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 quantit