT-III concentrate supplementation during L-asparaginase therapy, by the achievement of high levels of antithrombin III, is associated with a lack of activation of the haemostatic system and appears to overcome the complex coagulopathy associated with L-asparaginase. Anal Biochem, 1994 Feb 1, 216(2), 345 - 51 Enzymatic synthesis of folate and antifolate polyglutamates with Escherichia coli folylpolyglutamate synthetase; Hanlon MH et al.; Escherichia coli folylpolyglutamate synthetase was used to synthesize micromole quantities of polyglutamyl conjugates of folic acid, methotrexate, and other analogs of folic acid . The products of the enzymatic reactions were purified by semipreparative C18 HPLC . The position of each amide linkage (gamma or alpha carboxyl) in the polyglutamated products was determined by limited and exhaustive hydrolyses with hog kidney folylpolyglutamate hydrolase and with yeast carboxypeptidase Y . Under standard reaction conditions, the E . coli enzyme added up to five glutamyl residues to each monoglutamated substrate, primarily at the gamma carboxyl position . Thus, an enzyme which naturally adds only two glutamates to naturally occurring folates can be used synthetically to make higher polyglutamates of a wide range of synthetic substrates . The products of the reactions are valuable tools for the study of the metabolism of antifolate drugs as well as metabolic reactions involving folate cofactors. Virus Res, 1994 Feb, 31(2), 245 - 54 Tick-borne flavivirus NS1 gene: identification of conserved peptides and antigenic analysis of recombinant louping ill virus NS1 protein; Venugopal K et al.; The nucleotide sequence of the NS1 gene of louping ill (LI) virus has been determined . The sequence shows a high degree of homology with other members of the tick-borne serocomplex of flaviviruses and a lower homology with the mosquito-borne flaviviruses . Alignment of the deduced NS1 amino acid sequences with all tick-borne flavivirus NS1 sequences, identified four peptide regions which were conserved for all tick-borne flaviviruses, but were variable amongst mosquito-borne flaviviruses . A dendrogram, derived from the alignment of the NS1 protein sequences, indicated an evolutionary relationship that quite closely reflects the recognised serological classification . The LI virus NS1 protein expressed in Escherichia coli and baculoviruses showed similar antigenic reactivity to the authentic virus-coded protein when tested with NS1-specific monoclonal antibodies, but did not form high molecular weight complexes and was not secreted from cells. Scand J Gastroenterol, 1994 Feb, 29(2), 122 - 7 Localization of a pernicious anaemia autoantibody epitope on the alpha-subunit of human H,K-adenosine triphosphatase; Song YH et al.; Four cDNA fragments encoding different portions of the alpha-subunit of human H,K-adenosine triphosphatase (ATPase) were amplified by means of the polymerase chain reaction technique, ligated into the plasmid pGEX-2T, and expressed as glutathione S-transferase fusion proteins in Escherichia coli . The fragments A (residues 163-313), Ba (residues 360-797), Bb (residues 526-797), and C (residues 822-1031) together encompass 77% of the alpha-subunit and cover most of its cytosolic part . The reactivities of autoantibodies in the sera from patients with pernicious anaemia with the recombinant fusion proteins were analysed by immunoblotting . One autoantigenic epitope was found in the NH2-terminal part of the Ba fragment--that is, between residues 360 and 525 . No epitope was detected in the other fragments . The Ba fragment was cleaved off from the glutathione S-transferase fusion protein by the action of thrombin and was then further purified . By means of enzyme-linked immunosorbent assay, 28 of 42 sera (67%) from patients with pernicious anaemia were positive against the purified Ba fragment . The present results provide a final proof that the human H,K-ATPase alpha-subunit is a major autoantigen in the parietal cell and that the major epitope is located between residues 360 to 525 on the cytosolic side of the secretory membrane. Diabetologia, 1994 Feb, 37(2), 145 - 9 DNA damage by the glycation products of glyceraldehyde 3-phosphate and lysine; Mullokandov EA et al.; In order to evaluate whether base modifications, apurinic/apyrimidinic site formation, strand breaks, or a combination of these lesions results from the interaction of glycation products with DNA, plasmid DNA was first reacted with these products, and then subjected to digestion with endonuclease III and endonuclease IV of Escherichia coli . Analysis of the differential effects of digestions with these enzymes by electrophoresis on agarose gels demonstrated that reactive glycation products produce both base modification and apurinic/apyrimidinic sites in DNA, in addition to the strand breaks observed after incubation with glycation products alone . These types of DNA damage may occur in specific diabetic cells where elevated levels of glycating sugars are associated with pathologic dysfunction. Immunology, 1994 Feb, 81(2), 211 - 5 Differential induction of nitric oxide synthase in various organs of the mouse during endotoxaemia: role of TNF-alpha and IL-1-beta; Cunha FQ et al.; BALB/c mice injected intraperitoneally with bacterial lipopolysaccharide (LPS) developed lethal septic shock . This was accompanied by significantly elevated concentrations of nitrite and nitrate in the plasma and expression of high levels of nitric oxide (NO) synthase activity in the lungs, heart, spleen and peritoneal macrophages . Mice pretreated with anti-tumour necrosis factor-alpha (TNF-alpha) monoclonal antibody or anti-interleukin-1 beta (IL-1 beta) polyclonal antibody were protected, in a dose-dependent manner, from endotoxin-induced mortality . This effect was accompanied by a significant reduction in plasma levels of nitrite and nitrate . Antibody treatment also reduced the level of NO synthase activity in peritoneal macrophages, spleen and heart but had no effect on enzyme expression in the lung . These results demonstrate that TNF-alpha and IL-1 beta play an important role in the induction of NO following administration of LPS and in the development of endotoxin-induced shock . In addition, NO synthase activity is differentially expressed in various organs and this may not always require TNF-alpha and IL-1 beta. Immunology, 1994 Feb, 81(2), 205 - 10 Synergistic stimulation of human B lymphocytes by anti-CD40 monoclonal antibodies and synthetic lipopeptide analogues from Escherichia coli lipoprotein; Edinger M et al.; Human tonsillar B lymphocytes were stimulated with synthetic lipopeptide analogues of Escherichia coli lipoprotein alone or together with anti-CD40 and/or interleukin-4 (IL-4) . While lipopeptides alone or lipopeptides plus IL-4 did not include proliferation of B lymphocytes, synergistic stimulation was observed when anti-CD40 antibodies were added . Proliferation was even more pronounced in the presence of Fc receptor type II (FcRII)-transfected L cells . Compared to the stimulus anti-CD40 plus IL-4 plus FcRII-transfected fibroblasts exerted, the addition of lipopeptides induced a more rapid maximal response which peaked on day 4 . Antibody production could also be enhanced by lipopeptides . Our data provide evidence that lipopeptides, which act as mitogens toward murine B lymphocytes, also stimulate human B lymphocytes, provided that co-signals are added. Mol Microbiol, 1994 Feb, 11(3), 589 - 604 Evidence for a regulatory function of the histone-like Escherichia coli protein H-NS in ribosomal RNA synthesis; Tippner D et al.; We have isolated a small Escherichia coli protein which stably interacts with ribosomal RNA P1 promoter DNA . We present evidence showing that the protein is identical to the histone-like E . coli protein, H-NS (H1) . Binding of H-NS to the P1 promoter region is dependent on the DNA curvature . Mapping the H-NS-DNA contact sites by nuclease protection and high-resolution footprinting techniques reveal three H-NS-binding domains, and contacts of the protein in the major groove of the bent DNA . The binding region extends from position -18 to -89, relative to the P1 transcription start site, and shows an overlap with the known binding sites for Fis, another E . coli protein, which acts as transcriptional activator of P1 . The binding of H-NS does not displace Fis; instead, heterologous complexes are formed . Apparently, H-NS and Fis bind to separated curved DNA segments, with the planes of the curves pointing into different directions . In vitro transcriptional analyses demonstrate that H-NS represses rRNA P1 promoter-directed transcription . Repression is most pronounced in the presence of Fis . Thus, H-NS seems specifically to antagonize Fis-dependent activation . No comparable inactivation is observed for the second rRNA promoter P2. J Clin Microbiol, 1994 Feb, 32(2), 489 - 500 Identification of a highly cross-reactive outer surface protein B epitope among diverse geographic isolates of Borrelia spp . causing Lyme disease; Shoberg RJ et al.; The outer surface lipoprotein B (OspB) of Borrelia burgdorferi is a major component of the borrelial protein profile and has been shown to be highly immunogenic in experimentally immunized and infected mammals . However, the ospB loci of different strains show considerable heterology at the nucleic acid sequence level, and the progeny of a clonal strain of B . burgdorferi exhibited OspB polymorphisms with respect to apparent molecular weights and reactivities with monoclonal antibodies . These data suggest that OspB is not a good candidate for vaccination or diagnostic purposes . The present study describes a monoclonal antibody, designated 84C, directed against a very highly conserved domain of the OspB lipoprotein . Western immunoblot analysis with 84C demonstrated reactivity in 84.2% of human, tick, and other vertebrate isolate strains examined from widely diverse geographic regions, including strains of B . burgdorferi sensu stricto and two closely related species, B . garinii and B . afzelii . The 84C-binding region was delimited to a highly conserved 11-amino-acid region in the carboxyl terminus of OspB as demonstrated by (i) DNA sequence analysis of wild-type and 84C-resistant mutant ospB alleles and (ii) deletion mutagenesis of a recombinant ospB gene in Escherichia coli . Finally, the 84C epitope was demonstrated to be exposed on the borrelial surface in situ as (i) the monoclonal antibody 84C was able to agglutinate borrelias in culture and (ii) 84C-resistant escape variants were isolated . These data suggest that the potential value of OspB as a vaccine candidate or diagnostic tool be examined more closely, in the context of the 84C-reactive domain. Am J Physiol, 1994 Feb, 266(2 Pt 2), R419 - 25 Whole body and splanchnic leucine, phenylalanine, and glucose kinetics during endotoxemia in humans; Fong Y et al.; To examine the whole body and splanchnic tissue substrate handling during endotoxemia, an intravenous bolus of endotoxin was given to six healthy volunteers during primed, continuous infusions of {1-13C}leucine, {ring-2H5}phenylalanine, and {6,6-2H2}glucose . Whole body protein breakdown, based on whole body Leu and Phe appearance rates (Ra), increased in response to endotoxin given at time 0 (RaLeu 77 +/- 2 mol.kg-1 x h-1, t = 0 h; 88 +/- 6, t = 4 h; P < 0.05) (RaPhe 39 +/- 2 mol.kg-1 x h-1, t = 0; 46 +/- 3, t = 4 h; P < 0.05) . Splanchnic amino acid balance (Bal) increased (BalLeu 7 +/- 4 mol.kg-1 x h-1, t = 0; 21 +/- 5, t = 2 h; P < 0.05) (BalPhe 3 +/- 2 mol.kg-1 x h-1, t = 0; 16 +/- 4, t = 2 h; P < 0.05) and can be accounted for by increased splanchnic uptake (Rd) of Phe and Leu (RdLeu 21 +/- 3 mol.kg-1 x h -1, t = 0; 37 +/- 7, t = 120 min; P < 0.05) (RdPhe 10 +/- 3 mol.kg-1 x h-1, t = 0; 24 +/- 5, t = 120 min; P < 0.05) . Splanchnic conversion of Leu to ketoisocaproate increased with endotoxin administration (0.7 +/- 0.6 mol.kg-1 x h-1, t = 0; 8 +/- 3, t = 360 min; P < 0.05).(ABSTRACT TRUNCATED AT 250 WORDS) FEMS Microbiol Lett, 1994 Feb 1, 116(1), 95 - 100 Localization of promoters in the fim gene cluster and the effect of H-NS on the transcription of fimB and fimE; Olsen PB et al.; The expression of type 1 fimbriae in Escherichia coli undergoes phase variation in which individual bacteria switch between a fimbriated and non-fimbriated state . The transition from one state to the other is caused by inversion of a DNA segment containing the promoter for the fimA gene . The orientation of the invertible segment is controlled by two proteins, FimB and FimE, which mediate an on/off and off only orientation of the segment, respectively . In this study we have mapped the 5' termini of the fimB, fimE and fimA transcripts . Furthermore, we show that expression of fimB and fimE is strongly influenced by the H-NS nucleoid protein. J Clin Invest, 1994 Feb, 93(2), 692 - 702 Lipopolysaccharide (LPS)-binding protein and soluble CD14 function as accessory molecules for LPS-induced changes in endothelial barrier function, in vitro; Goldblum SE et al.; Bacterial LPS induces endothelial cell (EC) injury both in vivo and in vitro . We studied the effect of Escherichia coli 0111:B4 LPS on movement of 14C-BSA across bovine pulmonary artery EC monolayers . In the presence of serum, a 6-h LPS exposure augmented (P < 0.001) transendothelial 14C-BSA flux compared with the media control at concentrations > or = 0.5 ng/ml, and LPS (10 ng/ml) exposures of > or = 2-h increased (P < 0.005) the flux . In the absence of serum, LPS concentrations of up to 10 micrograms/ml failed to increase 14C-BSA flux at 6 h . The addition of 10% serum increased EC sensitivity to the LPS stimulus by > 10,000-fold . LPS (10 ng/ml, 6 h) failed to increase 14C-BSA flux at serum concentrations < 0.5%, and maximum LPS-induced increments could be generated in the presence of > or = 2.5% . LPS-binding protein (LBP) and soluble CD14 (sCD14) could each satisfy this serum requirement; either anti-LBP or anti-CD14 antibody each totally blocked (P < 0.00005) the LPS-induced changes in endothelial barrier function . LPS-LBP had a more rapid onset than did LPS-sCD14 . The LPS effect in the presence of both LBP and sCD14 exceeded the effect in the presence of either protein alone . These data suggest that LBP and sCD14 each independently functions as an accessory molecule for LPS presentation to the non-CD14-bearing endothelial surface . However, in the presence of serum both molecules are required. Mol Gen Genet, 1994 Feb, 242(3), 337 - 45 Arabidopsis homologs of the shaggy and GSK-3 protein kinases: molecular cloning and functional expression in Escherichia coli; Bianchi MW et al.; The conservation in evolution of fundamental signal transduction modules offers a means of isolating genes likely to be involved in plant development . We have amplified by PCR Arabidopsis cDNA and genomic sequences related to the product of the shaggy/zeste-white 3 (sgg) segment polarity gene of Drosophila . This regulatory protein is functionally homologous to glycogen synthase kinase-3 in mammals (GSK-3), which regulates, among others, the DNA-binding activity of the c-jun/AP1 transcription factor . Analysis of PCR products led to the identification of five genes; for two of which, corresponding full-length cDNAs, ASK-alpha and gamma (for Arabidopsis shaggy-related protein kinase), were characterized . The encoded proteins were 70% identical to GSK-3 and sgg over the protein kinase catalytic domain and, after production in Escherichia coli, autophosphorylated mainly on threonine and serine residues, but phosphotyrosine was also detected . ASK-alpha and ASK-gamma also phosphorylated phosphatase inhibitor-2 and myelin basic protein, on threonine and serine, respectively . The high conservation of the protein kinases of GSK-3 family, and their action at the transcriptional level, suggest that the ASK proteins have important functions in higher plants. EMBO J, 1994 Feb 1, 13(3), 699 - 703 Evidence of selection for protein introns in the recAs of pathogenic mycobacteria; Davis EO et al.; Protein introns are recently discovered genetic elements whose intervening sequences are removed from a precursor protein by an unusual protein splicing reaction . This involves the excision of a central spacer molecule, the protein intron, and the religation of the amino- and carboxy-terminal fragments of the precursor . The recA gene of Mycobacterium tuberculosis contains one such element and we now show that the other major mycobacterial pathogen, Mycobacterium leprae, also possesses a protein intron in its recA, although other mycobacterial recA genes do not . However, these two protein introns are different in size, sequence and location of insertion of their coding sequences into the recAs of M . tuberculosis and M . leprae, indicating that acquisition of the protein introns has occurred independently in the two species, and thus suggesting that there has been selection for splicing in the maturation of RecA in the pathogenic mycobacteria . The M . leprae protein intron provides an example of conditional protein splicing, splicing occurring in M . leprae itself but not when expressed in Escherichia coli, unlike most previously described protein introns . These observations suggest that protein introns may perform a function for their host, rather than being just selfish elements. Arch Surg, 1994 Feb, 129(2), 165 - 71 Differential induction of nitric oxide synthase in hepatocytes during endotoxemia and the acute-phase response; Geller DA et al.; OBJECTIVE: Nitric oxide (NO) is a potent biologic mediator produced by hepatocytes following exposure to cytokines and lipopolysaccharide (LPS) . These cytokines are also known to regulate induction of the hepatic acute-phase response . The objective of this study was to determine whether inducible nitric oxide synthase (iNOS), the enzyme that produces NO, is expressed as part of the hepatic acute-phase response . DESIGN: The gene expression for inducible NOS (iNOS) as well as alpha 1-acid glycoprotein (AGP), an established acute-phase reactant, was measured by Northern blot analysis in rat hepatocytes in vivo during endotoxemia (LPS injection) and during the acute-phase response produced by hindlimb turpentine injection . Hepatocyte iNOS messenger RNA (mRNA) levels were correlated with iNOS activity and circulating plasma nitrite and nitrate levels . In vitro, iNOS and AGP mRNA levels were determined in cultured hepatocytes stimulated with interleukin 6 (IL-6), interleukin 1 beta (IL-1 beta), tumor necrosis factor alpha (TNF-alpha), or dexamethasone . RESULTS: The AGP mRNA levels were increased in vivo following both LPS and turpentine injection, while iNOS expression was induced only by LPS injection . Hepatocyte iNOS activity and plasma nitrite and nitrate levels also increased after LPS treatment . In vitro, the cytokine combination IL-6, IL-1 beta, and TNF-alpha induced hepatocyte iNOS expression but had minimal effects on AGP in the absence of dexamethasone . Addition of dexamethasone alone markedly increased AGP mRNA levels, with further increases seen with TNF-alpha or IL-1 beta addition . In contrast, dexamethasone decreased iNOS expression . CONCLUSION: The results show that hepatocyte iNOS expression is not part of the acute-phase response induced by remote inflammation and indicates that iNOS is differentially regulated from the acute-phase reactant, AGP. Arch Surg, 1994 Feb, 129(2), 149 - 55; discussion 155-6 Detrimental hemodynamic effects of nitric oxide synthase inhibition in septic shock; Robertson FM et al.; OBJECTIVE: To investigate the physiologic effects of nitric oxide synthase inhibition with N-nitro-L-arginine methyl ester in an acute resuscitated model of porcine septic shock . DESIGN: Randomized control trial . SETTING: Animal research facility . STUDY SUBJECTS: Domestic Yorkshire swine . INTERVENTIONS: Twenty-four animals were randomly divided into one of four treatment groups as follows: normal saline resuscitation (NSR) (control group); NSR plus 200 micrograms/kg of lipopolysaccharide (LPS) at 1 hour after baseline (LPS group); NSR, LPS, and a continuous infusion of 50 micrograms/kg per minute of N-nitro-L-arginine methyl ester (NAME) at 1 hour after baseline (LPS/NAME group); and NSR and NAME (NAME group) . All animals received NSR at 1 mL/kg per minute starting at baseline . MAIN OUTCOME MEASURES: Mean arterial pressure (MAP), systemic vascular resistance index (SVRI), mean pulmonary arterial pressure (MPAP), and pulmonary vascular resistance index (PVRI) were measured at baseline and hourly for 4 hours . Values at baseline and 3 hours are given below as mean (+/- SE) . RESULTS: All variables remained unchanged in the control group . The administration of LPS produced a systemic hyperdynamic response characterized by a decrease in MAP and SVRI from 66.0 +/- 3.9 to 55.0 +/- 2.8 mm Hg (P < .05) and from 422.0 +/- 22.0 to 272.0 +/- 29.0 mm Hg.min.kg/L (P < .05), respectively . The administration of LPS produced an increase in MPAP and PVRI from 16.3 +/- 0.8 to 30.0 +/- 1.3 mm Hg (P < .05) and from 37.0 +/- 5.3 to 119.0 +/- 13.0 mm Hg.min.kg/L (P < .05), respectively . In the LPS/NAME group, NAME infusion normalized MAP and increased SVRI from 506.0 +/- 40.0 to 642.0 +/- 72.0 mm Hg.min.kg/L (P < .05) . Infusion of NAME potentiated LPS-induced pulmonary hypertension, increasing MPAP and PVRI from 16.8 +/- 0.6 to 36.0 +/- 2.8 mm Hg (P < .05) and from 59.0 +/- 3.5 to 319.0 +/- 64.0 mm Hg.min.kg/L (P < .05), respectively . Infusion of NAME alone increased MAP from 74.0 +/- 1.3 to 100.0 +/- 4.1 mm Hg (P < .05) and had no significant effect on MPAP and PVRI . CONCLUSIONS: The potentiation of LPS-induced pulmonary hypertension following NAME infusion suggests that inhibition of nitric oxide synthase may have a limited role in the treatment of septic shock. J Bacteriol, 1994 Feb, 176(3), 804 - 14 Genetic and molecular characterization of the Escherichia coli secD operon and its products; Pogliano KJ et al.; The secD operon of Escherichia coli is required for the efficient export of proteins . We have characterized this operon, and found that, in addition to secD and secF, it contains the upstream gene yajC, but not the genes queA or tgt, in contrast to previous reports . An analysis of yajC mutations constructed in vitro and recombined onto the chromosome indicates that yajC is neither essential nor a sec gene . The secD operon is not induced in response to either secretion defects or temperature changes . TnphoA fusions have been used to analyze the topology of SecD in the inner membrane; the protein contains six transmembrane stretches and a large periplasmic domain . TnphoA fusions to SecD and SecF have also been recombined onto the chromosome and used to determine the level of these proteins within the cell . Our results indicate that there are fewer than 30 SecD and SecF molecules per cell. Infect Immun, 1994 Feb, 62(2), 606 - 14 Excision of large DNA regions termed pathogenicity islands from tRNA-specific loci in the chromosome of an Escherichia coli wild-type pathogen; Blum G et al.; Uropathogenic Escherichia coli 536 (O6:K15:H31) carries two unstable DNA regions, which were shown to be responsible for virulence . These regions, on which the genes for hemolysin production (hly) and P-related fimbriae (prf) are located, are termed pathogenicity islands (PAI) I and II, and were mapped to positions 82 and 97, respectively, on the E . coli K-12 linkage map . Sequence analysis of the PAI region junction sites revealed sequences of the leuX and selC loci specific for leucine and selenocysteine tRNAs . The tRNA loci function as the targets for excision events . Northern (RNA) blot analysis revealed that the sites of excision are transcriptionally active in the wild-type strain and that no tRNA-specific transcripts were found in the deletion mutant . The analysis of deletion mutants revealed that the excision of these regions is specific and involves direct repeats of 16 and 18 nucleotides, respectively, on both sides of the deletions . By using DNA long-range mapping techniques, the size of PAI I, located at position 82, was calculated to be 70 kb, while PAI II, mapped at position 97, comprises 190 kb . The excision events described here reflect the dynamics of the E . coli chromosome. Infect Immun, 1994 Feb, 62(2), 449 - 56 Neutralizing antibodies and immunoprotection against pertussis and tetanus obtained by use of a recombinant pertussis toxin-tetanus toxin fusion protein; Boucher P et al.; The currently available diphtheria-tetanus-whole-cell pertussis (DTP) vaccines are associated with a variety of problems, including undesirable side effects and inconsistent efficacy . These problems are probably related to the poor definition of such vaccines, especially with respect to the whole-cell component against pertussis . Ideal vaccines should include only immunoprotective antigens with no toxin activity . As an initial step towards obtaining a well-defined and simplified DTP vaccine, a pertussis toxin-tetanus toxin chimeric protein was constructed . A soluble form of the pertussis toxin S1 subunit was fused to the protective fragment C of tetanus toxin, and the recombinant hybrid protein was produced in Escherichia coli . The 75-kDa fusion protein (p75) was overexpressed as a soluble molecule and purified to near homogeneity by two consecutive chromatographic steps . Purified p75 retained its ability to bind to ganglioside GT1b, the receptor for tetanus toxin, and to be recognized by protective and neutralizing anti-pertussis toxin antibodies specific for conformational epitopes . When administered to mice, the hybrid protein was found to be nontoxic but immunogenic . In addition, it was capable of inducing strong protection against tetanus and some protection against pertussis, as well as eliciting a pertussis toxin-neutralizing antibody response . Although the levels of anti-pertussis toxin antibodies were rather low, neutralizing titers of the immunized mice correlated well with anti-pertussis toxin titers, indicating that protective epitopes are conserved in the recombinant protein. Infect Immun, 1994 Feb, 62(2), 398 - 404 Septicemia-inducing Escherichia coli O115:K"V165"F165(1) resists killing by porcine polymorphonuclear leukocytes in vitro: role of F165(1) fimbriae and K"V165" O-antigen capsule; Ngeleka M et al.; Escherichia coli O115:K"V165":F165(1) wild-type strain 5131 survives in the bloodstream of experimentally inoculated gnotobiotic pigs and induces septicemia, whereas its afimbriate (F165(1)-negative) TnphoA mutant M48 and its acapsular (K"V165"-negative) spontaneous mutant 5131a are both nonpathogenic . We evaluated the role of the mannose-resistant F165(1) fimbrial system and of the O-antigen K"V165" capsule in resistance to phagocytosis by porcine polymorphonuclear leucocytes (PMNLs) in vitro . F165(1)-positive strains (5131 and 5131a) attached to and were ingested by PMNLs at a significantly higher level than afimbrial mutant M48 (P < 0.001) after 1 h of incubation . During incubation of these strains with PMNLs for up to 6 h, parental strain 5131 resisted killing whereas afimbriate mutant M48 and acapsular mutant 5131a were gradually killed and were found at significantly lower numbers than the parental strain 5131 at 2 (P < 0.05) and 6 (P < 0.001) h . When bacteria were opsonized with normal pig serum, the afimbriate and acapsular mutants survived less well than when the bacteria were nonopsonized . Upon examination by electron microscopy of PMNLs after 2 h of incubation with bacteria, structurally normal bacteria were observed more often within phagosomes of PMNLs incubated with the parental strain than within phagosomes of PMNLs incubated with the afimbriate or the acapsular mutant . The extracellular oxidative response (as tested by release of hydrogen peroxide) of PMNLs stimulated by phorbol myristate acetate was completely inhibited by F165(1)-positive strains but only partially inhibited by the afimbriate mutant . These results suggest that the F165(1) fimbrial system may mediate adherence of E . coli O115 to PMNLs . Survival of the parental strain in the presence of PMNLs, which may be intracellular, is at least partially due to the presence of the F165(1) fimbrial system and of the O-antigen capsule K"V165" . Furthermore, the presence of the F165(1) fimbrial system may contribute to the bacterial inhibition of the oxidative response of porcine PMNLs. Neurosci Lett, 1994 Jan 31, 166(2), 135 - 8 Immunohistochemical localization of substance P receptor in the superior colliculus . A light and electron microscope study in the rat; Ogawa-Meguro R et al.; The superficial layers of the superior colliculus (SC) have been known to contain many axons showing substance P-like immunoreactivity (SP-LI) . We, therefore, immunohistochemically examined the distribution of SP receptor (SPR) in the superficial layers of the SC in the rat by using a specific antibody against SPR . The majority of SC neurons with SPR-LI were distributed in the zonal and the superficial gray layers, the rest of them were in the optic layer . Electron microscopy revealed that SPR-immunoreaction products in SC neurons were distributed not only in postsynaptic sites, but also in non-synaptic regions of perikaryal and dendritic profiles. FEBS Lett, 1994 Jan 31, 338(2), 191 - 6 Different induction mechanisms of mRNA for inducible nitric oxide synthase in rat smooth muscle cells in culture and in aortic strips; Sirsjo A et al.; The expression of mRNA for the inducible form of nitric oxide synthase, (iNOS), was studied in rat aortic smooth muscle cells, (SMCs) in cell culture and in strips of rat aorta by reverse transcriptase coupled to the polymerase chain reaction . iNOS mRNA expression was weak in cultured SMCs when exposed to either interferon-gamma (IFN gamma) or lipopolysaccharide (LPS), but the combination LPS+IFN gamma enhanced the expression . In aortic strips LPS alone induced a pronounced expression, with no further increase by IFN gamma . Cycloheximide potentiated the expression of iNOS mRNA in SMCs in culture stimulated with LPS+IFN gamma but attenuated the response in aortic strips . The results indicate different cellular signaling pathways for the induction of iNOS mRNA by LPS and/or IFN gamma, in cultured SMCs and in rat aortic strips. J Mol Biol, 1994 Jan 28, 235(4), 1357 - 63 Arabidopsis thaliana NAPHP thioredoxin reductase . cDNA characterization and expression of the recombinant protein in Escherichia coli; Jacquot JP et al.; Using a clone characterized in the course of a random sequencing programme of Arabidopsis thaliana, two cDNAs encoding plant type cytosolic NADPH-dependent thioredoxin reductase (NTR) have been isolated . Their sequence homology with Escherichia coli NRT (the only thioredoxin reductase of known primary structure) is about 45% . In addition, analysis of the sequence of the encoded polypeptide (333 amino acids) reveals that several motifs are conserved in the FAD, central and NADPH binding domains, suggesting a similar folding of the protein . Definitive proof that the clone ATTHIREDB indeed encodes NTR was obtained by expressing the recombinant protein in E . coli cells . It was observed that plant type NTR was strongly overproduced (about 10 mg homogeneous protein could be purified per liter of culture) . The recombinant enzyme is homodimeric, each subunit containing an FAD prosthetic group . Recombinant plant type NTR is as effective as E . coli NTR in the DTNB (5,5'-dithiobis nitrobenzoic acid) reduction reaction, but its affinity for thioredoxin substrates was strikingly different . These results are discussed in relation to the primary structures of NADPH thioredoxin reductases. J Mol Biol, 1994 Jan 28, 235(4), 1326 - 41 Mutation of tyrosine-67 to phenylalanine in cytochrome c significantly alters the local heme environment; Berghuis AM et al.; The high resolution three-dimensional atomic structures of the reduced and oxidized states of the Y67F variant of yeast iso-1-cytochrome c have been completed . The conformational differences observed are localized directly in the mutation site and in the region about the pyrrole A propionate . Shifts in atomic positions are largely restricted to nearby amino acid side-chains, whereas little perturbation of the polypeptide chain backbone is observed . One prominent difference between the variant and wild-type structures involves a substantial increase in the size of an already existing internal cavity adjacent to residue 67 . This same cavity contains an internally bound water molecule (Wat166), which is found in all eukaryotic cytochromes c for which structures are available . In the reduced Y67F mutant protein a second water molecule (Wat300) is observed to reside in this enlarged internal cavity, assuming a position approximately equivalent to that of the hydroxyl group of Tyr67 in the wild-type protein . A further consequence of this mutation is the alteration of the hydrogen bond network between Tyr67, Wat166 and other nearby residues . This appears to be responsible for the absence of oxidation state dependent changes in polypeptide chain flexibility observed in the wild-type protein . Furthermore, loss of the normally resident Tyr67 OH to Met80 SD hydrogen bond leads to a significantly lower midpoint reduction potential . These results reaffirm proposals that both Tyr67 and Wat166 play a central role in stabilizing the alternative oxidation states of cytochrome c. J Mol Biol, 1994 Jan 28, 235(4), 1271 - 7 conformation and phasing of dystrophin structural repeats; Kahana E et al.; The presumptive rod domain of dystrophin contains a series of degenerate repeating sequences with homology to those of spectrin . To determine the relation of the implied structural repeating units to the sequence repeat (the phasing), recombinant fragments of the domain of dystrophin were prepared by expression in Escherichia coli . The phasing was established by identifying the minimum sequence element that would form a stable fold of high (approx . 75%) alpha-helicity: by contrast, incorrectly phased fragments had labile structure with an average alpha-helicity of about 40% . The isolated folded structural repeat showed high stability towards proteolysis and a urea-denaturation profile with a plateau at low denaturant concentration, indicative of a unique folded conformation . The phasing is consistent with a structure inferred from analysis of the amino acid sequence and also found in spectrin, in which each structural repeat comprises a three-stranded coiled-coil, made up of one short helix (approx . 30 residues) and the N and C-terminal halves of two separate long helices, such that each long helix participates in the formation of two contiguous structural units. J Mol Biol, 1994 Jan 28, 235(4), 1239 - 50 Visualization of protein-nucleic acid interactions involved in the in vitro assembly of the Escherichia coli 50 S ribosomal subunit; Tumminia SJ et al.; Protein-nucleic acid interactions which occur during Escherichia coli 50 S ribosomal subunit assembly between 23 S rRNA, 5 S rRNA and a complete set of 34 L-proteins were monitored by high resolution scanning transmission electron microscopy (STEM) . This approach made it possible to visualize and quantitatively analyze conformational changes induced in the rRNAs during E . coli 50 S ribosomal subunit assembly . The reconstituted RNA-protein complexes, the control 23 S rRNA and native 50 S subunits were characterized by their mass and morphology . Association of 23 S rRNA with the first assembly protein, L24, led to the formation of a distinct nucleus of mass ("cluster") on the filamentous and loosely coiled molecule of the 23 S rRNA . This structural feature was preserved and enhanced in 23 S rRNA after its association with the rest of the early assembly proteins, namely L3, L20, L13, L4 and L22 . Since the above proteins, with the exception of L3, bind to the 5' end of the 23 S rRNA, the cluster seems to be formed predominantly by interactions of L24, L13, L20, L22 and L4 with this segment of the 23 S rRNA molecule . Association with the rest of the primary binding proteins (L2, L23, L9, L1), which interact with the 3' end of the 23 S rRNA, appears to result in the formation of a second mass center . Binding of additional proteins led to the formation of compact particles with an apparent similarity to the 50 S subunit . However, particles with defined structural features characteristic of the native 50 S subunit requires the interaction of both 23 S rRNA and 5 S rRNA with all of the L-proteins . STEM image analysis demonstrated that 50 S subunit reconstitution proceeds by the immediate folding of the 23 S rRNA into a single mass center followed by the formation of a second mass center . These mass centers merge into one central body, which is gradually enhanced and decorated with structural elements characteristic of the 50 S subunit in the latter stages of assembly. J Biol Chem, 1994 Jan 28, 269(4), 3100 - 3 Cloning and identification of testis-specific transcription elongation factor S-II; Xu Q et al.; A new S-II cDNA clone was isolated from a rat testis library . This cDNA contained an open reading frame encoding 299 amino acid residues . The deduced amino- and carboxyl-terminal regions were very similar with those of Ehrlich cell S-II, which we reported previously, but the sequence of the intervening 46 amino acid residues was unique . This new S-II was expressed in the testis but not in the other rat tissues examined, suggesting that it was a testis-specific S-II . Recombinant testis-specific S-II produced in Escherichia coli was shown to stimulate RNA polymerase II. J Biol Chem, 1994 Jan 28, 269(4), 3095 - 9 Single amino acid insertions probe the alpha subunit of the Escherichia coli F1F0-ATP synthase; Wang S et al.; Single amino acid insertions of alanine or aspartate have been introduced into the alpha subunit of the F1F0-ATP synthase at seven different sites, after residues 187, 193, 198, 202, 212, 217, and 222 . These sites span a highly conserved region of the alpha subunit, parts of which are thought to be located in transmembrane spanning regions . Alanine insertions have little or no effect on function after positions 187, 193, 198, and 202, indicating that the region spanned by these residues is not essential for function . Alanine insertions after residues 212 and 217 disrupt ATP synthase function without grossly affecting the assembly of the enzyme, while the alanine insertion after residue 222 disrupts both ATP synthase function and assembly . All of the aspartate insertions are deleterious to ATP synthase function, except after residue 198 . At the other six sites, aspartate insertions prevent growth on succinate minimal medium, indicating an inability to synthesize ATP . Aspartate insertions after residues 187 and 193 result in alpha subunits that do not fractionate with membranes, as indicated by immunoblotting . These results support a model of the alpha subunit in which residues 187-193 and residues 212-222 are part of distinct transmembrane spans, separated by a short extramembrane loop . The results are consistent with an important interaction between residues 212-222 of the alpha subunit and b or c subunits . General aspects of "insertion scanning mutagenesis" are also discussed. J Biol Chem, 1994 Jan 28, 269(4), 2953 - 60 A phorbol ester binding domain of protein kinase C gamma . High affinity binding to a glutathione-S-transferase/Cys2 fusion protein; Quest AF et al.; Cysteine-rich regions of protein kinase C (PKC) are implicated in diacylglycerol-dependent regulation of kinase activity . The second cysteine-rich region (residues 92-173) of PKC gamma was expressed as a fusion protein with glutathione-S-transferase in Escherichia coli and purified to homogeneity by affinity chromatography . This fusion protein displayed high affinity phorbol dibutyrate (PDBu) binding (Kd 23 nM) . The phosphatidylserine dependence of PDBu binding was highly cooperative with Hill numbers (near 4.5) similar to those previously reported for PKC gamma (Burns, D . J., and Bell, R . M . (1991) J . Biol . Chem . 266, 18330-18338) . The fusion protein specifically bound 4 beta-hydroxy-PDBu but not the 4 alpha-stereoisomer . Furthermore, sn-1,2-dioctanoylglycerol (diC8) stereoselectively competed for PDBu binding . The cysteine-rich region was sufficient for association of the fusion protein to liposome preparations containing phosphatidylserine and phosphatidylcholine . Association was significantly enhanced in a stereospecific manner by the presence of PDBu as well as diC8 . These results establish that a single cysteine-rich domain (residues 92-173) of PKC gamma contains regions necessary and sufficient for lipid-dependent stereospecific interactions with PDBu and diC8 . Furthermore, the region is sufficient to confer translocation of a fusion protein to liposomes in a PDBu- and diC8-dependent fashion . Thus, a single cysteine-rich region of PKC gamma displays many of the properties characteristic of PKC. J Biol Chem, 1994 Jan 28, 269(4), 2921 - 8 Sequence and function of the aas gene in Escherichia coli; Jackowski S et al.; 2-Acylglycerophosphoethanolamine (2-acyl-GPE) acyltransferase and acyl-acyl carrier protein (acyl-ACP) synthase activities are encoded by the aas gene in Escherichia coli . The aas gene was cloned, and the DNA sequence of the aas gene and the region between aas and galR established the clockwise gene order in the 61.2 min of the E . coli chromosome as aas-orf-galR-lysA-lysR-orf-araE . The aas gene consists of a single open reading frame of 2,157 base pairs predicted to encode a protein of 80.6 kDa . Strains harboring multiple copies of the aas gene overproduced both 2-acyl-GPE acyltransferase and acyl-ACP synthetase activities in vitro and had higher specific activities for the incorporation of exogenous fatty acids and lysophospholipids into the membrane in vivo . Specific expression of the aas gene yielded a protein with an apparent molecular mass of 81 kDa . Comparison of the predicted amino acid sequence of the aas gene with mammalian, yeast, and bacterial long chain acyl-coenzyme A synthetases revealed three domains of high similarity which are postulated to form the acyl-AMP binding pocket . These data verify that 2-acyl-GPE acyltransferase and acyl-ACP synthetase are reactions carried out by the same gene product, verify the role of 2-acyl-GPE acyltransferase/acyl-ACP synthetase in the acylation of endogenous 2-acyl-GPE, and establish the product of the aas gene as the rate-limiting enzyme in the uptake and incorporation of exogenous 2-acyllysophospholipids. J Biol Chem, 1994 Jan 28, 269(4), 2895 - 901 An Escherichia coli protein consisting of a domain homologous to FK506-binding proteins (FKBP) and a new metal binding motif; Wulfing C et al.; Initially detected as a persistent contaminant in immobilized metal affinity chromatography of recombinant proteins in Escherichia coli, a 196-amino acid protein was isolated, cloned, overexpressed, and characterized . It consists of two domains, of which the first (146 amino acids) shows some homology to the FK506-binding proteins . The second domain (50 amino acids) is extremely rich in potentially metal-binding amino acids, such as histidine, cysteine, and acidic amino acids . The protein binds Ni2+ and Zn2+ tightly with 1:1 stoichiometry, Cu2+ and Co2+ with lower affinity, and Mn2+, Fe2+, Fe3+, Mg2+, and Ca2+ hardly at all. J Biol Chem, 1994 Jan 28, 269(4), 2782 - 9 Concerted action of three distinct domains in the DNA cleaving-joining reaction catalyzed by relaxase (TraI) of conjugative plasmid RP4; Pansegrau W et al.; The TraI protein of plasmid RP4 (IncP alpha) catalyzes a site- and strand-specific cleaving-joining reaction on form I or single-stranded DNA . Thus, TraI is one of the key components involved in the initiation and termination of horizontal DNA transfer by bacterial conjugation . Amino acid sequence comparison revealed three motifs in the TraI sequence conserved in relaxases from different origins . Site-directed mutagenesis of the traI structural gene and application of purified mutant TraI proteins for in vitro assays served to evaluate the functional importance of conserved amino acid residues . Two regions of TraI designated as motifs I and III are involved in catalyzing the cleaving-joining reaction . Motif I carries the tyrosine residue (Tyr-22), which covalently attaches TraI in a transesterification reaction to the 5' terminus of the cleaved DNA . Motif III contains one histidine residue (His-116) essential for relaxase activity and therefore proposed to activate the aromatic hydroxyl group of tyrosine 22 by proton abstraction . Exchange of a serine residue (Ser-74), located in motif II, against alanine prevents formation of stable relaxosomes but strongly enhances topoisomerase activity of the combination TraI/TraJ on form I oriT DNA . Motif II therefore might represent the DNA recognition domain of TraI . Our studies allowed us to establish a model of the interplay of three motifs located in the N-terminal region (amino acid positions 19-124) of TraI. J Biol Chem, 1994 Jan 28, 269(4), 2707 - 11 Structural and functional aspects of basic helix-loop-helix protein folding by heat-shock protein 90; Shue G et al.; The assembly and folding of nascent polypeptides are mediated in vivo by molecular chaperones, many of which are members of the heat-shock family of proteins . We have shown previously that heat-shock protein 90 (HSP90) folds an inactive fraction of a recombinant basic helix-loop-helix (bHLH) protein generated in Escherichia coli (MyoD) into its active conformation . We show here that HSP90 also folds another bHLH protein (E12) and heterodimers of E12/MyoD into their active conformations . By purifying inactive heterodimers of E12/MyoD and subsequently rendering them active in binding DNA by treatment with HSP90, we show that one folding step mediated by HSP90 occurs after oligomerization of the bHLH protein monomers . A series of deletion mutants is used to identify the 48-amino acid region of HSP90 that confers bHLH folding activity, which lies near the COOH terminus . This region is required for activation of DNA binding of MyoD and E12 homodimers and E12/MyoD heterodimers. J Biol Chem, 1994 Jan 28, 269(4), 2613 - 8 Structure-activity studies of human sterol carrier protein 2; Seedorf U et al.; Recombinant human sterol carrier protein 2 (SCP2) variants were generated by site-directed mutagenesis and expression in Escherichia coli . The ability of the variants to stimulate microsomal conversion of 7-dehydrocholesterol to cholesterol (sterol carrier activity) and to transfer cholesterol and phosphatidylcholine from donor small unilamellar vesicles to acceptor membranes (cholesterol and phosphatidylcholine transfer activities) was compared with wild-type recombinant SCP2 . Our results indicate that all measured activities of recombinant human pre-SCP2 (including the 20-amino acid leader sequence) and mature SCP2 were similar . Expressed glutathione S-transferase fusion proteins (GST-SCP2 and GST-pre-SCP2) possessed considerable activity, suggesting that steric obstruction at the amino terminus causes only minor inactivation . The effect of progressive removal of peptides from the carboxyl terminus showed that amino acids between Lys100 and Asn104 are essential for SCP2 activity . This conclusion was substantiated by the observation that replacing Asn104 with Asp or Ile caused considerable inactivation, whereas replacing Met105 with Leu had almost no effect . Since N-ethylmaleimide is known to inhibit SCP2 activity, substitutions were also introduced in the vicinity of Cys71 . Whereas Val71 and Ser71 variants possessed wild-type activity, replacing Asp70 with Asn almost completely abolished SCP2 activity . Further, the importance of residues located close to the amino terminus was indicated by complete inactivation of a 10-amino-terminal amino acid deletion mutant and by replacing Leu20 with Glu . Circular dichroism results showed that Leu20 and Asp70 may serve to stabilize the overall fold, whereas residue 104 appears to play a role in the specific lipid binding and/or transfer activity of SCP2. J Biol Chem, 1994 Jan 28, 269(4), 2562 - 7 Transmembrane helix-helix interactions in F0 suggested by suppressor mutations to Ala24-->Asp/Asp61-->Gly mutant of ATP synthase subunit; Fraga D et al.; A mutant of ATP synthase subunit c was isolated in which the essential aspartate was exchanged from position 61 on transmembrane helix-2 to position 24 on transmembrane helix-1 (Miller, M . J., Oldenburg, M., and Fillingame, R . H . (1990) Proc . Natl . Acad . Sci . U . S . A . 87, 4900-4904) . The H+ transporting ATP synthase function of the Ala24-->Asp/Asp61-->Gly mutant is not optimal, and cells grow more slowly than wild type . Twenty-three third-site suppressor mutants with optimized function were isolated in this study . Ten of the optimizing mutations were located to helix-2 of subunit c, and seven of these fell in residues Phe53, Met57, and Met65 . The side chains of these three residues are proposed to form a hydrophobic surface on transmembrane helix-2, which participates in the presentation or occlusion of the essential aspartate carboxyl group during proton translocation . The other 13 optimizing mutations were located to subunit a, and 10 of these fell in residues Ala217, Ile221, and Leu224 . These three residues are proposed to lie on one face of a transmembrane alpha-helix that includes the essential Arg210 residue . This helix is proposed to interact with the transmembrane bihelical unit of subunit c during protonation and deprotonation of the essential Asp24 in the mutant or Asp61 in wild type. J Biol Chem, 1994 Jan 28, 269(4), 2541 - 9 Characterization of sequences within heparin-binding EGF-like growth factor that mediate interaction with heparin; Thompson SA et al.; Heparin-binding (HB) epidermal growth factor (EGF)-like growth factor (HB-EGF), a member of the EGF protein family, is a potent mitogen for fibroblasts, smooth muscle cells, and keratinocytes that was initially identified as a secreted product of macrophage-like cells . HB-EGF and EGF appear to act on target cells utilizing the same receptor, but HB-EGF is distinguishable from EGF by its strong affinity for heparin . To facilitate studies of structure-function relationships in HB-EGF, a bacterial recombinant expression system was established that produced biologically active HB-EGF with the expected disulfide bonding pattern . Mutagenesis and protease digestion studies of the recombinant HB-EGF, coupled with heparin-binding analyses of synthetic peptides, indicated that the sequences within HB-EGF mediating its interaction with heparin are located primarily in a stretch of 21 amino acids characterized by a high content of lysine and arginine residues . Most of this heparin-binding domain lies in an amino-terminal region of HB-EGF that has no counterpart in EGF, but a portion of the 21-residue sequence extends into the EGF-like region of HB-EGF . In addition, the mutagenesis and synthetic peptide studies indicated that sequences in HB-EGF lying outside of the 21-residue stretch can also influence the interaction with heparin . Finally, a synthetic peptide derived from the 21-residue stretch was found to compete with HB-EGF for binding to Chinese hamster ovary cells, suggesting that the heparin-binding sequences in HB-EGF may also mediate the interaction of this factor with cell surface heparan sulfate proteoglycan. J Biol Chem, 1994 Jan 28, 269(4), 2529 - 34 Reverse transphosphorylation by ribonuclease A needs an intact p2-binding site . Point mutations at Lys-7 and Arg-10 alter the catalytic properties of the enzyme; Boix E et al.; Bovine pancreatic ribonuclease A interacts with RNA along multiple binding subsites that essentially recognize the negatively charged phosphates of the substrate . This work gives additional strong support to the existence of the postulated phosphate-binding subsite p2 (Pares, X., Llorens, R., Arus, C., and Cuchillo, C . M . (1980) Eur . J . Biochem . 105, 571-579) and confirms the central role of Lys-7 and Arg-10 in establishing an electrostatic interaction with a phosphate group of the substrate . The effects of charge elimination by Lys-7-->Gln (K7Q) and/or Arg-10-->Gln (R10Q) substitutions in catalytic and ligand-binding properties of ribonuclease A have been studied . The values of Km for cytidine 2',3'-cyclic phosphate and cytidylyl-3',5'-adenosine are not altered but are significantly increased for poly(C) . In all cases, kcat values are lower . Synthetic activity, i.e . the reversion of the transphosphorylation reaction, is reduced for K7Q and R10Q mutants and is practically abolished in the double mutant . Finally, the extent of the reaction of the mutants with 6-chloropurine-9-beta-D-ribofuranosyl 5'-monophosphate indicates that the phosphate ionic interaction in p2 is weakened . Thus, p2 modification alters both the catalytic efficiency and the extent of the processes in which an interaction of the phosphate group of the substrate or ligand with the p2-binding subsite is involved. J Biol Chem, 1994 Jan 28, 269(4), 2433 - 9 Identification of a mutant human topoisomerase I with intact catalytic activity and resistance to 9-nitro-camptothecin; Rubin E et al.; Human U-937 myeloid leukemia cells were selected for resistance to increasing concentrations of the camptothecin derivative, 9-nitro-20(S)camptothecin (9-NC) . The isolated single cell clone, designated U-937/CR, was approximately 20-fold resistant to 9-NC . Analysis of topoisomerase I (topo I) gene expression in U-937/CR cells demonstrated similar mRNA levels as compared with U-937 cells . Immunoblotting with an anti-topo I serum revealed reactive proteins at 100, 75, and 67 kDa which were expressed at the same level in the parental and 9-NC-resistant clones . These cell lines also demonstrated similar levels of topo I catalytic activity as determined by assaying nuclear extracts for relaxation of supercoiled plasmid DNA . In contrast, catalytic assays performed in the presence of 9-NC demonstrated that topo I activity from U-937/CR cells was approximately 10-fold more resistant than that from U-937 cells . Nucleotide sequencing of topo I cDNAs revealed the substitution of phenylalanine (TTC) at residue 361 in U-937 cells with serine (TCC) in the 9-NC-resistant clone . Expression and partial purification of the mutant topo I protein in Escherichia coli demonstrated resistance of this enzyme to 9-NC in catalytic assays . Taken together, these findings identify a novel mutation in topo I which confers resistance to 9-NC and support the involvement of this region in the interaction between topo I and 9-NC. J Biol Chem, 1994 Jan 28, 269(4), 2377 - 9 The tyrosine B10 hydroxyl is crucial for oxygen avidity of Ascaris hemoglobin; Kloek AP et al.; The parasitic nematode Ascaris suum has a gene encoding a two-domain hemoglobin with remarkable oxygen avidity . The strong interaction with oxygen is a consequence of a particularly slow oxygen off-rate . The single polypeptide chain consists of two domains, each of which can be expressed separately in Escherichia coli as a globin-like protein exhibiting oxygen binding characteristics comparable with the native molecule . Site-directed mutagenesis was performed on the gene segment encoding domain one . The E7 position, involved in forming a hydrogen bond with the liganded oxygen in vertebrate globins, is a glutamine in both Ascaris domains . Conversion of this residue to leucine or alanine produced a hemoglobin variant with an oxygen off-rate 5- or 60-fold faster than that of unaltered domain one . Replacement of the tyrosine B10 with either phenylalanine or leucine (as found in vertebrate globins) yielded hemoglobin mutants with oxygen off-rates 280- or 570-fold faster, approaching rates found with vertebrate myoglobins . The data suggest that the distal glutamine hydrogen bonds with the liganded oxygen and that the tyrosine B10 hydroxyl contributes an additional hydrogen bond that appears substantially responsible for the extreme oxygen avidity of Ascaris hemoglobin. J Biol Chem, 1994 Jan 28, 269(4), 2365 - 8 Cloning and recombinant expression of a novel human low molecular weight Ca(2+)-dependent phospholipase A2; Chen J et al.; Extensive biochemical studies of phospholipase A2s (PLA2s) over the last two decades indicate that there are likely to be several distinct PLA2 genes in mammals . Here we report the cloning of a 1-kilobase pair cDNA encoding a novel human low molecular weight PLA2 . The cDNA appears to encode a 118-amino acid mature peptide (M(r) = 13,592) preceded by a 20-residue prepeptide . The deduced amino acid sequence encodes a protein that lacks one of the seven disulfide bridges found in similar PLA2s and, therefore, represents a class of enzymes distinct from the mammalian group I and group II enzymes . An RNA blot hybridized with the cDNA exhibited a putative 1.2-kilobase pair transcript in heart and, less abundantly, in lung, as well as multiple putative transcripts in placenta . When the cDNA was expressed using an Epstein-Barr virus-based vector in human 293s cells, PLA2 activity accumulated in the culture medium . Conditioned medium optimally hydrolyzed the phospholipids of {1-14C}oleate-labeled Escherichia coli at neutral to alkaline pH with 10 mM or greater Ca2+ . In assays done with individual substrates, L-alpha-1-palmitoyl-2-oleoyl phosphatidylcholine was more efficiently hydrolyzed than L-alpha-1-palmitoyl-2-arachidonyl phosphatidylcholine, L-alpha-1-palmitoyl-2-arachidonyl phosphatidylethanolamine, or L-alpha-1-stearoyl-2-arachidonyl phosphatidylinositol. Gene, 1994 Jan 28, 138(1-2), 9 - 15 Secretion of eukaryotic growth hormones in Escherichia coli is influenced by the sequence of the mature proteins; Cheah KC et al.; We report the construction of secretion plasmids expressing the fusion proteins, OmpA::pGH (pSpGH.01) and OmpA::hGH (phGH.01), and compare the secretion of mature porcine growth hormone (pGH) and human growth hormone (hGH) employing Escherichia coli . E . coli {phGH.01} secreted 10-15 micrograms hGH/ml/A600 cells into the periplasmic space, representing 30% of total periplasmic proteins . E . coli {pSpGH.01}, however, secreted 30-fold less mature pGH . On the basis that both pSpGH.01 and phGH.01 are stably maintained in E . coli and in vitro transcription/translation data showed equivalent expression of OmpA::pGH and OmpA::hGH precursors, we attribute the higher secretion of hGH to the translocation-competent OmpA::hGH protein configuration . Two OmpA::GHF (growth hormone fusion) precursors, OmpA::GHF.02 and OmpA::GHF.03, both with hGH helix 3/helix 4 together instead of the pGH equivalent, secreted mature proteins as efficiently as OmpA::hGH . We propose that hGH helices 3 and 4 in these OmpA::GHF precursors play a major role in the folding of the precursor to a translocation-competent state, mimicking the translocation-competent nature of the OmpA::hGH precursor. Gene, 1994 Jan 28, 138(1-2), 263 - 4 PCR cloning and sequencing of the coding portion of the thioredoxin-encoding gene from Streptomyces aureofaciens BMK; Labudova O et al.; The nucleotide sequence of the PCR-cloned coding portion of the thioredoxin-encoding gene from Streptomyces aureofaciens BMK was determined . The deduced 106-amino-acid sequence was compared with three other thioredoxins. Gene, 1994 Jan 28, 138(1-2), 261 - 2 Three tandemly repeated structural genes encoding tRNA(f1Met) in the metZ operon of Escherichia coli K-12; Kenri T et al.; The metZ operon of Escherichia coli K-12 consists of three tandemly repeated structural genes encoding tRNA(f1Met) separated by two well-conserved spacer sequences . A multicopy plasmid carrying an intact metZ operon is unstable, deleting tRNA(f1Met)-coding regions by RecA-dependent recombination during cell growth. Gene, 1994 Jan 28, 138(1-2), 259 - 60 Nucleotide sequence corrections of the uidA open reading frame encoding beta-glucuronidase; Schlaman HR et al.; Part of the open reading frame of uidA, encoding beta-glucuronidase, was sequenced and two differences were found with the previously reported nucleotide sequence {Jefferson et al., Proc . Natl . Acad . Sci . USA 83 (1986) 8447-8451} . One is a silent mutation, the other results in the Glu279-->Gln substitution. Gene, 1994 Jan 28, 138(1-2), 253 - 6 Isolation of a cDNA encoding a human rolipram-sensitive cyclic AMP phosphodiesterase (PDE IVD); Baecker PA et al.; cDNAs encoding human family-IV phosphodiesterase, subtype D (hPDE IVD) were isolated from a human heart cDNA library . The overlapping cDNAs encode a polypeptide of 604 amino acids (aa) with a predicted M(r) of 68,502, which is 91.4% identical to the rat homolog, rPDE IVD . hPDE IVD produced in Escherichia coli was inhibited by rolipram . Expression of the hPDE IVD mRNA is widespread in human tissues and most abundant in skeletal muscle. Gene, 1994 Jan 28, 138(1-2), 17 - 25 Involvement of RecE exonuclease and RecT annealing protein in DNA double-strand break repair by homologous recombination; Kusano K et al.; We demonstrated that a double-stranded (ds) gap in DNA is repaired by a gene conversion mechanism in an Escherichia coli recBC sbcA23 strain, as predicted by the ds break repair models for homologous recombination . The sbcA mutation is known to induce several gene products encoded on the Rac prophage present in most strains of E . coli K-12 . These include exonuclease VIII (Exo VIII), a 5' to 3' exonuclease working from the end of a duplex DNA, and RecT, an annealing protein . We found that a rac- strain (lacking the Rac prophage) cannot support this repair . A plasmid carrying part of the Rac prophage supported highly efficient ds gap repair activity in a rac- strain, but two ExoVIII+ recT- plasmids did not . The recE159 mutation that blocks ds gap repair was found to be recT+, since these ExoVIII+ recT- plasmids complemented the recE159 mutation in repair of ultraviolet light damage . From these observations, we conclude that both ExoVIII and RecT are essential for ds gap repair . We discuss their possible roles in the ds break repair reaction. Gene, 1994 Jan 28, 138(1-2), 109 - 14 pKSS--a second-generation general purpose cloning vector for efficient positive selection of recombinant clones; Kast P; A new small plasmid vector (pKSS) for the direct selection of insert-containing plasmid clones is presented . The selection strategy is based on the acquired sensitivity of Escherichia coli cells to p-chloro-phenylalanine (p-Cl-Phe) if they carry a pheS allele encoding a phenylalanyl-tRNA synthetase alpha subunit with relaxed substrate specificity . This pheS allele is present on pKSS . Insertion into, or replacement of, the plasmidial pheS gene by a cloned fragment enables transformed pheS wild-type cells to survive on agar plates containing p-Cl-Phe plus ampicillin . This host strain-independent positive selection of recombinant clones proved to be highly efficient (> 99%) and did not require purification of the vector fragment prior to cloning . The high-copy-number vector pKSS offers a multitude of restriction sites and all of the features for analysis of cloned fragments that stem from the cloning vector pBluescript (Stratagene, La Jolla, CA, USA) . Thus, pKSS represents a valuable alternative to previously reported positive-selection vectors; it should prove particularly useful for cloning when expecting a high fraction of cells transformed with non-recombinant vector, and for construction of DNA libraries. Gene, 1994 Jan 28, 138(1-2), 105 - 8 A T7 expression vector for producing N- and C-terminal fusion proteins with glutathione S-transferase; Sharrocks AD; The pGEX system for protein production in E . coli is widely used in molecular biology . A bacterial expression vector, pETGEXCT, which incorporates features of the pGEX and pET expression systems was designed . pETGEXCT allows the production of N- and C-terminal fusions to glutathione S-transferase (GST) under the tight control of the T7 promoter . Use of this vector can circumvent problems associated with unstable or inactive fusions to the N terminus of GST . Indeed, it is demonstrated that fusions to the N terminus of the eukaryotic DNA-binding protein, RSRFC4, cannot be tolerated . Fusion of RSRFC4 to the N terminus of GST in the pETGEXCT vector is a prerequisite to purify the RSRFC4 DNA-binding domain in an active form using glutathione-agarose affinity chromatography. Gene, 1994 Jan 28, 138(1-2), 1 - 7 Use of the rep technique for allele replacement to construct new Escherichia coli hosts for maintenance of R6K gamma origin plasmids at different copy numbers; Metcalf WW et al.; Escherichia coli hosts were constructed for maintenance of vectors containing the gamma replication origin of the R6K plasmid (oriRR6K gamma) at different copy numbers (15 or 250/cell) . Such vectors require the trans-acting II protein (the pir gene product) for replication . New hosts carry pir+ or pir-116 on the chromosome within uidA, the E . coli gene encoding beta-glucuronidase . They were made using the rep technique for allele replacement and KmR M13 delta uid A::pir+ or M13 delta uidA::pir-116 phage . Because M13 cannot replicate in a rep mutant, KmR transductants arose by integration into the chromosomal uidA locus . Segregants lacking M13 sequences (which were selected as deoxycholate-resistant (DocR) ones) frequently contained delta uidA::pir+ or delta uidA::pir-116 on the chromosome . In principle, this procedure could be used for the introduction of any foreign gene into any nonessential gene on the E . coli chromosome . The delta uidA::pir+ and delta uidA::pir-116 loci were subsequently transferred to a variety of E . coli strains . One such strain is a suppressor-negative one that is especially useful for transposon (Tn) mutagenesis . This strain has an integrated RP4 derivative for conjugative transfer of oriRR6K gamma plasmids also containing oriT from RP4 . In addition, new oriRR6K gamma, oriT+ vectors carrying the TcR-encoding genes tetAR from Tn10 are described . These can be used for allele replacement by conjugative transfer of an oriRR6K gamma, oriT+, tetAR plasmid containing a mutated gene into a non-pir recipient and by subsequent selection for Tc-sensitive exconjugants. J Biol Chem, 1994 Jan 28, 269(4), 2485 - 90 Dissecting differential binding in the forward and reverse reaction of Escherichia coli maltodextrin phosphorylase using 2-deoxyglucosyl substrates; Becker S et al.; Substrate analogs are used in combination with site-directed mutagenesis to probe specific interactions between substrate and enzyme in the forward and reverse direction of the Escherichia coli maltodextrin phosphorylase reaction . In the phosphorolysis (degradation) mode, removal of the 2-OH group of the terminal glucose of the polysaccharide results in a 30-fold reduction of Km while similar changes were of no influence when the same polysaccharide was used for priming the synthesis . Mutation of active site residues Glu637 or Tyr538 does not change apparent affinity of substrates during degradation . In the synthesis mode, 2-deoxyglucose-1-P as substrate causes a 2-fold reduction of the wild-type kcat/Km while for the Y538F mutant a approximately 7-fold reduction is observed . In contrast, the mutation of Glu637 to Asp causes a 10-fold increase in kcat/Km . Therefore, different binding sites for the terminal glucose residue of the oligosaccharide and glucose-1-P exist . Glu637 and Tyr538 are part of the glucose-1-P binding site and do not interact with the terminal glucose residue . A 2-fold increase in rate was observed in both directions using the 2-deoxy derivatives . This confirms the role of intrinsic electronic effects in stabilizing the transition state . Uncompetitive substrate inhibition at high concentrations of maltoheptaose in the phosphorolysis direction is explained by inhibitory binding of the sugar in the synthesis mode. J Biol Chem, 1994 Jan 28, 269(4), 2447 - 51 Alteration of the quaternary structure of cpn60 modulates chaperonin-assisted folding . Implications for the mechanism of chaperonin action; Mendoza JA et al.; Chaperonin-mediated, in vitro folding of rhodanese by the intact protein cpn60 has previously been shown to require cpn10 and ATP hydrolysis (Martin, J., Langer, T., Boteva, R., Schramel, A., Horwich, A . L., and Hartl, F.-U . (1991) Nature 352, 36-42; Mendoza, J . A., Rogers, E., Lorimer, G . H., and Horowitz, P . M . (1991) J . Biol . Chem . 266, 13044-13049) . The present work demonstrates that the rhodanese-cpn60 complex can be dissociated by urea to allow folding to proceed, thus removing the obligatory requirement for cpn10 and ATP . Analytical ultracentrifugation and circular dichroism show that tetradecameric cpn60 can be disassembled into monomers that retain substantial secondary structure . Unfolded rhodanese induces the reassembly of tetradecameric cpn60 from monomers, and binding of rhodanese stabilizes cpn60 quaternary structure . Intermediate cpn60 species, possibly heptamers, are detected at intermediate urea concentrations after addition of unfolded rhodanese . The use of urea has demonstrated a functionally related loosening of subunit interactions in cpn60 that is not detectable under usual solution conditions . Our data suggest a highly dynamic role for the quaternary structure of cpn60 in chaperonin-mediated protein folding. Biochem Biophys Res Commun, 1994 Jan 28, 198(2), 459 - 65 Identification of a second poly(A) polymerase in Escherichia coli; Kalapos MP et al.; A second poly(A) polymerase (PAP II) has been identified in Escherichia coli using a strain carrying a deletion of pcnB (the structural gene for PAP I; Cao and Sarkar, 1992b) and pnp-7 (a null mutation in the structural gene for polynucleotide phosphorylase) . While PAP I has a M(r) of 53,000, PAP II is a smaller protein with a native M(r)-35,000 . PAP II differs from PAP I in preferring poly(A) over tRNA primers and being more thermolabile . The presence of multiple poly(A) polymerases in E . coli raises interesting questions regarding the role of polyadenylation in mRNA synthesis and decay. J Biol Chem, 1994 Jan 28, 269(4), 2659 - 66 The somatomedin B domain of vitronectin . Structural requirements for the binding and stabilization of active type 1 plasminogen activator inhibitor; Seiffert D et al.; We recently localized the high affinity binding site for activated type 1 plasminogen activator inhibitor (PAI-1) to the somatomedin B domain (i.e . amino acid (aa) 1-51) of vitronectin (Vn) . In this study to further define this site, N-terminal Vn fragments of various lengths were expressed in Escherichia coli and tested for PAI-1 binding activity . Vn polypeptides containing aa 1-52 and 1-40 retained PAI-1 binding activity and stabilized PAI-1 to a similar extent as intact Vn, but polypeptides containing aa 1-30 did not bind to PAI-1 nor stabilize its activity . The effects of monoclonal antibodies (mAbs) to Vn on PAI-1 binding was also determined . One mAb bound to Vn and blocked its ability to bind to PAI-1 . It also dissociated pre-existing PAI-1.Vn complexes, and prevented the incorporation of PAI-1 into extracellular matrix of HT 1080 cells . This mAb bound to the recombinant peptide containing aa 1-40, but not to the peptide consisting of aa 1-30 . A second randomly chosen mAb with similar affinity for Vn was inactive in these assays and bound to the region between aa 52 and 239 . These results indicate that the high affinity binding site for active PAI-1 in Vn is between aa 1 and 40, and that this domain may also stabilize active PAI-1. Biochemistry, 1994 Jan 25, 33(3), 840 - 7 Mutagenesis of the phosphorylation site (serine 19) of smooth muscle myosin regulatory light chain and its effects on the properties of myosin; Kamisoyama H et al.; A full-length cDNA of smooth muscle regulatory light chain was obtained and the recombinant regulatory light chain was expressed in an Escherichia coli expression system . The recombinant regulatory light chain was introduced into myosin or HMM using a subunit exchange strategy {Morita, J., Takashi, R., & Ikebe, M . (1991) Biochemistry 30, 9539-9545} . The recombinant wild-type regulatory light chain exhibited the same biological properties as the natural isolate, i.e., phosphorylation at Ser-19 by myosin light-chain kinase and phosphorylation-activated actomyosin ATPase activity . To clarify whether or not the activation of the ATPase by phosphorylation is simply due to the introduction of negative charge, we produced three mutant light chains . Two of them contain Ser-19 substituted by either Asp or Ala and the third contains Asp substituted for both Thr-18 and Ser-19 . Incorporation of the Asp mutant partially activated actomyosin ATPase activity but the activation level was significantly lower than that by phosphorylation . The Asp/Asp mutant further activated actomyosin ATPase activity . On the other hand, the Ala mutant did not affect the ATPase activity . Incorporation of Asp mutant slightly affected the 10S-6S conformational transition and filament formation of myosin . The Asp/Asp mutant more significantly affected the 10S-6S conformational transition and filament formation of myosin . These results suggested that the activation of smooth muscle myosin requires the introduction of negative charge in the defined spacial position . Using Ser-19 deficient mutants, the effects of Thr-18 phosphorylation on myosin function was also studied . Actin-activated ATPase activity of myosin was significantly activated by phosphorylation of Thr-18.(ABSTRACT TRUNCATED AT 250 WORDS) Biochemistry, 1994 Jan 25, 33(3), 773 - 9 Acceptor helix interactions in a class II tRNA synthetase: photoaffinity cross-linking of an RNA miniduplex substrate; Musier-Forsyth K et al.; The 875 amino acid class II Escherichia coli alanine tRNA synthetase aminoacylates hairpin minihelices and miniduplexes comprising complementary base pairs that reconstruct the acceptor helix of alanine tRNA . Aminoacylation is dependent upon a G3:U70 base pair in the tRNA acceptor stem . A synthetic RNA miniduplex with a phosphorothioate internucleotide linkage on the 5'-side of U70 facilitated the stable attachment of a pendant benzophenone to the ribonucleotide backbone . The benzophenone-labeled duplex is active for aminoacylation . Irradiation of the labeled duplex produced a cross-linked RNA protein complex, in which the major site of RNA attachment is the segment between the class II defining sequence motifs 2 and 3 . This segment spans a putative zinc-binding motif, which has been implicated in acceptor helix recognition, and is within a 461 amino acid N-terminal fragment that was recently shown to have full activity for minihelix aminoacylation . These results, together with the X-ray crystallographic investigations of the class II aspartate tRNA synthetase-tRNA(Asp) complex, suggest that the segment between motifs 2 and 3 in the 10 class II synthetases contributes generally to the docking of tRNA acceptor helices . The sequence diversity of this segment implies that its mode of interaction with the acceptor helix is idiosyncratic to the class II enzyme. Biochemistry, 1994 Jan 25, 33(3), 665 - 74 Hairpin folding of subunit c of F1Fo ATP synthase: 1H distance measurements to nitroxide-derivatized aspartyl-61; Girvin ME et al.; Subunit c from the F1Fo ATP synthase of Escherichia coli folds in a hairpinlike structure of two alpha-helices in a solution of chloroform-methanol-H2O, and thus resembles the structure predicted for the folded protein in the membrane . The relevance of the structure in solution to the native structure was demonstrated . Asp61 in the second helical arm was shown to retain its unique reactivity with dicyclohexylcarbodiimide (DCCD) in chloroform-methanol-H2O solution . Further, the protein purified from the Ile28-->Thr DCCD-resistant mutant proved to be less reactive with DCCD in solution . This suggested that the protein folded with Ile28 of the first helical arm close to Asp61 in the second helical arm . Subunit c in wild-type E . coli membranes was specifically labeled with a nitroxide analog of DCCD (NCCD), and the derivative protein was purified . DQF COSY spectra were recorded, and the distances between the paramagnetic nitroxide and resolved protons in the spectra were calculated based upon paramagnetic broadening of the 1H resonances . The paramagnetic contribution to T2 relaxation in the NCCD-labeled sample was calculated by an iterative computer-fitting method, where a control spectrum of a phenylhydrazine-reduced sample was broadened until the line shape of one-dimensional slices through each COSY cross-peak maximally mimicked the line shape of the paramagnetic sample . The distances calculated from paramagnetic broadening indicate that Ala24 and Ala25 in helix-1 lie close (ca . 12 A) to the derivatized Asp61 in helix-2 . A model for the interaction of helices in the NCCD-modified protein was generated by restrained molecular mechanics and molecular dynamics using 25 distances of < 10-20 A derived from paramagnetic broadening in combination with 15 long-range nuclear Overhauser enhancement (NOE) restraints (2-5 A) for distances between helices and the 89 intrahelical NOEs that defined helical structure in the DCCD-modified protein. Biochemistry, 1994 Jan 25, 33(3), 644 - 50 NMR analysis reveals a positively charged hydrophobic domain as a common motif to bound acetylcholine and d-tubocurarine; Fraenkel Y et al.; A complete 1H assignment of d-tubocurarine was carried out using 1D and 2D NMR techniques . Geometries of free acetylcholine (ACh) and d-tubocurarine were compared with those of the ligands bound to a recombinant cholinergic binding site (T alpha 184-200 expressed as a fusion protein in Escherichia coli) . The conformations of the free ligands were determined by NOESY experiments while those of the bound molecules were obtained by transferred NOESY . The complete relaxation matrix was solved yielding distance constraints which were further refined by a sigma back-calculation . ACh bound to recombinant T alpha 184-200 closely resembled the conformation previously reported for ACh bound to the intact receptor . d-Tubocurarine in the bound state undergoes extensive induced conformational rearrangements generating a "cup"-shaped structure . A unique positively charged hydrophobic domain is identified as characteristic of both bound cholinergic ligands. Nucleic Acids Res, 1994 Jan 25, 22(2), 247 - 50 Escherichia coli single-stranded DNA binding protein stimulates the DNA deoxyribophosphodiesterase activity of exonuclease I; Sandigursky M et al.; The E . coli single-stranded binding protein (SSB) has been demonstrated in vitro to be involved in a number of replicative, DNA renaturation, and protective functions . It was shown previously that SSB can interact with exonuclease I to stimulate the hydrolysis of single-stranded DNA . We demonstrate here that E . coli SSB can also enhance the DNA deoxyribophosphodiesterase (dRpase) activity of exonuclease I by stimulating the release of 2-deoxyribose-5-phosphate from a DNA substrate containing AP endonuclease-incised AP sites, and the release of 4-hydroxy-2-pentenal-5-phosphate from a DNA substrate containing AP lyase-incised AP sites . E . coli SSB and exonuclease I form a protein complex as demonstrated by Superose 12 gel filtration chromatography . These results suggest that SSB may have an important role in the DNA base excision repair pathway. Nucleic Acids Res, 1994 Jan 25, 22(2), 152 - 7 Missing-base and ethylation interference footprinting of P1 plasmid replication initiator; Papp PP et al.; RepA, the replication initiator protein of plasmid P1, binds to specific 19 bp sequences on the plasmid DNA . Earlier footprinting studies with dimethylsulfate identified the guanines that contact RepA through the major groove of DNA . In this study, base elimination was used to identify the contribution of all four bases to the binding reaction . Depurination and depyrimidation of any base in the neighborhood of the contacting guanines was found to decrease RepA binding . These results are consistent with the notion that RepA contacts bases of two consecutive major grooves on the same face of DNA . We also observed that depurination but not methylation of three guanines (G3, G8 and G9) affected binding . We identified the DNA phosphate groups (3 in the top strand, one of which mapped between G8 and G9, and 4 in the bottom strand, one of which was adjacent to C3) that strongly interfered with RepA binding upon ethylation . These results indicate that certain bases (e.g . G3, G8 and G9) may not contact RepA directly but contribute to base and backbone contacts by maintaining proper structure of the binding site. Biochemistry, 1994 Jan 25, 33(3), 682 - 6 Kinetic and mutagenic studies of the role of the active site residues Asp-50 and Glu-327 of Escherichia coli glutamine synthetase; Alibhai M et al.; The role of Asp-50 and Glu-327 of Escherichia coli glutamine synthetase in catalysis and substrate binding has been interrogated by construction of site-directed mutants at these positions . Steady-state and rapid-quench kinetic methods were used to elucidate contributions of Asp-50 and Glu-327 to the Km values of all three substrates, ATP, glutamate, and NH4+, as well as to the enzymatic kcat value . Kinetic constants were obtained for the D50A enzyme using both Mg2+ and Mn2+ as activating metal ions; the data reveal that Asp-50 has a significant role in both substrate binding and catalysis as reflected by the increases in the Km values for NH4+ and the destabilization of both the ground state and the transition state for phosphoryl transfer . The D50E mutant was found to have activity with Mn2+ but very low activity with Mg2+, the physiologically important metal ion . The kcat/Km values for all three substrates were substantially altered by changing Asp to Glu . The steady-state results for the E327A mutant indicate a decreased kcat/Km value for NH4+ compared to that of the wild-type enzyme . The E327A-Mg2+ enzyme destabilizes the ground state of the ternary complex (E-ATP-Glu-NH4+) and the transition state for phosphoryl transfer while the E327A-Mn2(+)-enzyme provides greater stabilization for the ATP and glutamate complexes but destabilizes phosphoryl transfer steps in the ternary complex . Overall, these results suggest that Asp-50 is likely involved in binding NH4+ and may also play a role in catalyzing deprotonation of NH4+ to form NH3.(ABSTRACT TRUNCATED AT 250 WORDS) J Biol Chem, 1994 Jan 21, 269(3), 2277 - 82 Analysis of acceptor stem base pairing on tRNA(Trp) aminoacylation and function in vivo; Pak M et al.; The role of acceptor stem base pairs in determining the identity of Escherichia coli tRNA(Trp) was examined by complementation of an E . coli strain containing a temperature-sensitive tRNA(Trp) gene (trpTts) and by monitoring aminoacylation levels in vivo . All derivatives of tRNA(Trp) containing substitutions at the first 3 base pairs in the acceptor stem complemented the trpTts mutation at the nonpermissive temperature (42 degrees C) . However, three acceptor stem derivatives (tRNA(Trp)/C1.G72, tRNA(Trp)/C2.G71, and tRNA(Trp)/A3.U70) required overexpression for growth at 42 degrees C . Northern analysis of these derivatives following acid/urea gel electrophoresis showed no defects in tRNA aminoacylation at the nonpermissive temperature . Instead, these tRNAs appear to be defective in translation . This was suggested by the weak opal suppressor activities of the corresponding tRNA(UCATrp) derivatives . These results demonstrate that the three terminal acceptor stem base pairs do not contribute to the identity of tRNA(Trp) . Substitution of the C1.A72 base pair in a methionine initiator tRNA containing the tryptophan anticodon and discriminator base (tRNA(CCAfMet)/G73) with A1.U72, the base pair found in tRNA(Trp), or G1.C72 resulted in the conversion of these tRNAs into tryptophan-inserting elongator tRNAs in vivo . However, changes to U1.A72 or C1.G72 in tRNA(CCAfMet)/G73 resulted in misaminoacylation and/or defects in translation . Our data indicate that the A1.U72 base pair is a context-dependent, negative identity element of tRNA(Trp). J Biol Chem, 1994 Jan 21, 269(3), 2241 - 4 Monomeric Re lipopolysaccharide from Escherichia coli is more active than the aggregated form in the Limulus amebocyte lysate assay and in inducing Egr-1 mRNA in murine peritoneal macrophages; Takayama K et al.; Using the equilibrium dialysis apparatus, an aqueous suspension of predominantly aggregated Re lipopolysaccharide (ReLPS) from Escherichia coli D31 m4 (99.9% at 82.5 microM) can be processed to yield a solution of monomeric ReLPS at a saturation concentration of 77 ng/ml (3.4 x 10(-8) M) . We compared the in vitro biological activities of these two physically distinct types of ReLPS preparations in two select assays, reaction in the Limulus amebocyte lysate (LAL) assay and induction of Egr-1 mRNA in macrophages . These assays were chosen for their rapid response times and relatively short incubation periods . The monomeric ReLPS was 179- and 1000-fold more active than the aggregated ReLPS preparation in the LAL assay and induction of Egr-1 mRNA by thioglycollate-elicited murine peritoneal macrophages, respectively . These results clearly showed that the monomeric ReLPS is the more active form . The lower biological activities of the aggregated ReLPS preparation might be due to the presence of a small amount of monomeric ReLPS (0.01-0.6%) produced during its preparation and the incubation periods in the biological assays . Thus, aggregated ReLPS may be relatively inactive. J Biol Chem, 1994 Jan 21, 269(3), 2093 - 9 Decatenating activity of Escherichia coli DNA gyrase and topoisomerases I and III during oriC and pBR322 DNA replication in vitro; Hiasa H et al.; oriC and pBR322 DNA replication, reconstituted with purified replication proteins, has been used to study the functional activities of Escherichia coli topoisomerase I, DNA gyrase, and topoisomerase III during the final stages of DNA replication . In the oriC system, DNA gyrase-catalyzed decatenation of daughter DNA molecules was very inefficient, whereas topoisomerase III could catalyze complete decatenation . In the pBR322 DNA replication system, almost all the daughter DNA molecules could be decatenated by DNA gyrase alone in the absence of salt . Decatenation by DNA gyrase in the pBR322 system was completely inhibited, without a concomitant inhibition of DNA synthesis, by the addition of physiological concentrations of salt . Topoisomerase III, however, could decatenate all of the daughter DNA molecules in the pBR322 system, even in the presence of high concentrations of salt . A similar effect could not be observed in the oriC system, because the addition of salt inhibited DNA synthesis . Topoisomerase I was incapable of catalyzing decatenation under any conditions examined in either the oriC or pBR322 replication system . The addition of topoisomerase I to the replication systems resulted only in an inhibition of DNA synthesis. J Biol Chem, 1994 Jan 21, 269(3), 2068 - 74 Hypernegative supercoiling of the DNA template during transcription elongation in vitro; Drolet M et al.; Supercoiled plasmid DNAs with negative superhelicity several times higher than normal have been isolated from Escherichia coli topA mutants . The formation of these hypernegatively supercoiled plasmid DNAs is apparently induced by transcription . We show that hypernegatively supercoiled plasmid DNAs isolated from topA mutants contain R-loop(s) . To study the mechanism of formation of hypernegatively supercoiled plasmid DNA, we have been able to reproduce hypernegatively supercoiled DNA in vitro using purified RNA polymerase and DNA gyrase . The formation of hypernegatively supercoiled plasmid DNA template in vitro is shown to require transcription elongation and is tightly linked to R-loop formation . We propose that one of the roles of topoisomerase I is to suppress R-loop formation during transcription elongation. J Biol Chem, 1994 Jan 21, 269(3), 1827 - 33 Membrane potential-linked reversed electron transfer in the beef heart cytochrome bc1 complex reconstituted into potassium-loaded phospholipid vesicles; Miki T et al.; The cytochrome bc1 complex purified from beef heart mitochondria was incorporated into potassium (K+)-loaded phospholipid vesicles by a cholate dialysis method to study the reverse reaction of electron transfer in the complex . The reduction of cytochrome b in the presence of sodium ascorbate was observed on addition of valinomycin to the K(+)-loaded proteoliposomes in a medium containing no external KCl; it was followed by the gradual oxidation . Nigericin accelerated the reoxidation of reduced cytochrome b, indicating that a K+ diffusion potential (negative inside) induced the reduction of cytochrome b . The extent of the cytochrome b reduction depended on the magnitude of the diffusion potential across the liposomal membranes, and its maximal reduction was attained at more than 210 mV of the diffusion potential . It was cytochrome b562 that was reduced during the establishment of the K+ diffusion potential in the presence of ascorbate, and about 90% of cytochrome b562 was estimated to be reduced . Antimycin A and myxothiazol inhibited the diffusion potential-induced reduction of cytochrome b562, and ubiquinone was proved to be essential for the reversed electron transfer . The K+ diffusion potential also induced the partial reduction of cytochrome b566 when cytochrome b562 had previously been reduced with ascorbate plus tetramethyl-p-phenylenediamine . These results were interpreted well based on the Q cycle scheme which assumed the energy-dependent reduction of ubiquinone at center o . Dicyclohexylcarbodiimide, which did not perturb the ability of proteoliposomes to generate the K+ diffusion potential, inhibited the energy-dependent reduction of cytochrome b562 without a significant loss in the catalytic activity of the complex . The half-inhibition was brought about by 200 mol of dicyclohexylcarbodiimide/mol of cytochrome c1 . These results strongly suggest the coupling of a proton flow with the reversed electron transfer in the bc1 complex. J Biol Chem, 1994 Jan 21, 269(3), 1794 - 803 Dual divalent cation requirement of the MutT dGTPase . Kinetic and magnetic resonance studies of the metal and substrate complexes; Frick DN et al.; Kinetic analyses of both the Mn(2+)- and Mg(2+)-activated hydrolysis of dGTP by MutT show the requirement for two divalent cations . Whereas Mn2+ supports a 20-fold lower kcat (0.19 s-1) than Mg2+ (4.0 s-1), the Km of Mn2+.dGTP (6.3 microM) is 45-fold lower than that of Mg2+.dGTP (284 microM) . Adenosine 5'-(alpha,beta-methylenetriphosphate) (AMPCPP) is a linear competitive inhibitor with respect to dGTP with a Ki for Mg2+.AMPCPP (42 microM) which is 57-fold lower than the Ki of Mg2+.AMPCPP (2.4 mM) . Such tightening suggests that a metal-bridge E.M2+.NTP.M2+ complex is the catalytically active species . The 12 dissociation constants describing the quaternary MutT.M2+.AMPCPP.M2+ complex were evaluated for both Mn2+ and Mg2+, using EPR and NMR methods . MutT binds a single Mn2+ with a Kd of 130 +/- 40 microM in reasonable agreement with the kinetically determined activator constant of Mn2+ of 230 +/- 72 microM . The MutT.AMPCPP complex binds two Mn2+ ions, the weaker of which has a Kd of 16 +/- 2 microM in agreement with the kinetically determined KmMn2+ of 26 +/- 10 microM . MutT.Mn2+ binds Mn2+.AMPCPP with Kd of 16 +/- 4 microM, whereas MutT alone binds Mn2+.AMPCPP with a Kd of 135 +/- 30 microM . The 17-fold enhanced paramagnetic effect of Mn2+ on the longitudinal relaxation rate of water protons found with the binary MutT.Mn2+ complex decreases to 4.7-fold upon binding of AMPCPP and to 8.7-fold upon binding of Mn2+.AMPCPP, further supporting a metal-bridge MutT.M2+.NTP.M2+ complex . By competition with Mn2+ MutT binds Mg2+ at one site with a Kd of 7.5 mM, and MutT.AMPCPP binds Mg2+ at two sites, the weaker of which has a Kd of 0.9 mM . These values are comparable to the kinetically determined KaMg of 15 +/- 7 mM and KmMg of 1.7 +/- 0.7 mM, respectively . Studies with the racemic, substitution-inert beta, gamma-bidentate tetraamminecobalt (III)-beta,gamma-phosphate-ATP (Co3+(NH3)4ATP) complex show that MutT slowly hydrolyzes only the lambda stereoisomer but requires Mg2+ or Mn2+ to do so, confirming a dual metal ion requirement. J Biol Chem, 1994 Jan 21, 269(3), 1692 - 8 trp repressor mutations alter DNA complex stoichiometry; Liu YC et al.; We have examined the interaction of a series of mutant trp repressors with various operator DNA sequences using gel retardation . Binding to 40 base pairs (bp) TrpEDCBA operator yielded patterns distinct from the wild-type protein for superrepressors EK13, EK18, and EK49, with a protein-DNA complex of higher stoichiometry (three dimers/operator) than observed for wild-type repressor (two dimers/operator) . This higher stoichiometry complex may contribute to the enhanced binding affinity and higher protein-operator stability observed for the superrepressors . In contrast, DN46 displayed the same complexes characteristic of the wild-type protein, although the complex of a single dimer with operator was more prominent in the DN46 binding pattern than wild-type despite higher apparent affinity of this protein for TrpEDCBA operator than wild-type protein . The binding of AV77 was indistinguishable from the wild-type protein . Similar patterns to that found for TrpEDCBA were also observed for the 40-bp aroH operator and symmetrized derivatives of TrpEDCBA for these superrepressors . Binding of EK13, EK18, and EK49 superrepressors to half-site DNAs, composed of 20 bp of TrpEDCBA sequence coupled with 20 bp of lac operator sequence, yielded 2:1 complex as the primary product with no detectable 3:1 complex; thus, two half-sites appear to be required for generation of the 3:1 complex . Mutation in the tryptophan-binding site can also generate higher order complexes with TrpEDCBA DNA as demonstrated by the binding of VA58; the presence of 3:1 complex with this protein was also dependent on the presence of two half-sites . In addition to effects of sequence changes in the protein, the ligand employed can influence the binding pattern, as demonstrated for EK49 and VA58 using 5-methyl-tryptophan; the 3:1 complex is produced more prominently and at lower protein concentration for both mutants . It is apparent from these data that binding of the trp repressor to DNA is influenced by the operator sequence, the nature of the corepressor, as well as interactions (perhaps involving the N-terminal regions) that occur within and between the dimeric structure of this protein. J Biol Chem, 1994 Jan 21, 269(3), 1648 - 53 Two distinct regions of the LamB signal sequence function in different steps in export; Wei SQ et al.; The hydrophobic core of the Escherichia coli LamB signal sequence contains two structurally distinct regions . One region forms a helix in nonpolar environments, and the other is less structured . These regions seem to be of special importance for export, as judged by the magnitude of the defect caused by their mutational inactivation . To gain insight into the mechanistic importance of these two regions, we examined the ability of precursors to pass partially through the export pathway when each region is mutated . The results demonstrate that mutations in the helical and unstructured regions of the signal sequence block different steps in the export pathway. J Mol Biol, 1994 Jan 21, 235(3), 848 - 54 Specificity of DnaK-peptide binding; Gragerov A et al.; The sequence specificity of DnaK substrate binding has been studied using a peptide display library . Based on the amino acid patterns that appeared in this selection, short peptides were synthesized for direct measurements of DnaK affinity . The results show that peptides enriched in internal hydrophobic residues are preferential DnaK substrates, and negatively charged peptides have poor affinity . The isolated C-terminal domain of DnaK binds peptides . Peptide dissociation studies indicate that bound peptides are released from the C-terminal fragment and from DnaK at identical rates . ATP stimulates peptide dissociation from DnaK but not from the C-terminal fragment. J Mol Biol, 1994 Jan 21, 235(3), 825 - 47 The twist, writhe and overall shape of supercoiled DNA change during counterion-induced transition from a loosely to a tightly interwound superhelix . Possible implications for DNA structure in vivo; Bednar J et al.; A cryo-electron microscopy study of supercoiled DNA molecules freely suspended in cryo-vitrified buffer was combined with Monte Carlo simulations and gel electrophoretic analysis to investigate the role of intersegmental electrostatic repulsion in determining the shape of supercoiled DNA molecules . It is demonstrated here that a decrease of DNA-DNA repulsion by increasing concentrations of counterions causes a higher fraction of the linking number deficit to be partitioned into writhe . When counterions reach concentrations likely to be present under in vivo conditions, naturally supercoiled plasmids adopt a tightly interwound conformation . In these tightly supercoiled DNA molecules the opposing segments of interwound superhelix seem to directly contact each other . This form of supercoiling, where two DNA helices interact laterally, may represent an important functional state of DNA . In the particular case of supercoiled minicircles (178 bp) the delta Lk = -2 topoisomers undergo a sharp structural transition from almost planar circles in low salt buffers to strongly writhed "figure-eight" conformations in buffers containing neutralizing concentrations of counterions . Possible implications of this observed structural transition in DNA are discussed. J Mol Biol, 1994 Jan 21, 235(3), 1147 - 51 Preliminary X-ray analysis of a truncated form of recombinant fructose-2,6-bisphosphatase; Lee YH et al.; The bisphosphatase domain of rat liver 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase and a C-terminal 30 amino acid truncated form were expressed in high yield in Escherichia coli and purified to homogeneity . The separately expressed bisphosphatase domain and its C-terminal truncated form had kinetic properties similar to the bisphosphatase of the intact bifunctional enzyme, but their turnover numbers were fourfold higher . The truncated enzyme crystallized in space group P1 with two molecules per asymmetric unit . The determined cell dimensions are: a = 41.9 A, b = 43.5 A, c = 57.6 A, alpha = 95.2 degrees, beta = 99.3 degrees, and gamma = 106.2 degrees . These crystals diffract to 2.0 A resolution when exposed to synchrotron radiation and are suitable for crystallographic structure analysis. J Biol Chem, 1994 Jan 21, 269(3), 2316 - 23 Glutamic acid 203 of the cAMP-dependent protein kinase catalytic subunit participates in the inhibition by two isoforms of the protein kinase inhibitor; Baude EJ et al.; Although the protein kinase inhibitors (PKIs) are known to be potent and specific inhibitors of the catalytic (C) subunit of cAMP-dependent protein kinase, little is known about their physiological roles . Glutamate 203 of the C alpha isoform (C alpha E203) has been implicated in the binding of the arginine 15 residue of the skeletal isoform of PKI (PKI alpha R15) (Knighton, D . R., Zheng, J., Ten Eyck, L . F., Xuong, N., Taylor, S.S., and Sowadski, J . M . (1991) Science 253, 414-420) . To investigate the role of C alpha E203 in the binding of PKI and in vivo C-PKI interactions, in vitro mutagenesis was used to change the C alpha E203 codon of the murine C alpha cDNA to alanine and glutamine codons . Initially, the C alpha E203 mutant proteins were expressed and purified from Escherichia coli . C alpha E203 is not essential for catalysis as all of the C subunit mutants were enzymatically active . The mutation of Glu203 did increase the apparent Km for Leu-Arg-Arg-Ala-Ser-Leu-Gly (Kemptide) severalfold but did not affect the apparent Km for ATP . The Vmax(app) was not affected by the mutation of C alpha E203 . The mutation of C alpha E203 compromised the ability of PKI alpha (5-24), PKI alpha, and PKI beta to inhibit phosphotransferase activity . PKI alpha was altered using in vitro mutagenesis to probe the role of Arg15 in interacting with C alpha E203 . The PKI alpha R15A mutant was reduced in its inhibition of C alpha . Preliminary studies of the expression of these C alpha mutants in COS cells gave similar results . These results suggest that the C alpha E203 mutants may be useful in assessing the role of PKI in vivo. J Biol Chem, 1994 Jan 21, 269(3), 1911 - 7 Interdependence of K+ and glutamate accumulation during osmotic adaptation of Escherichia coli; McLaggan D et al.; Escherichia coli responds to an increase in medium osmolarity by accumulating K+ and glutamate . At low osmolarity a large fraction of cytoplasmic K+ serves to balance charge on macromolecular anions . That fraction of K+ is here referred to as "bound," as distinguished from "free" K+ that serves to balance charge of small anions . At higher osmolarity where cytoplasmic K+ increases markedly, the bound fraction decreases but the absolute amount of bound K+ expressed per unit of dry weight increases . The increase in bound K+ can be explained largely by the reduction of cytoplasmic putrescine at high osmolarity . At high osmolarity, glutamate is the major cytoplasmic anion, equal to at least 70% of free cytoplasmic K+ . A sudden increase in the osmolarity of the medium stimulates glutamate synthesis with a lag of only about a minute; glutamate synthesis is almost totally dependent on K+ uptake . The high rate of flow of nitrogen through the glutamate pool under control conditions of growth at low osmolarity indicates that glutamate accumulation immediately after shift to high osmolarity must be due to inhibition of utilization of glutamate in the synthesis of other nitrogen-containing compounds rather than stimulation of glutamate synthesis . In agreement with this reasoning we find the kinetics of glutamate accumulation to be independent of the specific path of synthesis, whether by glutamate dehydrogenase or by glutamate synthase . Synthesis of glutamate appears to be required to attain normal values of the electrical membrane potential after shift to high osmolarity. Biochim Biophys Acta, 1994 Jan 20, 1210(3), 308 - 16 Site-directed mutagenesis studies on the iron-binding domain and the determinant for the substrate oxygenation site of porcine leukocyte arachidonate 12-lipoxygenase; Suzuki H et al.; cDNA for arachidonate 12-lipoxygenase of porcine leukocytes was expressed in Escherichia coli . The recombinant 12-lipoxygenase was purified by immunoaffinity chromatography to near homogeneity with a specific activity of about 1.5 mumol/min per mg protein . Each of eight histidine residues, which were well-conserved among various mammalian lipoxygenases and presumed as ligands for non-heme iron, was substituted with leucine by site-directed mutagenesis . Each mutant enzyme was immunoaffinity-purified to near homogeneity . Mutations of His-361, -366 and -541 caused a total loss of enzyme activity, and the iron content was much lower (0.10, 0.06 and 0.06 g atom/mol protein) than that of the wild-type enzyme (0.53) . Mutations of His-128 and -356 gave 159% and 162% specific activity of the wild-type enzyme, and the iron contents were 0.55 and 0.52 g atom/mol protein . Substitution of His-426 decreased the activity to 5%, but the iron content was 0.4 g atom/mol protein . The expression level of mutants at His-384 and -393 was too low to precisely determine the iron content . Taken together, His-361, -366 and -541 may play important roles for iron-binding in catalytically active 12-lipoxygenase . Since a high homology of amino acid sequence was known between porcine leukocyte 12-lipoxygenase and mammalian 15-lipoxygenases, we attempted to convert the 12-lipoxygenase to a 15-lipoxygenase . A double mutation of Val-418 and -419 to Ile and Met increased the ratio of 15- and 12-lipoxygenase activities from 0.1 to 5.7. Biochem Pharmacol, 1994 Jan 20, 47(2), 365 - 71 2'-Deoxy-2'-methylene derivatives of adenosine, guanosine, tubercidin, cytidine and uridine as inhibitors of L1210 cell growth in culture; Cory AH et al.; The 2'-deoxy-2'-methylene derivatives of adenosine (MdAdo), guanosine (MdGuo), tubercidin (MdTu), cytidine (MdCyd) and uridine (MdUrd) were synthesized as mechanism-based inhibitors directed at ribonucleotide reductase . It was shown that MdCyd 5'-diphosphate irreversibly inactivated ribonucleotide reductase from Escherichia coli (Baker et al., J Med Chem 34: 1879-1884, 1991) . In studies reported here, MdAdo/EHNA, MdGuo and MdCyd inhibited L1210 cell growth with IC50 values of 3.4, 10.6 and 1.4 microM, respectively . Since MdAdo is a substrate for adenosine deaminase, the presence of EHNA was required to give maximal growth inhibition . 8-Aminoguanosine was not required to maximize the cytotoxic effects of MdGuo . The 2'-deoxy-2'-methylene derivatives of tubercidin and uridine did not inhibit L1210 cell growth at concentrations as high as 50 microM (MdTu) or 100 microM (MdUrd) . L1210 cell lines resistant to hydroxyurea (directed at the non-heme iron subunit of ribonucleotide reductase) or deoxyadenosine (directed at the effector binding subunit of ribonucleotide reductase) were not resistant to MdCyd . An L1210 cell line that was highly resistant to dGuo due to the loss of a relatively specific deoxyribonucleoside kinase (Cory et al., J Biol Chem 268: 405-409, 1993) had a 6.6-fold increase in the IC50 value toward MdCyd, but showed only a 2-fold increase in resistance to MdGuo . Another L1210 cell line that was markedly deficient in adenosine kinase activity was highly resistant to MdAdo . Analysis by flow cytometry showed that MdCyd showed the transit of the cells through the G2/M phase of the cell cycle resulting in the buildup of the G2/M population . MdAdo, MdGuo and MdCyd inhibited the incorporation of {14C}cytidine into DNA without an effect on RNA synthesis or total cellular uptake of {14C}cytidine . The conversion of {14C}cytidine to deoxycytidine nucleotides was partially inhibited by MdGuo, but not by MdAdo or MdCyd . These data show that the 2'-deoxy-2'-methylene derivatives of adenosine, guanosine and cytidine are activated via specific nucleoside kinases and that the modes of action of these compounds are not identical. Biochim Biophys Acta, 1994 Jan 19, 1189(2), 225 - 32 Effect of divalent cations on lipid organization of cardiolipin isolated from Escherichia coli strain AH930; Killian JA et al.; Escherichia coli strain AH930 is a lipid biosynthetic mutant, which is unable to synthesize phosphatidylethanolamine . Instead it produces large amounts of phosphatidylglycerol and cardiolipin and has an absolute requirement for certain divalent cations . Cardiolipin was isolated from this mutant strain and its interaction with divalent cations was studied by various biophysical techniques . Monolayer measurements showed that the cations decrease the molecular surface area of cardiolipin in the order Ca2+ approximately Mg2+ > Sr2+ > Ba2+ . 31P-NMR and X-ray diffraction measurements demonstrated a comparable sequence for the ability of the cations to promote HII phase formation in dispersions of the E . coli cardiolipin: Ca2+ and Mg2+ induced HII phase formation at 50 degrees C, Sr2+ at 75 degrees C, while Ba2+ was found to be unable to promote HII phase formation in the temperature range measured . Furthermore, all divalent cations were found to increase the temperature at which the transition to the liquid-crystalline phase takes place, which was below 5 degrees C for the lipid in the absence of divalent cations . In the presence of Sr2+, Mg2+ and Ba2+ and at 25 degrees C two lamellar phases were observed, one corresponding to a liquid-crystalline phase, the other to either a gel or a crystalline phase . In the presence of Ca2+ at 25 degrees C and even at 45 degrees C no evidence for a liquid-crystalline phase was obtained and only a crystalline phase could be observed . The ability of the different cations to promote HII phase formation in the isolated E . coli cardiolipin was found to correlate with their ability to support growth of the mutant strain (De Chavigny, A., Heacock, P.N., Dowhan, W . (1991) J . Biol . Chem . 266, 5323-5332), suggesting that cardiolipin with divalent cations can replace the role of phosphatidylethanolamine in the mutant strain, and that this role involves the preference of these lipids for organization in non-bilayer lipid structures. Proc Natl Acad Sci U S A, 1994 Jan 18, 91(2), 733 - 7 Foot-and-mouth disease virus particles contain replicase protein 3D; Newman JF et al.; An antibody against the Escherichia coli-expressed RNA polymerase of foot-and-mouth disease virus (FMDV) reacts with the virus in ELISA and radioimmunoprecipitation experiments and with a protein of the disrupted virus particle in an immunoblot analysis . Treatment of the virus with trypsin, which cleaves capsid protein VP1 and a 56-kDa polypeptide present in trace amount in the particles, reduces the level of the reaction in ELISA and radioimmunoprecipitation and eliminates the immunoblot reaction . Electron microscopy showed that only approximately 20% of the virus particles reacted with the anti-polymerase antibody, whereas most reacted with an antibody against the immunodominant G-H loop of the virus . In the presence of ammonium ions, the expressed polymerase degrades the RNA of the virus into molecules sedimenting at approximately 12 S, indicating that it can act as a hydrolytic as well as a polymerizing enzyme . Moreover, the RNA in trypsin-treated virus particles is degraded when incubated at 37 degrees C, suggesting that the cleaved 56-kDa protein still possesses hydrolytic activity . In addition, the anti-polymerase antibody, which inhibits the polymerase activity of the E . coli-expressed protein, also partially inhibits the hydrolytic activity of the previously described endonuclease of the virus particle, suggesting that this enzyme is identical with the polymerase or forms part of it. Proc Natl Acad Sci U S A, 1994 Jan 18, 91(2), 689 - 93 Photoaffinity labeling of Arabidopsis thaliana plasma membrane vesicles by 5-azido-{7-3H}indole-3-acetic acid: identification of a glutathione S-transferase; Zettl R et al.; We used 5-azido-{7-3H}indole-3-acetic acid (5-azido-{7-3H}IAA), a photoaffinity analogue of the plant hormone indole-3-acetic acid (IAA), to search for auxin-binding proteins in Arabidopsis thaliana membranes . We identified an auxin-binding protein with a molecular mass of 24 kDa (Atpm24) in microsomes as well as in plasma membrane vesicles . Atpm24 was solubilized by 1% Triton X-100 and partially purified . A cDNA clone (Atpm24.1) corresponding to Atpm24 was isolated . The amino acid sequence predicted from the Atpm24.1 cDNA contains 212 amino acid residues with a relative molecular mass of 24,128 Da . Data base searches revealed that the predicted protein has homology to glutathione S-transferases (GSTs; EC 2.5.1.18) . When Atpm24.1 was expressed in Escherichia coli, we found a high level of GST activity in the bacterial extracts . We have analyzed the substrate specificity of this protein and found that cumene hydroperoxide and trans-stilbene oxide but not trans-cinnamic acid or IAA-CoA were substrates . A role for this GST in physiological processes of plants is discussed. Biochim Biophys Acta, 1994 Jan 18, 1217(1), 29 - 30 In vitro conversion of recombinant human proapolipoprotein A-I to apolipoprotein A-I; Saku K et al.; We recently investigated the in vivo conversion of recombinant human proapolipoprotein A-I (rh-Met-proapo A-I) from E . coli to apolipoprotein (apo) A-I in rabbits . In vitro incubation of rh-Met-proapo A-I with rabbit serum produced mature apo A-I3 isoproteins, as determined by the immunoblotting method . However, at the time we were unable to chemically confirm a newly produced protein band which appeared at the position of human apo A-I3 . Since then, we have confirmed the amino acid sequence of the protein using a membrane protein sequence technique, and have concluded that it corresponds to human apo A-I3. Biochim Biophys Acta, 1994 Jan 18, 1217(1), 16 - 22 Synthesis and expression of a gene coding for Erythrina trypsin inhibitor (ETI); Teixeira AV et al.; A gene coding for Erythrina trypsin inhibitor (ETI) was designed, based on the published N-terminal sequence of the protein, and synthesized by an oligonucleotide-directed single strand break-repair mechanism . Direct expression from the expression vector pBtac1 was unsuccessful . A construct, encoding an extended methionyl N-terminal amino acid was expressed from the vector pET12a which supplies a signal sequence for export to the periplasm . Most of the expressed protein was located in the cytoplasm but because the periplasm is an environment conducive to the formation of disulphide bridges, only periplasmic protein was extracted . Cyanogen bromide cleavage at the sole methionyl residue removed the undesired amino acid residues that remained after signal sequence peptidase processing . The resultant ETI was assayed against trypsin and tissue plasminogen activator and found to have activity similar to that of natural ETI. Biochemistry, 1994 Jan 18, 33(2), 562 - 9 A covalently trapped folding intermediate of subtilisin E: spontaneous dimerization of a prosubtilisin E Ser49Cys mutant in vivo and its autoprocessing in vitro; Hu Z et al.; The propeptide of subtilisin E (the N-terminal 77 amino acid extension) is required for the proper folding of the nascent mature protein and is also a potent and specific inhibitor of the active enzyme . Previous studies have demonstrated that the propeptide can renature denatured mature sequence either in cis or in trans and can be considered an intramolecular chaperone, since it is not required for activity of the mature enzyme . In this paper it is shown that a prosubtilisin-S49C mutant can be expressed in Escherichia coli either as a monomer or as a disulfide-linked dimer, (prosubtilisin-S49C)2, depending on the vector selected . Interconversion between (prosubtilisin-S49C)2 and prosubtilisin-S49C could be readily achieved by reduction and oxidation in denaturing solutions, such as guanidine hydrochloride or urea . While the monomer can undergo autoprocessing in vitro under refolding conditions, the dimer is trapped in an intermediate state which could not be processed into active enzyme . Remarkably, the autoprocessing of this trapped intermediate could be induced readily upon reduction by dithiothreitol . This disulfide-linked (prosubtilisin-S49C)2 is fairly stable, but does tend to aggregate when the ionic strength of the solution is reduced below 0.1 M . The disulfide-linked (prosubtilisin-S49C)2 has far- and near-UV CD spectra revealing the presence of both secondary and tertiary structures, respectively, similar to those of the active mature monomer . Hence this autoprocessing-competent state appears to be a "late" folding intermediate, arising after the "molten globule" state formed in the absence of the prosequence, that has no discernible tertiary structure.(ABSTRACT TRUNCATED AT 250 WORDS) Biochemistry, 1994 Jan 18, 33(2), 535 - 41 Bacteriorhodopsin can function without a covalent linkage between retinal and protein; Schweiger U et al.; Light energy is transferred from retinal to the protein in bacteriorhodopsin after absorption of a photon resulting in changes of protein conformation . To examine whether the covalent bond, formed by the carbonyl group of retinal and the epsilon-amino group of lysine 216, is essential for this process, a mutant with lysine 216 replaced by alanine was expressed in Halobacterium salinarium L33 (BO-, retinal+) . Reconstitution of the chromoprotein with varying retinylidene-n-alkylamines was possible in isolated membranes as well as in whole cells . When the protein in membranes with retinylidene Schiff bases of n-alkylamines of different lengths was reconstituted, the most stable chromoprotein was formed with retinylideneethylamine . The absorbance maximum was at 475 nm in alkaline solution and 620 nm in acidic solution . At neutral pH values both species equilibrate with a third one absorbing maximally at 568 nm . Reconstitution of whole cells with retinylideneethylamine led to a specific proton pump activity of 30 mol of protons per mol of BR per minute . This value indicates a lower limit of transport; no light saturation could be reached in these measurements in contrast to wild-type BR where transport activities of 162 mol of protons per mol of BR per minute under identical conditions can be achieved . Action spectra from flash photolysis experiments revealed that only the 568-nm form led to a M-intermediate with a half-time of decay of 17 ms . In summary, it could be shown that the covalent linkage between retinal and the protein is basically not required for the function of bacteriorhodopsin as a light-driven proton pump. Biochemistry, 1994 Jan 18, 33(2), 459 - 67 Adenosine 5'-diphosphate binding and the active site of nucleoside diphosphate kinase; Morera S et al.; The X-ray structure of nucleoside diphosphate kinase (NDP kinase) from the slime mold Dictyostelium discoideum has been determined to 2.2-A resolution and refined to an R-factor of 0.19 with and without bound ADP-Mg2+ . The nucleotide binds near His 122, a residue which becomes phosphorylated during the catalytic cycle . The mode of binding is different from that observed in other phosphokinases, and it involves no glycine-rich sequence . The adenine base makes only nonpolar contacts with the protein . It points outside, explaining the lack of specificity of NDP kinase toward the base . The ribose 2'- and 3'-hydroxyls and the pyrophosphate moiety are H-bonded to polar side chains . A Mg2+ ion bridges the alpha- to the beta-phosphate which approaches the imidazole group of His 122 from the N delta side . The geometry at the active site in the ADP-Mg2+ complex suggests a mechanism for catalysis whereby the gamma-phosphate of a nucleoside triphosphate can be transferred onto His 122 with a minimum of atomic motion. Biochemistry, 1994 Jan 18, 33(2), 439 - 42 Dynamics of a flexible loop in dihydrofolate reductase from Escherichia coli and its implication for catalysis; Falzone CJ et al.; Apo-dihydrofolate reductase from Escherichia coli samples two distinct environments slowly on the NMR time scale at room temperature . Several assigned resonances belong to residues in, or proximal to, a loop (loop I) which is comprised of residues 9-24 . This exchange process was altered (either removed or made fast on the NMR time scale) by deleting three hairpin turn forming residues from the loop and filling the gap with a single glycine {Li, L., Falzone, C . J., Wright, P . E., & Benkovic, S . J . (1992) Biochemistry 31, 7826-7833} . An approximate value of 35 s-1 for the exchange rate associated with loop I in apo-DHFR was obtained in two-dimensional nuclear Overhauser spectra by analyzing the time dependence of the cross-peak volume for N epsilon H of Trp-22, a residue which is located in this loop and which has resolved cross-peaks . Owing to the critical role that this loop plays in catalysis, the correspondence between this rate of conformational exchange and off-rates for tetrahydrofolate and the reduced nicotinamide cofactor from product and substrate complexes suggests that loop movement may be a limiting factor in substrate turnover. Biochemistry, 1994 Jan 18, 33(2), 403 - 7 Human ferrochelatase is an iron-sulfur protein; Dailey HA et al.; Recombinant human ferrochelatase has been expressed in Escherichia coli and purified to homogeneity . Metal analyses revealed approximately 2 mol of non-heme Fe per mol of the purified enzyme (M(r) = 40,000) . The UV-visible absorption spectrum of the purified enzyme consists of a protein absorption at 278 nm (epsilon approximately 90,000 M-1 cm-1) and bands at 330 nm (epsilon approximately 24,000 M-1 cm-1), 460 nm (shoulder, epsilon approximately 11,000 M-1 cm-1), and 550 nm (shoulder, epsilon approximately 9000 M-1 cm-1) that are indicative of a {2Fe-2S}2+ cluster . The spectra show an additional band at 415 nm that varied in intensity for different preparations and is attributed, at least in part, to a minor component of enzyme-associated high-spin Fe(III) heme . The presence of a single {2Fe-2S}2+,+ cluster as a redox active component of human ferrochelatase was confirmed by variable-temperature MCD and EPR studies of the dithionite-reduced enzyme which showed the presence of a S = 1/2 {2Fe-2S}+ cluster in addition to residual high spin Fe(II) heme . The reduced enzyme exhibits a S = 1/2 EPR signal, g = 2.00, 1.94, 1.91 accounting for 0.75 +/- 0.25 spins/molecule, that readily saturates at low microwave powers below 10 K but is observable without significant broadening at temperatures up to 100 K . The Fe-S cluster is labile and gradually disappears over period of 24 h, with concomitant loss of enzyme activity, when the enzyme is stored aerobically at 4 degrees C.(ABSTRACT TRUNCATED AT 250 WORDS) Biochemistry, 1994 Jan 18, 33(2), 398 - 402 Probing the metal binding sites of Escherichia coli isoleucyl-tRNA synthetase; Xu B et al.; The metal binding properties of isoleucyl-tRNA synthetase (IleRS) from Escherichia coli were studied by in vivo substitution of the enzyme-bound metals . Purified E . coli IleRS was shown to have two tightly bound zinc atoms per active site . Cobalt- and cadmium-substituted IleRS were also found to contain two tightly bound Co2+ and Cd2+ atoms per polypeptide chain, respectively . The d-d transitions in the low energy absorption spectrum of Co(2+)-substituted IleRS were characteristic of that expected for two tetrahedrally coordinated Co2+ metals . Apo-IleRS was found to be inactive in both the aminoacylation of tRNA(Ile) and in the isoleucine-dependent ATP-pyrophosphate exchange reactions . Both Co(2+)- and Cd(2+)-substituted IleRS were found to have kcat/Km values in the isoleucine-dependent ATP-pyrophosphate exchange assay approximately 5-fold lower than the native Zn2+ enzyme . A single enzyme-bound Zn2+ or Co2+ atom per polypeptide chain could be removed by dialysis of Zn(2+)- or Co(2+)-substituted IleRS against 1,10-phenanthroline . Removal of one of the two enzyme-bound Zn2+ atoms per polypeptide chain with 1,10-phenanthroline was found to decrease (kcat/Km)Ile by approximately 130-fold . The dependence of the kinetic parameters on the identity and number of enzyme-bound metals in the isoleucine-dependent ATP-pyrophosphate exchange reaction suggests that at least one enzyme-bound metal is indirectly involved in aminoacyladenylate formation . Metal substitution or removal of one of the two enzyme-bound metals in IleRS was found to have little effect on the Km value for tRNA(Ile) or the kcat value for aminoacylation of tRNA(Ile).(ABSTRACT TRUNCATED AT 250 WORDS) FEBS Lett, 1994 Jan 17, 337(3), 255 - 8 Escherichia coli PII protein: purification, crystallization and oligomeric structure; Vasudevan SG et al.; The Escherichia coli signal transduction protein PII, product of the glnB gene, was overproduced and purified . The predicted molecular weight of the protein based on the correct nucleotide sequence is 12,427 and is very close to the value 12,435 obtained by matrix-assisted laser desorption mass spectrometry . Hexagonal crystals of the unuridylylated form of PII with dimensions 0.2 x 0.2 x 0.3 mm were grown and analysed by X-ray diffraction . The crystals belong to space group P6(3) with a = b = 61.6 A, c = 56.3 A and Vm of 2.5 for one subunit in the asymmetric unit . A low-resolution electron density map showed electron density concentrated around a three-fold axis, suggesting the molecule to be a trimer . A sedimentation equilibrium experiment of the meniscus depletion type was used to estimate a molecular weight of 35,000 +/- 1,000 for PII in solution . This result is consistent with the native protein being a homotrimer. Mutat Res, 1994 Jan 16, 304(2), 271 - 84 The structural basis of the genotoxicity of nitroarenofurans and related compounds; Mersch-Sundermann V et al.; The CASE (Computer-Automated Structure Evaluation) methodology has been applied to an investigation of the basis of the genotoxicity (sfiA induction) of 79 nitroarenofurans and related molecules examined with the E . coli PQ37 genotoxicity assay (SOS chromotest) . CASE identified 9 major activating structural fragments (biophores) responsible for the probability of genotoxicity (SAR) . With respect to quantitative features, CASE identified 8 major molecular subunits related to the genotoxic potency (QSAR) . Both the SAR as well as the QSAR analysis indicate that a nitro group on position 2 of the furan ring is important for activity provided one or more aromatic rings are attached to the furan ring, i.e . 2-nitrobenzofuran, 2-nitronaphthofuran, 2-nitroanthrafuran and 8-nitropyrenofuran . Additionally, a small substituent at position 3 of the furan ring, i.e . the methyl group of R7371, the ethyl group of R7427 and the butyl group of R7429, enhance the activity of 2-nitronaphtho{2,1-b}furan (R6597), whereas longer aliphatic chains decrease activity . Moreover, the activity of the nitro group at position 2 of the furan ring was increased by substitution of a methoxy group at position 7 of the R6597 structure . Additionally the n-octanol/water partition coefficient (log P) was found to be an important descriptor for the genotoxic potency in E . coli PQ37 . Using the identified descriptors CASE correctly predicted the probability of genotoxicity of all of the genotoxicants and non-genotoxicants in the data base . The calculated genotoxic potency was equally good: 94% of all predicted results were within plus/minus one order of magnitude of the experimental result . Using CASE in the predictive mode, the program correctly predicted the probability of sfiA induction in E . coli of 95.8% of 24 "unknown" nitroarenofurans which were not part of the learning set (QSAR with r = 0.88-0.97). Mutat Res, 1994 Jan 16, 304(2), 265 - 9 Base incorporation and extension at a site-specific ethenocytosine by Escherichia coli DNA polymerase I Klenow fragment; Simha D et al.; Ethenocytosine (epsilon C) is a highly mutagenic exocyclic DNA lesion induced by carcinogens vinyl chloride and urethane . We have examined base incorporation and extension at a site-specific epsilon C residue by a quantitative gel electrophoretic assay using an exonuclease-deficient version of Escherichia coli DNA polymerase I (Klenow fragment) as the model enzyme . The data show that the KM for incorporation of adenine or thymine opposite epsilon C by is about 5 orders of magnitude higher than that for the incorporation of guanine opposite normal cytosine . The KM for base extension past epsilon C:A and epsilon C:T pairs is 1-2 orders of magnitude higher than that observed for a C:G pair . Although adenine misinsertion is favored over that of thymine, base extension occurs more readily when the base incorporated opposite epsilon C is thymine. Mutat Res, 1994 Jan 16, 304(2), 181 - 5 A simple assay for monitoring the mutagenic effects of 5-methylcytosine deamination in Escherichia coli; Petropoulos L et al.; We have developed and tested a simple phenotypic assay which monitors C to T transition mutations at the second C of a CCAGG sequence in the lacZ gene of Escherichia coli . The assay is based on new data concerning amino acid requirements on either side of a crucial active site residue in beta-galactosidase, glutamic acid 461 . We show that the frequency of occurrence of the mutation is influenced by two genes: dcm, the cytosine methylase gene, and vsr, one of the genes involved in very short patch repair . The assay has been used to evaluate the function of vsr cloned from a potential very short patch repair mutant. EMBO J, 1994 Jan 15, 13(2), 367 - 72 Mutagenesis supports water mediated recognition in the trp repressor-operator system; Joachimiak A et al.; High resolution crystallographic analysis of the trp repressor-operator complex indicates that the principal determinants of specificity are water mediated hydrogen bonds between the helix-turn-helix and the identity elements of the operator . One such hydration site involves a conserved G-C base pair (designated G6) six nucleotides away from the dyad which, if changed symmetrically to any other pair (e.g . G6-->A) reduces affinity to nonspecific levels . This same water site also contacts the conserved A5 which, if changed to G (mutation A5-->G), also diminishes affinity . The stereochemistry of the water mediated hydrogen bonding system predicts that the severe deterioration of in vitro binding caused by G6-->A should be reverted by a second deleterious mutation A5-->G . This proved to be the case . No other second mutation at conserved operator position 5 or 7 (flanking the G6-->A) reversed the effect of G6-->A. Eur J Biochem, 1994 Jan 15, 219(1-2), 83 - 7 Cloning and expression of three abrin A-chains and their mutants derived by site-specific mutagenesis in Escherichia coli; Hung CH et al.; DNAs encoding of three abrin A-chains were obtained from the cDNA library of Abrus precatorius by polymerase chain reaction and ligated into the expression vector, pGEX-2T . The mature A-chains of abrins a, b and d have been expressed in the cytoplasm of Escherichia coli, and the yield of the soluble recombinant proteins was 7 mg/l induced culture . Three recombinant abrin A-chains were purified to be homogeneity and their N-glycosidase ability to inhibit protein biosynthesis in a cell-free system and to depurinate 28S rRNA in rat liver ribosomes was demonstrated in vitro . The recombinant abrin-a A-chain had the highest N-glycosidase activity among three recombinant abrin A-chains while the recombinant abrin-b A-chain, the least . Three mutants, glutamic-acid-to-alanine replacement (E164A), arginine to leucine (R167L) or double mutation (E164A and R167L) were constructed and expressed . The protein-biosynthesis-inhibitory activity of mutant (E164A), mutant (R167L) and the double mutant was found to be 25-fold, 625-fold and 1250-fold lower than that of wild type, respectively . The results indicated that Arg167 was essential for abrin toxin A-chain catalysis. Eur J Biochem, 1994 Jan 15, 219(1-2), 707 - 12 Secondary structure and signal assignments of human-immunodeficiency-virus-1 protease complexed to a novel, structure-based inhibitor; Yamazaki T et al.; We report comprehensive NMR studies in solution of the human-immunodeficiency-virus (HIV)-1 protease . Stable solutions of the protease were obtained by complexing the protein to a designed cyclic urea inhibitor DMP 323 . A variety of triple-resonance experiments provided essentially complete 1H, 13C and 15N NMR signal assignments of the protease . These assignments, together with short-range NOE constraints, coupling constants and hydrogen-exchange data, yielded the secondary structure of the protease in solution . The results reported herein open the way to the determination of the high-resolution three-dimensional solution structures of protease/inhibitor complexes, as well as to studies of protease dynamics and solvent interactions. Eur J Biochem, 1994 Jan 15, 219(1-2), 65 - 71 Undecagold cluster modified tRNA(Phe) from Escherichia coli and its activity in the protein elongation cycle; Blechschmidt B et al.; An undecagold cluster (Au11) of molecular mass 6200Da was attached to the 3-(3-amino-3-carboxypropyl)uridine at position 47 of tRNA(Phe) from Escherichia coli . This modified tRNA can be enzymically aminoacylated with phenylalanine in the reaction catalyzed by phenylalanyl-tRNA synthetase . Au11-labeled Phe-tRNA(Phe) forms a ternary complex with the elongation factor Tu.GTP and is active in poly(U)-dependent poly(phe) synthesis . The Au11 modification does not hinder the specific binding of tRNA to distinct ribosomal binding sites or the precise positioning of the aminoacyl and peptidyl residues in the peptidyltransferase center, and does not impair the translocation . The modified tRNA is suitable for the identification of ribosomal binding sites by scanning transmission electron microscopy and for crystallographic studies of the 70S ribosome at different states of the protein-elongation cycle. Eur J Biochem, 1994 Jan 15, 219(1-2), 627 - 39 Proteinase yscD (oligopeptidase yscD) . Structure, function and relationship of the yeast enzyme with mammalian thimet oligopeptidase (metalloendopeptidase, EP 24.15); Buchler M et al.; The yeast PRD1 gene, encoding proteinase yscD, was cloned by complementation of the prd1-6 point mutation . Sequencing of the gene revealed an open reading frame of 2.136 kb, encoding a protein of 712 amino acids with a calculated molecular mass of 81.8 kDa . The sequence HEGLG beginning at residue 501 represents the HEXXH motif, unique for the zinc metallo-peptidases . Sequence comparison revealed complete identity of the proteinase yscD gene with a recently published open reading frame of yeast chromosome III . We found 34.8% identity between proteinase yscD and rat metalloendopeptidase (thimet oligopeptidase, EP 24.15) . Proteinase yscD hydrolyzes several chromogenic and fluorogenic peptides that are substrates of thimet oligopeptidase . N-{1-(RS)-carboxy-3-phenylpropyl}-Ala-Ala-Phe-p-aminobenzoic acid, a compound designed as specific inhibitor of EP 24.15, is also a strong inhibitor of the yeast enzyme . Proteinase yscD is a nonvacuolar enzyme . 3-5% of the total enzyme activity can be detected in the intermembrane space of mitochondria . In a mutant carrying a deletion of the PRD1 gene no proteinase yscD activity is detectable in the cytoplasm and in mitochondria of these cells . They do not show any grossly altered phenotype but exhibit a decrease in the intracellular degradation of peptides . This suggests a function of proteinase yscD in the late stages of protein degradation. Eur J Biochem, 1994 Jan 15, 219(1-2), 595 - 602 Magnetic-circular-dichroism studies of Escherichia coli cytochrome bo . Identification of high-spin ferric, low-spin ferric and ferryl {Fe(IV)} forms of heme o; Cheesman MR et al.; Room-temperature (295 K) magnetic-circular-dichroism spectra at 280-2500 nm have been recorded for Escherichia coli cytochrome bo in its fast form (which has a g = 3.7 EPR signal and reacts rapidly with cyanide) and for its formate, fluoride, cyanide and hydrogen-peroxide derivatives . The spectra of all forms are dominated by signals from low-spin ferric heme b . These include a porphyrin-to-ferric ion charge-transfer transition in the near-infrared region (the near-infrared charge-transfer band) at 1610 nm . High-spin ferric heme o gives rise to a negative magnetic-circular-dichroism feature at 635, 642 and 625 nm (corresponding to a shoulder observed in the electronic absorption spectra) and a derivative charge-transfer feature at 1100, 1180 and 940 nm for the fast, formate and fluoride forms, respectively . The energies of these bands confirm that fluoride and formate are ligands to heme o . The energies of the analogous bands in the spectrum of fast cytochrome bo are typical for high-spin ferric hemes with histidine and water axial ligands . Addition of cyanide ion to fast cytochrome bo causes a red shift in the position of the Soret absorption peak, from 406.5 nm to 413 nm, and results in the loss of the 635-nm feature from the magnetic-circular-dichroism spectrum and of the corresponding shoulder in the electronic absorption spectrum . In the magnetic-circular-dichroism spectrum, the intensities of the Soret and alpha, beta bands are significantly increased . New near-infrared charge-transfer intensity is observed at 1000-2300 nm with a peak near 2050 nm . These changes are interpreted as resulting from a high-spin to low-spin transition at ferric heme o brought about by the binding of cyanide ion . The energy of the near-infrared charge-transfer band suggests that the cyanide ion is bridged to the CuB of the binuclear site . Treatment of fast cytochrome bo with hydrogen peroxide also causes a red shift in the position of the Soret absorbance, to 412 nm, and a loss of the 625-nm absorption shoulder . Changes in the magnetic-circular-dichroism spectrum at 450-600 nm are observed, but there is no significant increase in the intensity of the magnetic-circular-dichroism Soret band and no new near-infrared charge-transfer bands are detected, ruling out a similar high-spin to low-spin transition at heme o.(ABSTRACT TRUNCATED AT 400 WORDS) Eur J Biochem, 1994 Jan 15, 219(1-2), 497 - 502 Expression and characterization of recombinant human and rat liver 6-pyruvoyl tetrahydropterin synthase . Modified cysteine residues inhibit the enzyme activity; Burgisser DM et al.; 6-Pyruvoyl-tetrahydropterin synthase is the rate-limiting enzyme in the synthesis of human tetrahydrobiopterin, a cofactor for several hydroxylases involved in catecholamine and serotonin biosynthesis . The human and rat liver cDNAs encoding the 16-kDa subunit of 6-pyruvoyl tetrahydropterin synthase were expressed as maltose-binding-6-pyruvoyl-tetrahydropterin-synthase fusion proteins . After cleavage from the fusion protein, the human and rat enzymes were purified to homogeneity . Apparent Km for the substrate dihydroneopterin triphosphate (8.5 microM for the human and 8.0 microM for the rat enzyme), pI (4.6 and 4.8) and heat stability of the recombinant enzymes were similar to the native enzymes . The specific activity of the enzymes was enhanced up to fourfold in the presence of dithiothreitol during purification . The modification of the only cysteine residue in rat 6-pyruvoyl tetrahydropterin synthase, which is conserved in the human enzyme, inhibited its activity up to 80% . Modification under non-reducing conditions of both cysteine residues of the human enzyme by N-ethylpyridine resulted in a 95% loss of enzyme activity . This demonstrates that the two cysteines are not linked by disulfide bridges but rather involved in catalysis . Cross-linking experiments and analysis by gel electrophoresis showed predominantly trimeric and hexameric forms of the recombinant enzymes from both species suggesting that the native form is a homohexamer of 98 kDa, for the human, and 95 kDa, for the rat enzyme, composed of two trimeric subunits. Eur J Biochem, 1994 Jan 15, 219(1-2), 455 - 62 Expression, purification and characterization of the recombinant kringle 2 and kringle 3 domains of human plasminogen and analysis of their binding affinity for omega-aminocarboxylic acids; Marti D et al.; The kringle 2 (E161T/C162S/EEE{K2HPg/C169S}TT) and the kringle 3 (TYQ{K3HPg}DS) domains of human plasminogen (HPg) were expressed in Escherichia coli in an expression vector with the phage T5 promotor/operator element N250PSN250P29 and the cDNA sequence for a hexahistidine tail to facilitate the isolation of the recombinant protein . A coagulation factor Xa (FXa)-sensitive cleavage site was introduced to remove the N-terminal histidine tag . In r-K2, mutations E161T and C162S were introduced to enhance the FXa cleavage yield and C169S to replace the cysteine residue, participating in the inter-kringle disulfide bridge between kringles 2 and 3 . Recombinant proteins were isolated by affinity chromatography on Ni(2+)-nitrilotriacetic acid/agarose and refolded under denaturing and reducing conditions followed by a non-denaturing and oxidising environment . The free thiol group in position 297 in r-K3 was selectively alkylated with iodoacetamide . The hexahistidine tail was successfully removed with FXa . The N-terminal sequence, the amino acid composition and the molecular mass analyses are in agreement with the expected data . The correct arrangement of the disulfide bonds was verified by sequence analysis of the corresponding thermolytic and subtilisin fragments . r-K2 exhibits weak binding to lysine-Bio-Gel . The weak binding affinity of r-K2 for omega-aminocarboxylic acids is confirmed by intrinsic fluorescence titration with 6-aminohexanoic acid (NH2C5COOH) indicating a Kd of approximately 401 microM . In contrast, r-K3 seems to be devoid of a binding affinity for omega-aminocarboxylic acids . Considering earlier determined Kd values of kringle 1, kringle 4 and kringle 5, the binding affinity of HPg kringle domains for NH2C5COOH is proposed to decrease in the following order, kringle 1 > kringle 4 > kringle 5 > kringle 2 > kringle 3. Eur J Biochem, 1994 Jan 15, 219(1-2), 435 - 9 Discrimination against misacylated tRNA by chloroplast elongation factor Tu; Stanzel M et al.; Chloroplast elongation factor Tu was purified from Pisum sativum and the binding properties of glutamylated chloroplast tRNAs were studied by gel-permeation chromatography . Whereas chloroplast Glu-tRNA(Glu) is efficiently bound by this factor, the misacylated Glu-tRNA(Gln) does not interact with chloroplast elongation factor Tu.GTP and is thus efficiently excluded from protein synthesis . Comparison with the behaviour of Escherichia coli elongation factor Tu.GTP shows that this factor, which is not confronted with the in vivo misacylation phenomenon of organelles, binds both Glu-tRNA(Glu) and Glu-tRNA(Gln) from chloroplasts with approximately equal efficiency. Eur J Biochem, 1994 Jan 15, 219(1-2), 415 - 23 Purification, cDNA cloning and heterologous expression of human phosphomannose isomerase; Proudfoot AE et al.; Phosphomannose isomerase catalyses the interconversion of fructose-6-P and mannose-6-P and has a critical role in the supply of D-mannose derivatives required for many eukaryotic glycosylation reactions . Three classes of enzymes possessing phosphomannose-isomerase activity have been identified in bacteria and lower eukaryotes . We have purified human phosphomannose isomerase to homogeneity from placental tissue . Protein sequence information obtained from internal fragments of the protein was used to design degenerate oligonucleotides which were used to amplify a fragment of a human phosphomannose-isomerase cDNA . A full-length cDNA was isolated from a human testes lambda gt11 library using this fragment as a probe . The cDNA encoded a protein with significant sequence identity to fungal and some bacterial phosphomannose isomerases but was unrelated to those from other bacteria . Based on amino acid sequence identity we propose a classification system for enzymes with phosphomannose-isomerase activity . The cDNA, under the control of the GAL1 promoter, was expressed in a Saccharomyces cerevisiae strain from which the native gene encoding phosphomannose isomerase had been deleted . The human enzyme was found to be able to functionally substitute for the yeast enzyme . Phosphomannose-isomerase mRNA was found in all human tissues tested but was more highly expressed in heart, brain and skeletal muscle . The cDNA was expressed in Escherichia coli permitting the isolation of pure recombinant protein which will be used for kinetic and structural studies. Eur J Biochem, 1994 Jan 15, 219(1-2), 365 - 73 Characterisation of a trisulphide derivative of biosynthetic human growth hormone produced in Escherichia coli; Jespersen AM et al.; A novel protein derivative has been found during process development of biosynthetic human growth hormone; it has been characterised as human growth hormone with a Cys182-Cys189 trisulphide bridge . We have not been able to find a previous report in the literature about this kind of derivative . The characterisation was obtained partly on the full-length derivative and partly on a tryptic fragment of the derivative . The full-length derivative was characterised by reduction with 1,4-dithiothreitol followed by electrospray mass spectrometry, treatment with cysteine and measurement of hydrogen sulphide liberation upon cysteine treatment . The tryptic fragment from peptide mapping was characterised by amino acid analysis, amino acid sequencing and mass spectrometry . All data indicated an extra sulphur atom in the Cys182-Cys189 cystine bridge. Eur J Biochem, 1994 Jan 15, 219(1-2), 277 - 86 Molecular cloning and expression of a cDNA encoding human electron transfer flavoprotein-ubiquinone oxidoreductase; Goodman SI et al.; Electron-transfer flavoprotein-ubiquinone oxidoreductase (ETF-QO) in the inner mitochondrial membrane accepts electrons from electron-transfer flavoprotein which is located in the mitochondrial matrix and reduces ubiquinone in the mitochondrial membrane . The two redox centers in the protein, FAD and a {4Fe4S}+2,+1 cluster, are present in a 64-kDa monomer . We cloned several cDNA sequences encoding the majority of porcine ETF-QO and used these as probes to clone a full-length human ETF-QO cDNA . The deduced human ETF-QO sequence predicts a protein containing 617 amino acids (67 kDa), two domains associated with the binding of the AMP moiety of the FAD prosthetic group, two membrane helices and a motif containing four cysteine residues that is frequently associated with the liganding of ferredoxin-like iron-sulfur clusters . A cleavable 33-amino-acid sequence is also predicted at the amino terminus of the 67-kDa protein which targets the protein to mitochondria . In vitro transcription and translation yielded a 67-kDa immunoprecipitable product as predicted from the open reading frame of the cDNA . The human cDNA was expressed in Saccharomyces cerevisiae, which does not normally synthesize the protein . The ETF-QO is synthesized as a 67-kDa precursor which is targeted to mitochondria and processed in a single step to a 64-kDa mature form located in the mitochondrial membrane . The detergent-solubilized protein transfers electrons from ETF to the ubiquinone homolog, Q1, indicating that both the FAD and iron-sulfur cluster are properly inserted into the heterologously expressed protein. Eur J Biochem, 1994 Jan 15, 219(1-2), 261 - 6 Alteration of the cleavage mode and of the transglycosylation reactions of the xylanase A of Streptomyces lividans 1326 by site-directed mutagenesis of the Asn173 residue; Moreau A et al.; The amino acid replacement of Asn173 by Asp in the xylanase A (Xln A) of Streptomyces lividans significantly altered its enzymic properties . A time-course hydrolysis of xylan showed that the altered xylanase ({N173D} Xln A) initially produced larger amounts of xylose (X1), xylobiose (X2) and xylotriose (X3) than Xln A, but less xylotetraose (X4) . The bond-cleavage frequencies were determined for both enzymes using xylopentaose (X5), xylotetraose (X4) and xylotriose (X3) labelled at the reducing end of the molecule . Xln A hydrolysed X5, yielding 56% X2 and 44% X3, while {N173D}Xln A liberated 90% X2 and only 10% X3 . Both enzymes hydrolysed X4 into 100% X2 and X3 into 100% X1 . Transglycosylation reactions were detected in HPLC hydrolysis patterns using high substrate concentrations, where larger products than the starting substrates were formed . Their subsequent degradation also affected the yield of hydrolysis products . Using X5 as substrate, products from xylohexaose (X6) up to xylooligosides larger than xylooctaose (X8) were synthesized by Xln A, while {N173D}Xln A produced only a small amount of xyloheptaose (X7) and X8 . Xln A hydrolysed X5 into an equivalent amount of X4 and X2 and 1.5-fold more X3 . However, {N173D}Xln A yielded the same amount of X3 and X2 but half as much X4 . With X4 as substrate, Xln A synthesized twofold more X7 and X6 than {N173D}Xln A . Xln A liberated 1.4-fold more X3 than X2, while {N173D}Xln A yielded twofold more X2 than X3 . Xln A liberated almost fourfold more X2 than X1 from X3, while {N173D}Xln A produced only twofold more X2 than X1 . These results indicated that the negative charge introduced by the mutation greatly affected the transglycosylation reaction catalysed by this xylanase. Eur J Biochem, 1994 Jan 15, 219(1-2), 253 - 60 Metallobiochemistry of the magnesium ion . Characterization of the essential metal-binding site in Escherichia coli ribonuclease H; Huang HW et al.; Ribonuclease H (Escherichia coli) contains one strong magnesium-binding site, as determined by metal-titration experiments monitored by high field 1H-NMR and also by direct titration calorimetry . Kinetic and thermodynamic parameters were evaluated by 25Mg-NMR and were as follows: dissociation constant Kd, approximately 60 +/- 10 microM; activation free energy delta G*, approximately 49.8 +/- 0.9 kJ; on/off-rate for magnesium binding Kon, approximately 1.8 x 10(8) M-1 s-1, koff, approximately 1.1 x 10(4) s-1; quadrupole coupling constant chi B, 1.2 +/- 0.2 MHz . The dissociation constant was independently determined by standard analysis of 1H chemical shifts in magnesium-titration experiments and by microcalorimetry (Kd approximately 200 +/- 20 microM) . Cobalt hexaamine, which also activates RNase H {Jou, R . & Cowan, J . A . (1991) J . Am . Chem . Soc . 113, 6685-6686}, appears to bind at the same location as Mg2+(aqueous) . Assignments of C2H and C4H protons to specific histidine residues have been made by two-dimensional correlated spectroscopy experiments . Direct 25Mg-NMR pH titrations show that an ionizable residue (pKa approximately 5.8), most likely one of the carboxylates in the active site, influences magnesium binding . On the basis of the magnesium coordination chemistry elucidated herein, recent proposals on active-site chemistry are critically assessed and general physicochemical aspects of magnesium-binding sites on proteins and enzymes are discussed. Eur J Biochem, 1994 Jan 15, 219(1-2), 201 - 8 Intracellular expression of Vitreoscilla hemoglobin alters Escherichia coli energy metabolism under oxygen-limited conditions; Kallio PT et al.; An earlier stoichiometric analysis of oxygen-limited metabolism of Escherichia coli expressing cloned Vitreoscilla hemoglobin (VHb) suggested improved efficiency of ATP production relative to wild-type controls {Khosla, C., Curtis, J . E., DeModena, J., Rinas, J . & Bailey, J . E . (1990) BioTechnol . 8, 849-853} . This hypothesis has been further examined by determining several energetic parameters of different VHb-expressing E . coli (VHb+) strains relative to controls not expressing VHb (VHb-) . The H+/O ratio, the transmembrane delta pH, and the ATP content of VHb+ constructs are 1.5, 1.6 and 2 times, respectively, corresponding values in VHb- controls . VHb was expressed using a low-copy-number vector in E . coli mutant strains lacking cytochrome o, cytochrome d, or both terminal oxidases; significant growth enhancement due to VHb expression was observed only in the strain having functional cytochrome o and lacking cytochrome d . All of these data obtained using different E . coli strains are consistent with a model of VHb action that hypothesizes enhancement by VHb of activity of the lower oxygen-affinity, higher proton-pumping-efficiency cytochrome o terminal oxidase under oxygen-limited growth conditions. Eur J Biochem, 1994 Jan 15, 219(1-2), 11 - 23 Heat-shock proteins as molecular chaperones; Becker J et al.; Functional proteins within cells are normally present in their native, completely folded form . However, vital processes of protein biogenesis such as protein synthesis and translocation of proteins into intracellular compartments require the protein to exist temporarily in an unfolded or partially folded conformation . As a consequence, regions buried when a polypeptide is in its native conformation become exposed and interact with other proteins causing protein aggregation which is deleterious to the cell . To prevent aggregation as proteins become unfolded, heat-shock proteins protect these interactive surfaces by binding to them and facilitating the folding of unfolded or nascent polypeptides . In other instances the binding of heat-shock proteins to interactive surfaces of completely folded proteins is a crucial part of their regulation . As heat shock and other stress conditions cause cellular proteins to become partially unfolded, the ability of heat-shock proteins to protect cells against the adverse effects of stress becomes a logical extension of their normal function as molecular chaperones. Biochem J, 1994 Jan 15, 297 ( Pt 2), 389 - 97 Molecular characterization of hNRP, a cDNA encoding a human nucleosome-assembly-protein-I-related gene product involved in the induction of cell proliferation; Simon HU et al.; We have isolated from a human thymus cDNA library a cDNA clone encoding a potential protein with 54% amino acid similarity to that encoded by a previously identified cDNA for yeast nucleosome assembly protein I (NAP-I) . The deduced amino acid sequence for this newly identified cDNA, designated hNRP (human NAP-related protein), contains a potential seven-residue nuclear localization motif, three clusters of highly acidic residues and other structural features found in various proteins implicated in chromatin formation . When expressed as a fusion protein in Escherichia coli, hNRP reacted specifically with a monoclonal antibody raised against human NAP-I . The hNRP transcript was detected in all tissues and cell lines studied, but levels were somewhat increased in rapidly proliferating cells . Moreover, levels of both hNRP mRNA and protein increased rapidly in cultured T-lymphocytes induced to proliferate by incubation with phorbol ester and ionomycin . Phorbol 12-myristate 13-acetate/ionomycin-induced increases in both hNRP mRNA and mitogenesis, as measured by thymidine incorporation, were markedly inhibited, however, in cells treated with an hNRP antisense oligonucleotide . These results demonstrate a correlation between induction of hNRP expression and mitogenesis and taken together with the structural similarities between hNRP and yeast NAP-I suggest that the hNRP gene product participates in DNA replication and thereby plays an important role in the process of cell proliferation. Am J Epidemiol, 1994 Jan 15, 139(2), 193 - 205 Proportional hazards analysis of diarrhea due to enterotoxigenic Escherichia coli and breast feeding in a cohort of urban Mexican children; Long KZ et al.; Ninety-eight women-infant pairs were followed for up to 50 weeks in the northern part of Guadalajara, Mexico, from August 1986 to July 1987 as part of a community-based, prospective study of the relation between infant feeding patterns and enterotoxigenic Escherichia coli producing heat-labile toxin (LT-ETEC) diarrheal disease . Strictly formula-fed children had an incidence of diarrhea over three times that of strictly breast-fed infants and twice that of breast-fed and supplementally fed children . Strictly formula-fed infants colonized by LT-ETEC were symptomatic for diarrhea nearly three times as often as strictly breast-fed infants and twice as often as infants receiving a mixed diet . The fitting of parametric hazard models to durations until LT-ETEC colonization revealed that the hazard for the first colonization was time invariant . The hazard of diarrhea increased by 400-500% during the rainy season or among children 3 months of age or older who received avena, a barley drink . The best-fitting hazard models to durations until symptomatic expression of LT-ETEC infection all increased through time . This hazard was inversely impacted by the overall amount of LT-ETEC-specific, immunoglobulin A antibodies the infant received via the mother's breast milk and by the provision of traditional medicinal teas. Blood, 1994 Jan 15, 83(2), 446 - 51 Differential effects of anti-tumor necrosis factor monoclonal antibodies on systemic inflammatory responses in experimental endotoxemia in chimpanzees; van der Poll T et al.; Tumor necrosis factor (TNF) is considered to be a pivotal mediator of endotoxin-induced lethality . To assess the intermediate role of TNF in specific systemic inflammatory responses known to contribute to tissue injury in endotoxemia, eight healthy adult chimpanzees were intravenously injected with Escherichia coli endotoxin (4 ng/kg) . In four of these animals the administration of endotoxin was followed immediately by a bolus intravenous injection of an anti-TNF monoclonal antibody (15 mg/kg) . Treatment with anti-TNF completely prevented the endotoxin-induced increase in serum TNF activity, and profoundly reduced the appearance of interleukin-6 and -8 (both P < .05) . Neutrophilia and lymphopenia were not affected by anti-TNF, whereas neutrophil degranulation, as measured by the plasma concentrations of elastase-alpha 1-antitrypsin complexes, was only slightly reduced (peak levels after endotoxin alone 31.0 +/- 3.4 ng/mL, versus 25.5 +/- 3.4 ng/mL after endotoxin with anti-TNF; P < .05) . Anti-TNF did not influence endotoxin-induced activation of the coagulation system, as reflected by unchanged increases in the plasma concentrations of the prothrombin fragment F1 + 2 and thrombin-antithrombin III complexes . In contrast, anti-TNF strongly attenuated the activation of the fibrinolytic system, ie, peak plasma levels of plasmin-alpha 2-antiplasmin were 33.8 +/- 11.1 nmol/L after endotoxin alone and 17.0 +/- 2.9 nmol/L after endotoxin with anti-TNF (P < .05) . These results suggest that TNF is not the common mediator of systemic inflammatory changes in low-grade endotoxemia . Moreover, the finding that in this mild model anti-TNF specifically inhibited fibrinolysis suggests that treatment with anti-TNF potentially may enhance the tendency towards microvascular thrombosis in sepsis. Genomics, 1994 Jan 15, 19(2), 376 - 8 Chromosome mapping of the human (RECA) and mouse (Reca) homologs of the yeast RAD51 and Escherichia coli recA genes to human (15q15.1) and mouse (2F1) chromosomes by direct R-banding fluorescence in situ hybridization; Takahashi E et al.; We mapped the human (RECA) and mouse (Reca) homologs of the yeast RAD51 and Escherichia coli recA genes to human and mouse chromosomes by direct R-banding fluorescence in situ hybridization . This gene was assigned to human chromosome 15q15.1 and to mouse chromosome 2F1, respectively . This is the first report on the precise localization of this gene to human and mouse chromosomes . This gene was mapped to a region on human chromosome 15q15.1 and mouse 2F1 that is believed to be a conserved syntenic group. FEMS Microbiol Lett, 1994 Jan 15, 115(2-3), 151 - 5 Tn 5Map, a transposon for the rapid mapping of restriction sites in plasmids; Hiller B et al.; A transposon was constructed allowing the rapid restriction mapping of plasmids . This transposon, Tn 5Map, contains a cleavage site for the I-SceI endonuclease which recognizes an 18-mer . After in vivo transposition of Tn5Map into the plasmid of interest, the plasmid is isolated and linearized with I-SceI . Splinkers labelled with digoxygenin and complementary to the left and right end of the linearized molecule are added and ligated . After partial digestion of the splinkered molecules with the restriction enzyme of interest, separation of the cleavage products in an agarose gel, and Southern transfer, the labelled fragments are visualized by the addition of the chemiluminescent substrate AMPPD and alkaline phosphatase . The restriction map can be directly read from the bottom to the top of the gel. FEMS Microbiol Lett, 1994 Jan 15, 115(2-3), 223 - 8 Effect of various analogues of D-glutamic acid on the D-glutamate-adding enzyme from Escherichia coli; Pratviel-Sosa F et al.; Twenty-four analogues of D-glutamic acid were tested as substrates or inhibitors of the D-glutamate-adding enzyme from Escherichia coli . The best substrates were, in decreasing order of specific activity, D-erythro-4-methylglutamic acid, D-erythro-3-methylglutamic acid, DL-homocysteic acid, (+/-)-trans-1-amino-3-carboxy-cyclopentanecarboxylic acid and (+/-)-trans-1-amino-3-carboxy-cyclohexanecarboxylic acid . Among the different stereoisomers, only the D-erythro isomers for methylglutamic acids, and the trans isomers for the cyclic analogs, were substrates . Apart from the D-erythro-3- and 4-methylglutamic acids and DL-homocysteic acid, none of the examined compounds significantly inhibited the addition of radioactive D-glutamic acid to UDP-N-acetylmuramyl-L-alanine. Eur J Biochem, 1994 Jan 15, 219(1-2), 357 - 63 Cloning and expression of cDNA of human delta 4-3-oxosteroid 5 beta-reductase and substrate specificity of the expressed enzyme; Kondo KH et al.; The enzyme delta 4-3-oxosteroid 5 beta-reductase (3-oxo-5 beta-steroid: NADP+ oxidoreductase and 4,5 beta-dihydrocortisone: NADP+ delta 4-oxidoreductase) catalyzes the reduction of the delta 4 double bond of bile acid intermediates and steroid hormones carrying the delta 4-3-one structure in the A/B cis configuration . Human delta 4-3-oxosteroid 5 beta-reductase cDNA was isolated from a liver cDNA library by cross hybridization with a previously cloned rat cDNA, which was used as a probe {Onishi, Y . Noshiro, M., Shimosato, T . & Okuda, K.-I . (1991) FEBS Lett . 283, 215-218} . DNA sequence analysis of a hybridization-positive clone predicted the human delta 4-3-oxosteroid 5 beta-reductase to contain 326 amino acids . The amino acid sequence of the human delta 4-3-oxosteroid 5 beta-reductase had 79% overall identity to the rat enzyme sequence . It also showed 54% and 50% overall identity with rat 3 alpha-hydroxysteroid dehydrogenase and human aldose reductase, respectively . RNA blotting analysis demonstrated the existence of a single delta 4-3-oxosteroid 5 beta-reductase mRNA of approximately 2.7 kb in human liver . Transfection of the cDNA into COS cells resulted in the expression of an active enzyme with a high activity toward the bile acid intermediates 7 alpha,12 alpha-dihydroxy-4-cholesten-3-one and 7 alpha-hydroxy-4-cholesten-3-one . In addition, the expressed enzyme showed a small but significant 5 beta-reduction activity toward 11 beta,17 alpha,21-trihydroxy-delta 4-pregnene-3,20-dione (cortisol) and 17 beta-hydroxy-delta 4-androsten-3-one (testosterone) whereas no activity was observed toward delta 4-pregnene-3,20-dione (progesterone) or delta 4-androstene-3-17-dione (androstenedione) . The substrate specificity of the human enzyme is considerably narrower than that of the rat enzyme, and the enzyme seems to be more important for bile acid biosynthesis than for metabolism of steroid hormones. Biochem Biophys Res Commun, 1994 Jan 14, 198(1), 127 - 31 Ferric reductases in Escherichia coli: the contribution of the haemoglobin-like protein; Eschenbrenner M et al.; The haemoglobin-like protein (HMP) of E . coli previously isolated as a dihydropteridine reductase was shown to be also a ferric citrate reductase . We demonstrate that, in fact, HMP is a flavin reductase and that its ferric reductase activity is a result of its ability to reduce free flavins . However, when compared to the two main ferric/flavin reductases of E . coli, i.e., the NAD(P)H: flavin oxidoreductase and the sulfite reductase, one can conclude that the contribution of HMP to iron reduction is negligible. J Mol Biol, 1994 Jan 14, 235(2), 780 - 2 Crystallization and X-ray studies of the DNA-binding domain of OmpR protein, a positive regulator involved in activation of osmoregulatory genes in Escherichia coli; Kondo H et al.; The OmpR protein of Escherichia coli is a positive regulator involved in the activation of expression of ompC and ompF genes encoding the major outer membrane protein OmpC and OmpF, respectively . The C-terminal half domain of OmpR (OmpR-C), which is responsible for DNA-binding, has been crystallized using the hanging drop vapour diffusion method . X-ray studies show that the crystals belong to the trigonal space group P3(1)21 (or P3(2)21) with a = b = 60.4 A, c = 58.8 A and gamma = 120 degrees . The asymmetric unit contains one molecule . The crystals diffract to at least 3 A resolution and are suitable for X-ray structure analysis. J Mol Biol, 1994 Jan 14, 235(2), 774 - 6 Sequence and crystallization of Escherichia coli dethiobiotin synthetase, the penultimate enzyme of biotin biosynthesis; Alexeev D et al.; The enzyme dethiobiotin synthetase (EC 6.3.3.3) has been cloned and over-expressed in Escherichia coli in such a way that milligram quantities are available . The purified enzyme has been subjected to a number of physical and chemical studies, sequenced and most notably it has been crystallized in a form that is suitable for X-ray structure determination . The cell dimensions are a = 72.8 A, b = 49.2 A, c = 61.4 A, beta = 106.2 degrees . The systematic absences are consistent with the monoclinic space group C2 with one polypeptide chain in the asymmetric unit. J Mol Biol, 1994 Jan 14, 235(2), 635 - 56 Cytidine deaminase . The 2.3 A crystal structure of an enzyme: transition-state analog complex; Betts L et al.; We have solved the structure of Escherichia coli cytidine deaminase (CDA) complexed to the transition state analog, 5-fluoroprimidin-2-one riboside . The monomer of the alpha 2 CDA dimer is composed of a small N-terminal alpha-helical domain with no obvious connection to the active sites, and two, larger, core domains . The two core domains have nearly identical tertiary structures and are related by approximate 2-fold symmetry, but lack internal amino acid sequence homology . Comparison of the core domain structure with known structures by sequence homology and structural compatibility searches suggests that the CDA tertiary structure cannot be superimposed on any known protein structure . The two active sites per dimer are formed across the subunit interface . The N-terminal core domain provides a pyrimidine nucleoside and zinc-binding pocket and the structurally homologous C-terminal core domain in the other monomer covers this active-site cleft, completely sequestering the ligand from solvent . The deeply buried zinc-binding site is formed by a novel "topological switch point" at the amino termini of two alpha-helices in consecutive alpha-beta-alpha-beta segments . The transition state analog is bound as a covalent hydrate at C4 . The inhibitor hydroxyl oxygen atom interacts both with the zinc atom and the Glu104 carboxylate group, affording high differential affinity for the hydroxyl group relative to a hydrogen atom, in a manner reminiscent of that observed in adenosine deaminase (ADA) . Unlike the latter enzyme, the zinc atom is coordinated in a tetrahedral ligand field to two cysteine and one histidine ligands, plus the hydroxyl group . Moreover, the inhibitor stereochemistry is of the opposite hand from that of the corresponding ADA inhibitor at C4(R), but is the same at the hydroxyl group O4(S) . A consequence of these stereochemical differences is that in CDA a single conserved carboxylate side-chain, Glu104, can provide all of the necessary proton transfer functions involved in generating the zinc hydroxide nucleophile, and protonating the pyrimidine ring nitrogen atom and leaving amino group . The differences in zinc ligands, ligand-binding stereochemistry, and tertiary structures of CDA and ADA strongly suggest that the common features of transition state stabilization arose by convergent evolution. J Mol Biol, 1994 Jan 14, 235(2), 465 - 71 Accuracy of replication past the T-C (6-4) adduct; Horsfall MJ et al.; The thymine-cytosine pyrimidine-pyrimidone (6-4) adduct has variously been predicted to be among the most and among the least mutagenic of the ultraviolet light photoproducts . We have therefore investigated the frequency and accuracy of DNA replication past this lesion, using a single-stranded M13mp7-based vector with a uniquely located example of this lesion transfected into SOS-induced and uninduced cells of a uvr A6 strain of Escherichia coli . Both the UVC T-C (6-4) adduct and its Dewar valence (UVB) photoisomer were studied . Random samples from non-selective collections of progeny phage were sequenced to determine the nature of the replication events that occurred at or near the site of template damage under SOS conditions . The UVC (6-4) adduct was found to be much less mutagenic than its T-T counterpart, but still much more mutagenic than a cyclobutane dimer; 34% (71 out of 206) of all bypass events yielded mutations, of which all were targeted and 80% (57 out of 71) were 3' C-->T transitions . The Dewar valence photoisomer exhibited reduced specificity and enhanced mutagenicity; 79% (183 out of 233) of the phage progeny were mutants, of which all but one were targeted and 45% (83 out of 183) were 3' C-->T transitions . For the most part, these results are consistent with a model postulating base-pairing between the pyrimidinone (of either the C or T variety) and guanine, via hydrogen bonds at N-3 and O-2 in the UVC, but not the Dewar, isomer . The occurrence of the 3' C-->T transitions, not predicted by this model, shows however that the absence of a methyl group at C-5 also has a significant influence on mutation induction . Both isomers were efficient blocks to replication; less than 1% of these vectors could be replicated in uninduced cells . Following SOS induction the frequency of bypass increased to 24.5% and 12.5% for the UVC and the Dewar isomers, respectively. J Mol Biol, 1994 Jan 14, 235(2), 424 - 35 A dominant negative allele of the Escherichia coli uvrD gene encoding DNA helicase II . A biochemical and genetic characterization; George JW et al.; A site-specific lysine to methionine mutation has been engineered at the invariant Lys35 residue in the ATPase A binding site of the Escherichia coli uvrD gene encoding DNA helicase II . The mutant protein (UvrDK35M) has been purified to apparent homogeneity and characterized . The kcat for DNA-dependent ATP hydrolysis was less than 0.5% that of the wild-type enzyme with no change in the apparent Km for ATP . No unwinding of partial duplex DNA substrates could be detected using the mutant protein . Moreover, the mutant protein inhibited the unwinding reaction catalyzed by the wild-type protein at ratios of mutant enzyme to wild-type enzyme < 1 . We conclude that the K35M mutation renders helicase II catalytically inactive as a DNA helicase with little or no effect on the ability of the enzyme to bind ATP, DNA, or other proteins . In vivo complementation assays indicate that the mutant protein cannot substitute for the wild-type protein in methyl-directed mismatch repair, suggesting that the ATPase and/or helicase activity of helicase II is required in this repair pathway . Additional genetic characterization of the uvrDK35M allele, supplied on a plasmid, suggests that expression of the mutant protein, at levels equivalent to that of the wild-type protein, results in a dominant negative phenotype . Expression of lower levels of the mutant protein, both in the presence and absence of wild-type helicase II, results in a constitutive induction of the cellular SOS response and extensive filamentation of cells . This induction of the SOS response is not due to a defect in methyl-directed mismatch repair . Taken together, these data are consistent with the notion that E . coli helicase II may have a role in DNA replication. J Mol Biol, 1994 Jan 14, 235(2), 414 - 23 recA-independent and recA-dependent intramolecular plasmid recombination . Differential homology requirement and distance effect; Bi X et al.; We show that recombination between directly repeated sequences in plasmids occurs via both recA-independent and recA-dependent mechanisms in Escherichia coli . They are differentially affected by two factors, the distance separating the homologies and the size of the homology . Recombination between tandem duplications up to 300 base-pairs shows virtually no recA dependence . Increasing the size of the duplications beyond 300 base-pairs gradually increases the recA dependence . Furthermore, insertion of a sizable DNA sequence in between the duplications, substantially increases the recA dependence . We conclude that increasing the distance separating the homologous regions preferentially inhibits the recA-independent recombination . On the other hand, shortening of the homology preferentially inhibits recA-dependent recombination . Consequently, recombination between short tandem duplications is totally recA-independent. J Mol Biol, 1994 Jan 14, 235(2), 405 - 13 Role of the sigma 70 subunit of Escherichia coli RNA polymerase in transcription activation; Kumar A et al.; The role of the sigma 70 subunit of Escherichia coli RNA polymerase in transcription activation by positive transcription factors was investigated . For this purpose, we constructed a nested set of E . coli rpoD deletion mutants generating carboxy-terminally truncated sigma 70 subunits of RNA polymerase in a high-expression plasmid . The purified mutant sigma 70 subunits were reconstituted into holoenzymes and examined in vitro for their promoter selectivity . As expected, since the -35 recognition helix of sigma 70 was deleted in all cases, the mutant enzymes were unable to initiate at factor-independent promoters, except for the special case of perfect "extended minus 10" promoters, at which the need for -35 sequence recognition by RNA polymerase is replaced by recognition of additional base-pairs in the -10 region . However, two factor-dependent promoters, PhoB-dependent PpstS and cAMP receptor protein (CRP)-dependent P1gal, could be activated for transcription by different subsets of the mutant holoenzymes, although these promoters do not contain the perfect extended -10 sequences . These results establish that -35 DNA recognition by sigma 70 is not essential for these cases . Presumably it is replaced by protein-protein contacts between RNA polymerase and the activator, which in both cases is bound to the DNA in a position overlapping the -35 region . Further, the detailed results support the view that the contact and/or activation sites for these two factors may lie on the sigma 70 subunit, within a "contact site II", which extends at least from conserved region 3.2 to the upstream end of region 4.2 . Moreover, as in the case of contact site I on the alpha subunit, it appears that contact site II contains various different subsites for interaction with specific class II activators, and that PhoB and CRP require distinct subsites. J Biol Chem, 1994 Jan 14, 269(2), 944 - 54 Molecular mechanism of the synergistic phosphorylation of phosphatase inhibitor-2 . Cloning, expression, and site-directed mutagenesis of inhibitor-2; Park IK et al.; Inhibitor-2 (I-2) is the regulatory subunit of the ATP-Mg-dependent phosphatase, a cytosolic form of type 1 protein phosphatase . Phosphorylation of I-2 at Thr-72 by the protein kinase glycogen synthase kinase-3 (GSK-3) leads to activation of the enzyme . Casein kinase II action was shown to synergistically enhance phosphorylation and activation by GSK-3 (DePaoli-Roach, A.A . (1984) J . Biol . Chem . 259, 12144-12152) . Rabbit skeletal muscle and liver I-2 cDNA clones have been isolated . Rabbit skeletal muscle cDNAs could be placed in two subtypes, differing in the length of the 3'-untranslated region . The coding sequence of 612 nucleotides was identical in the two skeletal muscle and the liver cDNAs and predicted a protein of 204 amino acids, consistent with analysis of the purified protein . Northern hybridization analysis indicated that the two mRNAs of 1.7 and 2.7 kilobase pairs were present in all rabbit tissues examined, except in liver, where only the larger transcript was detected, and in testis, where additional transcripts were present . Expression in Escherichia coli of wild-type and phosphorylation site mutants resulted in the production of I-2 polypeptides with apparent M(r) values of approximately 31,000 on sodium dodecyl sulfate-polyacrylamide gel electrophoresis . The inhibitory activity of the recombinant proteins was similar to that of native rabbit skeletal muscle I-2 and was unaffected by the substitution of alanine for the GSK-3 site (Thr-72) and for the casein kinase II sites (Ser-86 and Ser-120/121) or by substitution of glutamic acid and aspartic acid for Thr-72 and Ser-86 . Recombinant wild-type I-2 and the Ala-120/121 mutant were phosphorylated synergistically by GSK-3 and casein kinase II . The Thr-72 and Ser-86 mutants, however, did not undergo this synergistic phosphorylation . Our studies indicate that Thr-72 is the only GSK-3 site and that Ser-86 is the casein kinase II site required for the potentiation of GSK-3 action . Furthermore, acidic residues cannot substitute for the phosphate group either in enhancing GSK-3 phosphorylation or in activating the phosphatase. J Biol Chem, 1994 Jan 14, 269(2), 868 - 71 Identification of Lys277 at the active site of Escherichia coli glycogen synthase . Application of affinity labeling combined with site-directed mutagenesis; Furukawa K et al.; Lys15 in Escherichia coli glycogen synthase, which is specifically labeled by adenosine diphosphopyridoxal, is mainly involved in binding of the substrate ADP-glucose (Furukawa, K., Tagaya, M., Tanizawa, K., and Fukui, T . (1993) J . Biol . Chem . 268, 23837-23842) . We have found that the mutant glycogen synthase in which Lys15 is replaced by Gln via site-directed mutagenesis is inactivated by adenosine diphosphopyridoxal at concentrations higher than those required for the inactivation of the wild-type enzyme . ADP and ADP-glucose offered protective effects on inactivation, suggesting that the label binds to the ADP-glucose-binding site in the mutant enzyme . Sequence analysis of the labeled peptide revealed that the labeled residue is Lys277 . This lysyl residue is conserved in maize starch synthase, which shows about 30% amino acid identity to E . coli glycogen synthase . Substitution of Gln for Lys277 by site-directed mutagenesis resulted in a 140-fold decrease in the kcat value with little changes in the Km values for ADP-glucose and glycogen . These results suggest that Lys277 at the active site participates in the catalytic reaction rather than binding of substrate . The present study shows the usefulness of the combined application of affinity labeling and site-directed mutagenesis. J Biol Chem, 1994 Jan 14, 269(2), 1560 - 3 A dual role for phosphatidylglycerol in protein translocation across the Escherichia coli inner membrane; Kusters R et al.; The involvement of phosphatidylglycerol in the SecA-independent translocation of M13 procoat in Escherichia coli was demonstrated . Processing of procoat to mature coat protein was retarded when the level of phosphatidylglycerol was reduced . In vitro translocation experiments using inner membrane vesicles isolated from a strain with inducible synthesis of phosphatidylglycerol, showed that translocation of procoat and of a SecA-dependent procoat analog was proportional to the content of phosphatidylglycerol . Moreover, introduction of phosphatidylglycerol by means of a lipid transfer method into phosphatidylglycerol-depleted inner membrane vesicles, efficiently restored procoat translocation . The phosphatidylglycerol dependence in both the SecA-dependent and -independent translocation pathway indicates that phosphatidylglycerol plays a dual role in translocation . We suggest that besides membrane binding of SecA this lipid has a direct interaction with the M13 procoat in translocation across the inner membrane. J Biol Chem, 1994 Jan 14, 269(2), 1501 - 5 Coupled replication-translation of amplifiable messenger RNA . A cell-free protein synthesis system that mimics viral infection; Ryabova L et al.; Amplifiable messenger RNAs (Wu, Y., Zhang, D . Y., and Kramer, F . R . (1992) Proc . Natl . Acad . Sci . U.S.A . 89, 11769-11773) were used as templates in coupled replication-translation reactions . These amplifiable mRNAs contained a preselected messenger sequence embedded within the sequence of MDV-1 RNA, which is a small, naturally occurring template for Q beta replicase . When these recombinant mRNAs were incubated in vitro in reactions that contained both an Escherichia coli cell-free translation system and Q beta replicase, the encoded protein was synthesized more efficiently than in corresponding reactions that did not contain Q beta replicase . Moreover, when coupled replication-translation reactions were carried out in a continuous-flow format (Spirin, A . S., Baranov, V . I., Ryabova, L . A., Ovodov, S . Yu., and Alakhov, Yu . B . (1988) Science 242, 1162-1164), the synthesis of biologically active protein continued for a prolonged period . The results suggest that the mechanism of replication and translation in coupled reactions is similar to the mechanism by which Q beta phage genomic RNA is simultaneously replicated and translated in Q beta-infected E . coli: protein synthesis occurs on nascent RNA strands; many more sense strands are synthesized than antisense strands; and the integrity of the messenger sequence is preserved because a relatively small number of antisense strands serve as master templates for the synthesis of new messenger strands. J Biol Chem, 1994 Jan 14, 269(2), 1380 - 7 Identification of discrete structural domains in the retinoblastoma protein . Amino-terminal domain is required for its oligomerization; Hensey CE et al.; To characterize the protein product of the retinoblastoma tumor suppressor gene biochemically, a recombinant human protein was produced in an Escherichia coli expression system . The full-length protein, p110RB, and an amino-terminal truncated form, p56RB, were expressed and purified to near homogeneity by conventional chromatographic procedures . To probe the structural organization of the retinoblastoma protein the purified proteins were subjected to partial proteolysis by trypsin, chymotrypsin, and subtilisin . Four discrete structural domains were revealed in p110RB by this method . Two of these structural domains, found in both p56RB and p110RB, were mapped to the carboxyl-terminal half of the protein and corresponded to the SV40 large T binding domains defined previously by genetic methods . In addition two distinct domains in the amino-terminal half of the protein were also defined . A potential role for these newly defined amino-terminal domains was uncovered upon analysis of the purified proteins by nondenaturing polyacrylamide gel electrophoresis . p110RB revealed multiple bands by this method, suggesting the formation of oligomeric structures by the protein, while this property was not observed for p56RB . Electron microscopy of p110RB revealed linearly extended, macromolecular structures, further supporting the formation of homologous higher order structures by the full-length retinoblastoma protein . Analysis of the interactions between retinoblastoma protein molecules using the yeast two-hybrid system confirmed that the retinoblastoma protein could self-associate and that this association was mediated by interactions between the amino- and carboxyl-terminal ends of the protein . These observations suggest that the retinoblastoma protein contains multiple structural domains with the amino-terminal domains being required for oligomerization of the full-length protein. J Biol Chem, 1994 Jan 14, 269(2), 1243 - 8 Transport of an export-defective protein by a highly hydrophobic signal peptide; Rusch SL et al.; We have examined the sequence constraints on the amino-terminal region of the mature portion of alkaline phosphatase that are important for its efficient transport in Escherichia coli . Using a homopolymeric sequence of serines to replace 6 residues in this region, a transport-incompetent mutant was produced . Reintroduction of residues from the native sequence which restore charge and beta-turn potential resulted in little improvement . However, by replacing the hydrophobic core of the signal peptide with a homopolymeric series of leucines, not only was transport restored but precursor processing was more efficient than for the wild type and was insensitive to disruption of the protonmotive force . Moreover, we have titrated the signal peptide with leucine to alanine substitutions (Doud, S . K., Chou, M . M., and Kendall, D . A . (1993) Biochemistry 32, 1251-1256) and determined the minimum level of hydrophobicity necessary to achieve transport of the mutant protein . The results indicate that signal peptide hydrophobicity can completely override possible requirements for negatively charged residues and strong beta-turn forming potential in the mature protein and that the polyleucine-containing signal peptide may act as a generic signal sequence for the transport of non-native proteins in E . coli. J Biol Chem, 1994 Jan 14, 269(2), 1217 - 21 The active site of hamster 3-hydroxy-3-methylglutaryl-CoA reductase resides at the subunit interface and incorporates catalytically essential acidic residues from separate polypeptides; Frimpong K et al.; We employed site-directed mutagenesis based on sequence comparisons and characterization of purified mutant enzymes to identify Glu558 and Asp766 of Syrian hamster 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase (EC 1.1.1.34) as essential for catalysis . Mutant enzymes E558D, E558Q, and D766N had wild-type Km values for (S)-HMG-CoA and NADPH, but exhibited less than 0.5% of the wild-type catalytic activity . The inactive mutant polypeptides E558Q and D766N nevertheless can associate to generate an active enzyme . In vitro, 6% of the wild-type activity was observed when mutant polypeptides E558D and D766N were mixed in the absence of chaotropic agents . When mutant polypeptides E558Q and D766N were co-expressed in Escherichia coli, the resulting purified enzyme had 25% of wild-type activity . Hamster HMG-CoA reductase thus is a two-site, dimeric enzyme whose subunits associate to form an active site in which each monomer contributes at least one residue (e.g . Glu558 from one monomer and Asp766 from the other) . The wild-type enzyme behaves as a dimer during size exclusion chromatography and has one HMG-CoA binding site per monomer . Syrian hamster HMG-CoA reductase thus appears to be a homodimer with two active sites which are located at the subunit interface. J Biol Chem, 1994 Jan 14, 269(2), 1083 - 90 1H NMR study of the solution molecular and electronic structure of engineered distal myoglobin His64(E7) Val/Val68(E11) His double mutant . Coordination of His64(E11) at the sixth position in both low-spin and high-spin states; Qin J et al.; A genetically engineered human myoglobin (Mb) in which the distal His, His64(E7), and the distal Val, Val68(E11), are replaced by Val and His, respectively, has been expressed in Escherichia coli, for the purpose of assessing the potential role of a E11 residue in providing a hydrogen bond donor to the coordinated ligand . Molecular modeling indicates that such an interaction is possible . The 1H NMR spectrum of the ferric form of the double mutant Mb exhibits large hyperfine shifts and strong paramagnetic relaxation for which the temperature dependence of the hyperfine shifts reveals a thermal equilibrium between a low-spin and high-spin state (70, 30% at 25 degrees C, respectively) . Standard sequence specific two-dimensional (2D) NMR assignments of the E and F helical backbones allow the identification of the peptide protons for the proximal His93(F8) and substituted distal His68(E11) . Steady-state nuclear Overhauser effect from these peptide protons locate strongly hyperfine shifted His93(F8) and His68(E11) side chain protons which dictate that both the imidazole rings are coordinated to the iron . 2D bond correlation and one-dimensional and 2D dipolar correlation experiments locate and assign the resonances for the heme . The pattern of the heme contact shifts in both the low-spin and high-spin state, together with the nature of the temperature dependence of the His93(F8) and His68(E11) resonances, establish that the two His are ligated in the high-spin as well as low-spin forms . The pattern of heme methyl hyperfine shifts in the low-spin state, and the smaller hyperfine shifts for His68(E11) as compared to His93(F8) in the high-spin state, indicate that the axial bond to the distal His68(E11) is weakened or strained as compared with that for the proximal His93(F8) in both spin states . This weak ligation originates from a tilted iron-His68 bond, the only conformation in which His68 can place its imidazole group sufficiently close to bind to the heme iron in the conventional Mb folding . Not only do these results support the belief that distal His is indispensable for the control of the ligand binding in Mb and hemoglobin, but also reveal the significance of the evolution that the stereochemical disposition of both His64 and Val68 are unique and non-exchangeable for interacting with the bound ligand. J Biol Chem, 1994 Jan 14, 269(2), 1057 - 62 Stabilization of the EBNA1 protein on the Epstein-Barr virus latent origin of DNA replication by a DNA looping mechanism; Frappier L et al.; DNA replication from the Epstein-Barr virus latent origin of replication, oriP, is activated by Epstein-Barr nuclear antigen 1 (EBNA1) . This activation involves the binding of EBNA1 dimers to multiple sites present in the two noncontiguous functional elements of oriP, the dyad symmetry element (DS) from which replication initiates, and the family of repeats (FR) enhancer element . EBNA1 complexes formed on the FR and DS elements of oriP interact by a DNA looping mechanism . This interaction requires EBNA1 sequences in addition to those required for DNA binding and dimerization . To map the EBNA1 sequences required for the efficient interaction of FR- and DS-bound EBNA1 complexes, we have overproduced in Escherichia coli and purified a series of EBNA1 N-terminal truncation mutants, all of which retain the DNA binding and dimerization domains . The results of electron microscopy and ligation-enhancement assays using these mutants indicated that EBNA1 sequences between amino acids 350 and 361 are required for the efficient interaction of FR- and DS-bound EBNA1 complexes . EBNA1-mediated FR-DS interactions were shown to stabilize EBNA1 binding to the DS element, while EBNA1-mediated DS-DS interactions did not . These results suggest that the stabilization of EBNA1 on the DS element, which occurs as a result of EBNA1-mediate oriP looping, may be important for the activation of DNA replication from the DS element. Science, 1994 Jan 14, 263(5144), 227 - 30 inhA, a gene encoding a target for isoniazid and ethionamide in Mycobacterium tuberculosis; Banerjee A et al.; Isoniazid (isonicotinic acid hydrazide, INH) is one of the most widely used antituberculosis drugs, yet its precise target of action on Mycobacterium tuberculosis is unknown . A missense mutation within the mycobacterial inhA gene was shown to confer resistance to both INH and ethionamide (ETH) in M . smegmatis and in M . bovis . The wild-type inhA gene also conferred INH and ETH resistance when transferred on a multicopy plasmid vector to M . smegmatis and M . bovis BCG . The InhA protein shows significant sequence conservation with the Escherichia coli enzyme EnvM, and cell-free assays indicate that it may be involved in mycolic acid biosynthesis . These results suggest that InhA is likely a primary target of action for INH and ETH. J Biol Chem, 1994 Jan 14, 269(2), 992 - 1000 Estimation of genomic complexity, heterologous expression, and enzymatic characterization of mouse glutathione S-transferase mGSTA4-4 (GST 5.7); Zimniak P et al.; We have previously isolated a cDNA clone for a unique mouse lung glutathione S-transferase, mGSTA4-4 (GST 5.7) (Zimniak, P., Eckles, M . A., Saxena, M., and Awasthi, Y . C . (1992) FEBS Lett . 313, 173-176) . By genomic Southern blotting and polymerase chain reaction single strand conformation polymorphism analysis we have now demonstrated the presence of at least two mGSTA4-related genes in the mouse . The heterogeneity of mGSTA4-4 was further examined by comparing the structural and kinetic properties of mGSTA4-4 isolated from mouse lung with those of recombinant rec-mGSTA4-4 expressed in Escherichia coli . Except for the isoelectric point, the physical properties of the two proteins were indistinguishable . Western blots using antibodies against rec-mGSTA4-4 have shown selective expression of the enzyme in mouse tissues . Even though the substrate specificity profiles of the tissue-isolated and recombinant enzymes, which point to a role of mGSTA4-4 in the detoxification of lipid peroxidation products, were generally similar, significant differences were observed with selected substrates . The existence of functionally distinct forms of mGSTA4-4 and the presence of more than one gene strongly suggest that the previously observed differences in properties of mGSTA4-4 isolated from various mouse tissues (Awasthi, S., Singhal, S . S., Srivastava, S . K., and Awasthi, Y . C . (1993) Arch . Biochem . Biophys . 301, 143-150) may be due to tissue-specific expression of mGSTA4-related genes. J Biol Chem, 1994 Jan 14, 269(2), 986 - 91 Strand displacement activity of the human immunodeficiency virus type 1 reverse transcriptase heterodimer and its individual subunits; Hottiger M et al.; By using a DNA substrate with defined gap size, we found that human immunodeficiency virus type 1 reverse transcriptase (HIV-RT) was able to perform strand displacement DNA synthesis . This activity was not affected first by calf thymus proliferating cell nuclear antigen and replication factor C and second by Escherichia coli single-stranded DNA-binding protein, which together allow DNA polymerase delta to perform strand displacement DNA synthesis (Podust, V., and Hubscher, U . (1993) Nucleic Acids Res . 21, 841-846) . 3'-Azido-2',3'-dideoxythymidine triphosphate inhibited displacement completely, indicating that DNA synthesis is required for this reaction . The HIV-RT p66 polypeptide alone could perform limited strand displacement DNA synthesis, whereas the HIV-RT p51 polypeptide was completely inactive, likely due to its inability to replicate extensively on a M13 DNA template . On the other hand the HIV-RT p51 polypeptide enhanced the strand displacement activity of the HIV-RT p66 subunit at a molar ratio of 4:1, mainly by chasing short products into longer ones . Furthermore, kinetic experiments after complementation of HIV-RT p66 with HIV-RT p51 indicated that HIV-RT p51 can restore rate and extent of strand displacement activity by HIV-RT p66 compared with the HIV-RT heterodimer p66/p51, suggesting a function of the 51-kDa polypeptide. Science, 1994 Jan 14, 263(5144), 191 - 7 The transfer RNA identity problem: a search for rules; Saks ME et al.; Correct recognition of transfer RNAs (tRNAs) by aminoacyl-tRNA synthetases is central to the maintenance of translational fidelity . The hypothesis that synthetases recognize anticodon nucleotides was proposed in 1964 and had considerable experimental support by the mid-1970s . Nevertheless, the idea was not widely accepted until relatively recently in part because the methodologies initially available for examining tRNA recognition proved hampering for adequately testing alternative hypotheses . Implementation of new technologies has led to a reasonably complete picture of how tRNAs are recognized . The anticodon is indeed important for 17 of the 20 Escherichia coli isoaccepting groups . For many of the isoaccepting groups, the acceptor stem or position 73 (or both) is important as well. Biochemistry, 1994 Jan 11, 33(1), 235 - 40 Regiospecificity of the hydrolysis of diadenosine polyphosphates catalyzed by three specific pyrophosphohydrolases; Guranowski A et al.; The different patterns of enzymatic cleavage of diadenosine polyphosphates, ApnAs, where n = 3-5, have been established by fast atom bombardment mass spectrometry, FAB MS, of the nucleotide products formed in the presence of H2(18)O . The three specific pyrophosphohydrolases, Ap3A hydrolase (EC 3.6.1.29) and (asymmetrical) Ap4A hydrolase (EC 3.6.1.17) from lupin and the (symmetrical) Ap4A hydrolase (EC 3.6.1.41) from Escherichia coli, manifest three different regiospecificities . The Ap3A hydrolase cleaves all four substrates tested, Ap3A, Ap4A, ApCH2ppA, and ApCHFppA, to give {18O}AMP and the corresponding unlabeled adenosine nucleotide . In each case, the enzyme cleaves at the phosphate proximate to the bound adenosine moiety . The (asymmetrical) Ap4A hydrolase cleaves both Ap4A and Ap5A to give unlabeled ATP plus {18O}AMP and {18O}ADP, respectively, and is thus seen to add water at the fourth phosphate from the bound adenosine moiety . Lastly, the (symmetrical) Ap4A hydrolase from E . coli gives beta-{18O}ADP from Ap3A, Ap4A, and Ap5A along with the unlabeled nucleotide coproducts . In addition, with Ap4A alpha S (ApspppA) as substrate for the bacterial enzyme, the products are beta-{18O}ADP and unlabeled ADP alpha S . This symmetrical enzyme is thus characterized as cleaving the polyphosphate chain at the second phosphate from the bound adenosine moiety.
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