J Mol Endocrinol, 1994 Aug, 13(1), 77 - 86 Molecular cloning, sequence analysis and expression of human pituitary glutaminyl cyclase; Song I et al.; A cDNA clone for glutaminyl cyclase was isolated from a human pituitary cDNA library and the complete DNA sequence determined . The cDNA clone had 1573 bp and contained an open reading frame of 1086 bases, coding for a protein of 361 amino acids and molecular mass of 40,876 Da . The predicted amino acid sequence of the human cDNA showed 86% sequence identity to the previously reported bovine glutaminyl cyclase sequence . A comparison of the amino acid sequences derived from the human and bovine cDNAs showed that several glycosylation and phosphorylation sites as well as two cysteine residues (Cys139, Cys164) were conserved . The human cDNA was cloned into the Escherichia coli expression vectors pMALc2 and pET19b . Expression of this cDNA in either vector resulted in the production of a glutaminyl cyclase fusion protein which was enzymatically active and reacted with anti-bovine glutaminyl cyclase antisera . Substrate specificity studies with the recombinant enzyme suggested a bias against acidic and tryptophan residues adjacent to the N-terminal glutaminyl residue and a lack of importance of chain length after the second residue. Mol Microbiol, 1994 Aug, 13(4), 627 - 40 Partial suppression of an Escherichia coli TonB transmembrane domain mutation (delta V17) by a missense mutation in ExbB; Larsen RA et al.; Active transport of vitamin B12 and Fe(III)-siderophore complexes across the outer membrane of Escherichia coli appears to be dependent upon the ability of the TonB protein to couple cytoplasmic membrane-generated protonmotive force to outer membrane receptors . TonB is supported in this role by an auxiliary protein, ExbB, which, in addition to stabilizing TonB against the activities of endogenous envelope proteases, directly contributes to the energy transduction process . The topological partitioning of TonB and ExbB to either side of the cytoplasmic membrane restricts the sites of interaction between these proteins primarily to their transmembrane domains . In this study, deletion of valine 17 within the aminoterminal transmembrane anchor of TonB resulted in complete loss of TonB activity, as well as loss of detectable in vivo crosslinking into a 59 kDa complex believed to contain ExbB . The delta V17 mutation had no effect on TonB export . The loss of crosslinking appeared to reflect conformational changes in the TonB/ExbB pair rather than loss of interaction since ExbB was still required for some stabilization of TonB delta V17 . Molecular modeling suggested that the delta V17 mutation caused a significant change in the predicted conserved face of the TonB amino-terminal membrane anchor . TonB delta V17 was unable to achieve the 23 kDa proteinase K-resistant form in lysed sphaeroplasts that is characteristic of active TonB . Wild-type TonB also failed to achieve the proteinase K-resistant configuration when ExbB was absent . Taken together these results suggested that the delta V17 mutation interrupted productive TonB-ExbB interactions . The apparent ability to crosslink to ExbB as well as a limited ability to transduce energy were restored by a second mutation (A39E) in or near the first predicted transmembrane domain of the ExbB protein . Consistent with the weak suppression, a 23 kDa proteinase K-resistant form of TonB delta V17 was not observed in the presence of ExbBA39E . Neither the ExbBA39E allele nor the absence of ExbB affected TonB or TonB delta V17 export . Unlike the tonB delta V17 mutation, the exbBA39E mutation did not greatly alter a modelled ExbB transmembrane domain structure . Furthermore, the suppressor ExbBA39E functioned normally with wild-type TonB, suggesting that the suppressor was not allele specific . Contrary to expectations, the TonB delta V17, ExbBA39E pair resulted in a TonB with a greatly reduced half-life (approximately 10 min) . These results together with protease susceptibility studies suggest that ExbB functions by modulating the conformation of TonB. Mol Microbiol, 1994 Aug, 13(4), 609 - 18 Mutant Escherichia coli arginine repressor proteins that fail to bind L-arginine, yet retain the ability to bind their normal DNA-binding sites; Burke M et al.; The Escherichia coli arginine repressor (ArgR) is an L-arginine-dependent DNA-binding protein that controls expression of the arginine biosynthetic genes and is required as an accessory protein in Xer site-specific recombination at cer and related recombination sites in plasmids . Site-directed mutagenesis was used to isolate two mutants of E . coli ArgR that were defective in arginine binding . Results from in vivo and in vitro experiments demonstrate that these mutants still act as repressors and bind their specific DNA sequences in an arginine-independent manner . Both mutants support Xer site-specific recombination at cer . One of the mutant proteins was purified and shown to bind to its DNA target sequences in vitro with different affinity and as a different molecular species to wild-type ArgR. Mol Microbiol, 1994 Aug, 13(4), 599 - 608 Mutational analysis of the arginine repressor of Escherichia coli; Tian G et al.; Arginine biosynthesis in Escherichia coli is negatively regulated by a hexameric repressor protein, encoded by the gene argR and the corepressor arginine . By hydroxylamine mutagenesis two types of argR mutants were isolated and mapped . The first type is transdominant . In heterodiploids, these mutant polypeptides reduce the activity of the wild-type repressor, presumably by forming heteropolymers . Four mutant repressor proteins were purified . Two of these map in the N-terminal half of the protein . Gel retardation experiments showed that they bind poorly to DNA, but they could be precipitated by L-arginine at the same concentration as the wild-type repressor . The other two mutant repressors map in the C-terminal half of the protein . They are poorly precipitated by L-arginine and they bind poorly to DNA . In addition, one of these mutants appears to exist as a dimer . The second type of argR mutant repressor consists of super-repressors . Such mutants behave as arginine auxotrophs as a result of hyper-repression of arginine biosynthetic enzymes . They map at many locations throughout the argR gene . Three arginine super-repressor proteins were purified . In comparison with the wild-type repressor, two of them were shown to have a higher DNA-binding affinity in the absence of bound arginine, while the third was shown to have a higher DNA-binding affinity when bound to arginine. Mol Microbiol, 1994 Aug, 13(3), 513 - 23 Identification of a partition region carried by the plasmid QpH1 of Coxiella burnetii; Lin Z et al.; Coxiella burnetii is an intracellular bacterial pathogen which causes Q fever in humans and other animals . Most of the isolates found carry plasmids which share considerable homology . Unfortunately all of these plasmids remain cryptic . Initial attempts to look for secreted or membrane proteins encoded by these plasmids using TnphoA mutagenesis revealed an open reading frame on the EcoRI-fragment C of the plasmid QpH1 . Upstream DNA sequencing of the TnphoA insertions revealed a deduced peptide sequence with homology to the SopA protein which is encoded by the F plasmid in Escherichia coli . Maxicell analysis showed that fragment C encoded two proteins: one was 43.5 kDa in size and designated QsopA, and a second was 38 kDa in size . These proteins are similar in molecular weight to the SopA and SopB proteins, which are essential components of the partition mechanism of the F plasmid . The region appears to be conserved in plasmids QpRS, QpDV, and QpDG, but is absent in a plasmidless isolate in which plasmid sequences have integrated into the chromosomal DNA . Complementation studies demonstrated that fragment C has a plasmid partitioning function and can restore maintenance stability of the partition-defective mini-F plasmid . These data suggest that fragment C carries the plasmid partition region of the plasmid QpH1. Mol Microbiol, 1994 Aug, 13(3), 475 - 83 An analogue of the DnaJ molecular chaperone whose expression is controlled by sigma s during the stationary phase and phosphate starvation in Escherichia coli; Yamashino T et al.; The Escherichia coli CbpA protein appears to be an analogue of the molecular chaperone, DnaJ, as judged from not only its structure but also its possible function . The expression of cbpA, however, was not significantly affected by up-shift of the growth temperature . Remarkably, it was found that the expression of cbpA was induced under certain growth conditions, such as the entry of cells into stationary phase, or growth in a phosphate-limited medium . Such conditional expression of cbpA was regulated at the transcriptional level in a sigma s-dependent manner . The structure of this sigma s-dependent cbpA promoter was clarified by determining its transcription start site . The cbpA promoter region was found to contain an unusual DNA structure (i.e . DNA curvature) . From these results, it was suggested that, in contrast to DnaJ, CbpA may function as a molecular chaperone in an adaptive response to environmental stresses other than heat shock. Mol Microbiol, 1994 Aug, 13(3), 469 - 74 Neither absence nor excess of lambda O initiator-digesting ClpXP protease affects lambda plasmid or phage replication in Escherichia coli; Szalewska A et al.; Owing to rapid proteolysis of the coliphage lambda-coded initiator protein, lambda O, this protein is considered to carry a rate-limiting step in lambda DNA replication . The discovery of ClpXP protease responsible for lambda O protein turnover allowed an opportunity to verify this hypothesis . However, neither absence nor excess of this protease significantly affected the transformation efficiency and copy number of lambda plasmid, or the kinetics of the lambda phage growth . These results are also incompatible with the hypothesis that the stabilization of lambda O plays a role in the switch from early (circle-to-circle) to late (rolling-circle) lambda phage DNA replication . Transcriptional activation of ori lambda, probably assisted by the Escherichia coli DnaA function, remains as the possible rate-limiting step in lambda DNA replication. Mol Microbiol, 1994 Aug, 13(3), 459 - 67 The protein HU can displace the LexA repressor from its DNA-binding sites; Preobrajenskaya O et al.; The major bacterial histone-like protein HU is a small, basic, dimeric protein composed of two closely related subunits . HU is involved in several processes in the bacterial cell such as the initiation of replication, transposition, gene inversion and cell division . It has been suggested that HU could introduce structural changes to the DNA which would facilitate or inhibit the binding of regulatory proteins to their specific sites . In this study we investigated the effect of HU on the binding of LexA protein, the regulator of SOS functions, to three of its specific binding sites . We show that HU can displace LexA from its binding sites on the operators of the lexA, recA and sfiA genes . The lexA operator was the most sensitive while the higher affinity sfiA operator was the least sensitive . Since HU, like its homologue IHF, probably binds DNA in the minor groove we tested the effect of distamycin, a drug which binds to the minor groove, on LexA binding . Like HU, this drug disrupted LexA-operator complexes . These results suggest that distortion of the minor groove of the lexA operators excludes the binding of the repressor to the major groove. Mol Microbiol, 1994 Aug, 13(3), 445 - 57 EGTA induces the synthesis in Escherichia coli of three proteins that cross-react with calmodulin antibodies; Laoudj D et al.; Escherichia coli mutants, (verA, dilA) specifically resistant to the Ca2+ channel inhibitors verapamil and diltiazem, respectively, are hypersensitive to EGTA and BAPTA . We have shown, using 1-D and 2-D gel electrophoresis, that the synthesis of at least 25 polypeptides in the mutants was enhanced by treatment with Ca2+ chelators and the synthesis of at least 11 polypeptides was repressed . This pattern of induction was not observed in heat- or SDS-treated cells and therefore does not appear to be a general stress response . The majority of the induced proteins are low molecular weight, extremely heat stable and acidic, characteristic properties of calmodulin . Moreover, of the major induced species, three with apparent molecular masses of 12, 18, and 34 kDa all cross-reacted with polyclonal and monoclonal antibodies to eukaryote calmodulins or calerythrin, a heat-resistant Ca(2+)-binding protein from Saccharopolyspora erythraea . The verA, dilA mutants, in being hypersensitive to EGTA and to the Ca2+ ionophore A23187 + Ca2+, may be defective in the regulation of the level of free intracellular Ca2+. Mol Microbiol, 1994 Aug, 13(3), 435 - 44 Transmembrane signal transduction by the Escherichia coli osmotic sensor, EnvZ: intermolecular complementation of transmembrane signalling; Tokishita S et al.; The Escherichia coli regulatory proteins, EnvZ and OmpR, are crucially involved in expression of the outer membrane proteins OmpF/OmpC in response to the medium osmolarity . The EnvZ protein is presumably a membrane-located osmotic sensor (or signal transducer), which exhibits both kinase and phosphatase activities specific for the OmpR protein . To examine the functional importance of the membrane-spanning segments (named TM1 and TM2) of EnvZ molecules in transmembrane signalling, a set of EnvZ mutants, each having amino acid substitutions within the membrane-spanning regions, was characterized in terms of both their in vivo phenotype and in vitro catalytic activities . One of them, characterized further, has an amino acid change (Pro-41 to Ser or Leu) in TM1, and appeared to be defective in its phosphatase activity but not in its kinase activity . This EnvZ mutant conferred a phenotype of OmpF-/OmpC-constitutive . For this EnvZ(P41S or P41L) mutant, a set of intragenic suppressors, each exhibiting a wild-type phenotype of OmpF+/OmpC+, was isolated . These suppressor mutants were revealed to have an additional amino acid change within either TM1 or TM2 . Furthermore, they exhibited restored phosphatase activity (i.e., both kinase+ and phosphatase+ activities) . It was further demonstrated that one of the suppressors, EnvZ(Arg-180 to Trp in TM2), was able to suppress the defects in both the in vivo phenotype and the in vitro catalytic activities caused by EnvZ(P41S), through intermolecular complementation . These results are best interpreted as meaning that an intimate intermolecular interaction between the membrane-spanning segments of EnvZ is crucial for transmembrane signalling per se in response to an external osmotic stimulus. Dev Dyn, 1994 Aug, 200(4), 278 - 93 Murine PGK-1 promoter drives widespread but not uniform expression in transgenic mice; McBurney MW et al.; Pgk-1 is an X-linked gene encoding 3-phosphoglycerate kinase, an enzyme necessary in every cell for glycolysis . The regulatory sequences of the Pgk-1 gene were used to drive the E . coli lacZ reporter gene and 2 strains of transgenic animals created with this Pgk-lacZ transgene carried on autosomes . The levels of expression of Pgk-1 varied from one adult tissue to another and the transgene was similarly regulated . However, in situ staining of the beta-galactosidase encoded by the transgene indicated extensive cell-to-cell variability in its level of expression . A reproducible subset of cells stained darkly for the transgene product . Some of these beta-galactosidase positive cells were rapidly proliferating while others appeared to be metabolically very active, suggesting that the Pgk-1 promoter is regulated so as to be more active in cells requiring high levels of glycolysis . Although Pgk-1 is X-linked and subject to X chromosome inactivation, the transgenes were not inactivated in either female somatic or male germ cells . Thus, the Pgk-1 promoter drives transgene expression in all tissues but the levels of expression are not uniform in each cell. Surg Endosc, 1994 Aug, 8(8), 913 - 4 The lost gallstone . Complication after laparoscopic cholecystectomy; Gallinaro RN et al.; Laparoscopic cholecystectomy has become the treatment of choice for most patients with gallstones . During this procedure it is not uncommon for the gallbladder to be entered inadvertently, spilling gallstones freely into the peritoneal cavity . Finding and removing all of the spilled gallstones can be difficult and time consuming . The natural history of stones left in the peritoneal cavity, outside the gallbladder, bile ducts, or intestine, is not known . This is a case report of a complication related to several gallstones left in the peritoneal cavity after laparoscopic cholecystectomy . An abscess developed around them, which necessitated the drainage of purulent exudate from the right flank 8 months postoperatively . the abscess and sinus tract did not heal until the stones were removed . If possible, all stones should be removed during laparoscopic cholecystectomy to forestall the development of this type of complication. Eur J Pharmacol, 1994 Aug 1, 260(2-3), 265 - 8 Constitutive nitric oxide modulates the injurious actions of vasopressin on rat intestinal microcirculation in acute endotoxaemia; Laszlo F et al.; The administration of the nitric oxide (NO) synthase inhibitor, NG-nitro-L-arginine methyl ester (L-NAME, 5 mg/kg s.c.) concurrently with Escherichia coli endotoxin (3 mg/kg i.v.) increased vascular permeability and caused mucosal damage in the rat intestine 1 h later . The vasopressin V1 receptor antagonist, {Mca1,Tyr(Me)2, Arg8}vasopressin (0.01-0.2 microgram/kg s.c., 15 min before endotoxin) dose-dependently reduced this damage . These results suggest a beneficial role of NO, counteracting the injurious vascular actions of endogenous vasopressin, in maintaining intestinal mucosal integrity in acute endotoxaemic states. Protein Sci, 1994 Aug, 3(8), 1286 - 95 Determination of the structure of the DNA binding domain of gamma delta resolvase in solution; Liu T et al.; The DNA binding domain (DBD) of gamma delta resolvase (residues 141-183) is responsible for the interaction of this site-specific DNA recombinase with consensus site DNA within the gamma delta transposable element in Escherichia coli . Based on chemical-shift comparisons, the proteolytically isolated DBD displays side-chain interactions within a hydrophobic core that are highly similar to those of this domain when part of the intact enzyme (Liu T, Liu DJ, DeRose EF, Mullen GP, 1993, J Biol Chem 268:16309-16315) . The structure of the DBD in solution has been determined using restraints obtained from 2-dimensional proton NMR data and is represented by 17 conformers . Experimental restraints included 458 distances based on analysis of nuclear Overhauser effect connectivities, 17 phi and chi 1 torsion angles based on analysis of couplings, and 17 backbone hydrogen bonds determined from NH exchange data . With respect to the computed average structure, these conformers display an RMS deviation of 0.67 A for the heavy backbone atoms and 1.49 A for all heavy atoms within residues 149-180 . The DBD consists of 3 alpha-helices comprising residues D149-Q157, S162-T167, and R172-N183 . Helix-2 and helix-3 form a backbone fold, which is similar to the canonical helix-turn-helix motif . The conformation of the NH2-terminal residues, G141-R148, appears flexible in solution . A hydrophobic core is formed by side chains donated by essentially all hydrophobic residues within the helices and turns . Helix-1 and helix-3 cross with a right-handed folding topology . The structure is consistent with a mechanism of DNA binding in which contacts are made by the hydrophilic face of helix-3 in the major groove and the amino-terminal arm in the minor groove . This structure represents an important step toward analysis of the mechanism of DNA interaction by gamma delta resolvase and provides initial structure-function comparisons among the divergent DBDs of related resolvases and invertases. Protein Sci, 1994 Aug, 3(8), 1276 - 85 A negative electrostatic determinant mediates the association between the Escherichia coli trp repressor and its operator DNA; Guenot J et al.; The electrostatic potential surfaces were characterized for trp repressor models that bind to DNA with sequence specificity, without specificity, and not at all . Comparisons among the surfaces were used to isolate protein surface features likely to be important in DNA binding . Models that differ in protein conformation and tryptophan-analogue binding consistently showed positive potential associated with the protein surfaces that interact with the DNA major groove . However, negative potential is associated with the trp repressor surface that contacts the DNA minor groove . This negative potential is significantly neutralized in the protein conformation that is bound to DNA . Positive potential is also associated with the tryptophan binding-site surface, a consequence of the tryptophan- or tryptophan analogue-induced allosteric change . This protein region is complementary to the strongest negative potential associated with the DNA phosphate backbone and is also present in the isolated protein structure from the protein-DNA complex . The effects of charge-change mutation, pH dependence, and salt dependence on the electrostatic potential surfaces were also examined with regard to their effects on protein-DNA binding constants . A consistent model is formed that defines a role for long-range electrostatics early in the protein-DNA association process and complements previous structural, molecular association, and mutagenesis studies. Protein Sci, 1994 Aug, 3(8), 1236 - 44 Ligation alters the pathway of urea-induced denaturation of the catalytic trimer of Escherichia coli aspartate transcarbamylase; Bromberg S et al.; We have examined the pathway and energetics of urea-induced dissociation and unfolding of the catalytic trimer (c3) of aspartate transcarbamylase from Escherichia coli at low temperature in the absence and presence of carbamyl phosphate (CP; a substrate), N-(phosphonacetyl)-L-Asp (PALA; a bisubstrate analog), and 2 anionic inhibitors, Cl- and ATP, by analytical gel chromatography supplemented by activity assays and ultraviolet difference spectroscopy . In the absence of active-site ligands and in the presence of ATP, c3 dissociates below 2 M urea into swollen c chains that then gradually unfold from 2 to 6 M urea with little apparent cooperativity . Linear extrapolation to 0 M urea of free energies determined in 3 independent types of experiments yields estimates for delta Gdissociation at 7.5 degrees C of about 7-10 kcal m-1 per interface . delta Gunfolding of dissociated chains when modeled as a 2-state process is estimated to be very small, on the order of -2 kcal m-1 . The data are also consistent with the possibility that the unfolding of the dissociated monomer is a 1-state swelling process . In the presence of the ligands CP and PALA, and in the presence of Cl-, c3 dissociates at much higher urea concentrations, and trimer dissociation and unfolding occur simultaneously and apparently cooperatively, at urea concentrations that increase with the affinity of the ligand. Br J Haematol, 1994 Aug, 87(4), 700 - 7 Recombinant human interleukin-3: pharmacokinetics after intravenous and subcutaneous bolus injection and effects on granulocyte kinetics; Hovgaard DJ et al.; The pharmacokinetics of E . coli derived recombinant human interleukin-3 (rhIL-3) was studied following intravenous (i.v.) and subcutaneous (s.c.) bolus injection of rhIL-3 . After i.v . bolus injection in eight patients, serum peak levels of 34.5-135.0 ng/ml were reached, followed by a rapid decline with a t1/2 alpha of 17 +/- 2 min and a t1/2 beta of 59 +/- 7 min . After s.c . bolus injection in five patients, the absorption was more prolonged with peak serum levels reached at 2.8 +/- 0.4 h . Elimination was also more protracted, and serum base-line levels were reached at 14-24 h . The immediate effect of rhIL-3 on peripheral white blood cells was less pronounced and more variable than previously found for G- or GM-CSF . Following i.v . administration, neutrophils showed a moderate drop to median 64% of initial values (range 42-85%) at median 30 min after injection (range 15-60 min) followed by an increase at 24 h to 69-288% of initial values . Eosinophils dropped to a median nadir of 34% and then gradually increased to maximum values in the range 135-720% at 18-24 h . The effect of rhIL-3 was further examined following i.v . injection of autologous 111Indium-labelled granulocytes in six patients . In steady state, i.v . injection of rhIL-3 caused a moderate drop in 111Indium activity of peripheral blood within 20 min without tendency to subsequent recovery . No change occurred in the activity recorded over the lungs and liver . The activity over the spleen decreased moderately in two patients . These results are strikingly different from those previously obtained after i.v . injection of rhGM-CSF. Genetics, 1994 Aug, 137(4), 895 - 902 Roles of ruvA, ruvC and recG gene functions in normal and DNA damage-inducible replication of the Escherichia coli chromosome; Asai T et al.; Induction of the SOS response in Escherichia coli activates normally repressed DNA replication which is termed inducible stable DNA replication (iSDR) . We previously demonstrated that initiation of iSDR requires the products of genes, such as recA, recB and recC, that are involved in the early stages of homologous recombination . By measuring the copy number increase of the origin (oriM1) region on the chromosome, we show, in this study, that initiation of iSDR is stimulated by mutations in the ruvA, ruvC and recG genes which are involved in the late stages of homologous recombination . Continuation of iSDR, on the other hand, is inhibited by these mutations . The results suggest that Holliday recombination intermediates, left on the chromosome due to abortive recombination, arrest replication fork movement . Low levels of iSDR and sfiA (sulA) gene expression were also observed in exponentially growing ruvA, ruvC and recG mutants, suggesting that the SOS response is chronically induced in these mutants . We propose that replication forks are arrested in these mutants, albeit at a low frequency, even under the normal (uninduced) conditions. Anal Biochem, 1994 Aug 1, 220(2), 397 - 402 Use of a synthetic peptide as a selective substrate for glycogen synthase kinase 3; Wang QM et al.; Glycogen synthase kinase 3 (GSK-3) is involved in the regulation of several metabolic enzymes and transcription factors in response to extracellular signals . Here we report the use of a synthetic peptide derived from the sequence of the cyclic AMP responsive element binding protein (CREB) as a specific substrate for GSK-3 isoforms . The 13-amino acid peptide, KRREILSRRPSYR, was phosphorylated by the catalytic subunit of cAMP-dependent protein kinase (PKA) and purified on a C18 cartridge . Phosphorylation of the COOH-terminal serine of the peptide by PKA creates a phosphorylation site for GSK-3 since GSK-3 recognizes the consensus motif -S-X-X-X-S(P)- . Although the COOH-terminal serine of the peptide can be phosphorylated by PKA and several other kinases, the phospho-CREB peptide is specific for GSK-3 with Kms of 140 and 200 microM for GSK-3 alpha and GSK-3 beta isoforms, respectively . Using the phospho-CREB peptide, we have successfully purified GSK-3 activity from rabbit skeletal muscle and Escherichia coli cells transformed with a GSK-3 expression vector . The assay described provides a convenient and specific determination of GSK-3 activity. Acta Anaesthesiol Scand, 1994 Aug, 38(6), 612 - 4 Traumatic or septic aortic regurgitation: diagnosis by oesophageal echocardiography; Habre WN et al.; Severe aortic regurgitation was discovered in a young man 21 days after blunt chest trauma and after a prolonged febrile state with positive blood cultures . Using transoesophageal echocardiography (TEE), it was possible to make the differential diagnosis between traumatic rupture and endocarditis as the cause of valvular insufficiency . The use of TEE in the initial evaluation of severe thoracic trauma with an unclear clinical picture is recommended . This method is easy to use at the bedside and gives precise information on the aortic valve and the ascending aorta. Vaccine, 1994 Aug, 12(10), 953 - 7 Construction of recombinant Marek's disease virus type 1 (MDV1) expressing the Escherichia coli lacZ gene as a possible live vaccine vector: the US10 gene of MDV1 as a stable insertion site; Sakaguchi M et al.; This paper describes the construction of a recombinant Marek's disease virus serotype 1 (MDV1) in which the Escherichia coli lacZ gene was inserted into the open reading frame homologous to the US10 gene of herpes simplex virus 1 (HSV1) . The recombinant virus replicated as well in cell culture as the parental MDV1 K-554 strain . Chickens immunized with the virus were protected against challenge with virulent MDV1, and produced a high level of antibodies against beta-galactosidase as well as against MDV1 antigens . The antibody titres persisted for at least 16 weeks . These results demonstrate that the US10 gene of MDV1 is an effective site for the insertion of foreign genes from which to construct a polyvalent live vaccine for poultry. Vaccine, 1994 Aug, 12(10), 925 - 32 Impact of the saponin adjuvant QS-21 and aluminium hydroxide on the immunogenicity of recombinant OspA and OspB of Borrelia burgdorferi; Ma J et al.; The impact of the adjuvants QS-21 and aluminium hydroxide (alum) on the immunogenicity of recombinant outer surface proteins A (OspA) and B (OspB) of Borrelia burgdorferi was investigated . Both non-acylated OspA and OspB derived from strain B31 were expressed in Escherichia coli and purified by reversible citraconylation and anion-exchange chromatography . Antisera to OspA or OspB were prepared in mice with antigens formulated with QS-21 or alum, and evaluated for specific immunoglobulin G isotypes, agglutination and borreliacidal activity . QS-21 significantly enhanced IgG2a and IgG2b antibody responses to OspA and OspB, and IgG1 response to OspA when compared with the formulation containing antigen alone . In contrast, alum significantly inhibited the induction of IgG2a and IgG2b responses to OspA . Alum had no significant effect on IgG1 response to OspA, or IgG2a and IgG2b responses to OspB, but significantly enhanced IgG1 antibody response to OspB . Antisera to OspA or OspB formulated by QS-21 possessed higher titres of agglutinating antibody than antisera to OspA or OspB alone . Borreliacidal activity was eight- to 64-fold higher in antisera to OspA formulated with QS-21 than in antisera to OspA formulated with or without alum . These antisera were highly borreliacidal to New York strain B31, a California isolate CA-2-87, German isolate Fr, and Swedish isolate G25 . Antisera to OspB formulated with QS-21 were highly borreliacidal to strains B31 and Fr, but not to CA-2-87 and G25 . Antisera to OspB formulated with alum were borreliacidal only to B31 . Thus, OspA was superior to OspB and QS-21 superior to alum at eliciting functional antibody responses.(ABSTRACT TRUNCATED AT 250 WORDS) Gen Comp Endocrinol, 1994 Aug, 95(2), 155 - 68 Isolation and characterization of the cDNA encoding the channel catfish (Ictalurus punctatus) form of cytochrome P450arom; Trant JM; Cytochrome P450arom (aromatase) is responsible for the conversion of androgens to estrogens . A cDNA library was constructed from poly(A)-enriched mRNA isolated from channel catfish (Ictalurus punctatus) ovary and ligated into EcoRI-cut lambda arms . The amplified library was screened using a human aromatase DNA probe . The longest clone isolated (2.1 kb) contained 20 bp of the 5'-untranslated region, a 1572-bp open reading frame, and a 509-bp 3'-untranslated region . Northern blot analysis indicated a single 3.4-kb transcript . A putative polyadenylation signal (AATAAA) is 13 bp from the 3'-end of the clone . The deduced amino acid sequence of the catfish form of aromatase is 65% identical to the rainbow trout form . The catfish form is 50 to 53% identical to mammalian (rat, human, mouse, bovine) and chicken forms . There are large regions of extremely high identity shared among all the forms . The deduced catfish aromatase protein is 524, 517, or 502 residues in length, depending on the translation initiation site . Steroidogenic activity of gonadal tissue is correlated with mRNA and protein content . Nonsteroidogenic COS cells, transfected with the catfish aromatase cDNA, converted androstenedione to estrone, thus verifying its identify . The catfish P450arom was modified to facilitate production in transformed Escherichia coli and its subsequent purification by a single column technique . This purified protein was used to raise antisera for Western blot analysis . Western blot analysis indicates that the catfish form is similar in size (57 kDa) to other vertebrate forms. Protein Expr Purif, 1994 Aug, 5(4), 371 - 8 The hydroxymethyldihydropterin pyrophosphokinase domain of the multifunctional folic acid synthesis Fas protein of Pneumocystis carinii expressed as an independent enzyme in Escherichia coli: refolding and characterization of the recombinant enzyme; Ballantine SP et al.; The folic acid synthesis (Fas) protein of Pneumocystis carinii is a multifunctional enzyme containing dihydroneopterin aldolase, 6-hydroxymethyl-7,8-dihydropterin pyrophosphokinase (PPPK), and dihydropteroate synthase activities . Isolation of the stretch of fas cDNA shown by amino acid similarity to the bacterial counterparts to code for PPPK activity (fasC domain) is described . FasC was expressed to high levels in Escherichia coli inclusion bodies using an inducible tac promoter expression system . Solubilization of the inclusion bodies in 6 M guanidine hydrochloride and refolding of the recombinant protein yielded enzymatically active PPPK which was purified to homogeneity by anion-exchange and gel-filtration chromatography . Sequence analysis showed that the first 13 amino acids of the purified protein were in agreement with those predicted from the DNA sequence and, furthermore, that the amino-terminal methionine had been removed . The enzyme is active in the monomeric form, exhibiting maximum activity at around pH 8.0 . Isoelectric focusing gave a pI of 9.1 . The Km value for 6-hydroxymethyl-7,8-dihydropterin was 3.6 microM in 50 mM Tris buffer, pH 8.2 . The production of independently folded, active P . carinii PPPK will allow detailed biochemical and structural studies, increasing our understanding of this enzyme domain. Protein Expr Purif, 1994 Aug, 5(4), 357 - 63 A beta-turn rich barley seed protein is correctly folded in Escherichia coli; Tamas L et al.; Wild-type and cysteine-containing mutant C hordeins from barley were expressed in Escherichia coli at high levels (> or = 30mg/liter) . N-terminal sequence analysis, SDS-PAGE, RP-HPLC, cd spectroscopy, and small angle X-ray scattering demonstrated that their physicochemical properties were similar to those of C hordeins isolated from barley grain . This indicates that the expressed proteins were correctly folded . The cysteine-containing mutant showed evidence of polymer formation in E . coli, nonreduced preparations of the protein showing the presence of polymers that were replaced by a single protein when a reducing agent was added. Protein Expr Purif, 1994 Aug, 5(4), 324 - 30 Rapid isolation of nuclear transport-competent Xenopus nucleoplasmin produced in Escherichia coli strain BL21(DE3); Kalinich JF et al.; Nucleoplasmin is a thermostable karyophilic protein widely used in nuclear transport studies . An expression vector was constructed that contains a string of 10 histidine residues ligated, in frame, to the amino terminal end of the Xenopus nucleoplasmin gene . The vector was then transformed into Escherichia coli strain BL21(DE3) . This strain possesses the gene for T7 RNA polymerase under control of the lacUV5 promoter . The induction of the RNA polymerase and subsequent production of nucleoplasmin occurs after exposure to isopropyl-beta-D-thiogalactopyranoside . The nucleoplasmin, produced in milligram quantities per liter of culture, is then isolated by a rapid purification method that includes metal chelation chromatography to purify the oligohistidine-linked nucleoplasmin . Nuclear transport studies indicate that fluorescently labeled nucleoplasmin is translocated to the nuclear interior of permeabilized V79A03 cells, while nucleoplasmin that lacks a nuclear localization signal (core nucleoplasmin) is not imported . The use of this method to produce nuclear transport-competent nucleoplasmin avoids the lengthy purification procedure used to isolate nucleoplasmin from Xenopus laevis oocytes as well as the cost of purchasing and maintaining a toad colony. Protein Expr Purif, 1994 Aug, 5(4), 317 - 23 Periplasmic fractionation of Escherichia coli yields recombinant plastocyanin despite the absence of a signal sequence; Ybe JA et al.; Poplar plastocyanin has been expressed in E . coli from a synthetic gene cloned into the T7 expression system . Despite the absence of a signal sequence, large quantities of the recombinant protein were readily obtained by procedures typically used to isolate proteins from the bacterial periplasm . Several different fractionation methods were equally successful . The presence of plastocyanin in these fractions does not reflect wholesale leakage of intracellular proteins, since neither beta-galactosidase activity nor the bulk of Escherichia coli proteins were released by the fractionation . The identity of the overexpressed protein was unequivocally proven to be poplar plastocyanin by N-terminal amino acid sequence analysis and by spectroscopic characterization of the purified blue copper protein. Hum Gene Ther, 1994 Aug, 5(8), 987 - 95 Effect of exogenous nerve growth factor on neurotoxicity of and neuronal gene delivery by a herpes simplex amplicon vector in the rat brain; Pakzaban P et al.; We have previously shown that local destruction of neural tissue by wild-type herpes simplex virus type 1 (HSV-1) is attenuated by intracerebral infusion of nerve growth factor (NGF) . To investigate the effect of NGF on the extent of neurolysis and efficacy of neuronal gene transfer mediated by an HSV-1 amplicon vector system in vivo, rats were stereotaxically injected in the striatum with an amplicon preparation, pHSVlac . This amplicon contains the Escherichia coli lacZ gene under the transcriptional control of the HSV-1 immediate early 4/5 promoter and is packaged by an HSV-1 helper virus carrying a deletion in the immediate early 3 gene . Vector injection was followed by continuous intracerebral infusion of NGF-beta (total dose 5 micrograms) or vehicle solution over 7 days . Animals were sacrificed at the end of the 7-day infusion period for histological analysis of the brains . A distinct zone of inflammation and necrosis surrounded the injection site in all vector-inoculated animals . The volume of striatal tissue destruction was significantly smaller in NGF-treated animals (1.27 +/- 0.19 mm3; mean +/- SEM) than in the vehicle-treated controls (2.16 +/- 0.37 mm3; P < 0.05 by t-test) . Immunohistochemical staining for HSV and beta-galactosidase (beta-Gal) in vehicle-treated animals revealed that many striatal cells harbored HSV antigens (3,678 +/- 636), but only a small number expressed the reporter gene at 7 days post-injection (294 +/- 60) . NGF infusion did not significantly affect the number of HSV-immunoreactive cells (4,224 +/- 618), or the number of cells expressing beta-Gal (330 +/- 72) at this time.(ABSTRACT TRUNCATED AT 250 WORDS) Hum Gene Ther, 1994 Aug, 5(8), 969 - 78 Experimental tumor therapy in mice using the cyclophosphamide-activating cytochrome P450 2B1 gene; Wei MX et al.; Most malignant tumors of the central nervous system do not respond well to chemotherapy . The anticancer drug cyclophosphamide (CPA) is largely ineffective against these neoplasms as its conversion to DNA-alkylating, cytotoxic metabolites is restricted primarily to the liver and these metabolites do not readily cross the blood-brain barrier . Here, we show that brain tumor cells can be sensitized to the cytotoxic effects of CPA, both in culture and in vivo, by introduction of the hepatic enzyme responsible for the activation of CPA, cytochrome P450 2B1 . Stable transfection of rat C6 glioma cells with the P450 2B1 gene rendered the cultured tumor cells sensitive to CPA . Further, C6 cells bearing this gene were more sensitive than parental cells to the cytotoxic action of CPA when grown subcutaneously in the flanks of athymic mice . Murine fibroblasts producing a retrovirus vector encoding P450 2B1 and expressing this enzyme were then prepared and grafted into the brains of athymic mice seeded with rat C6 gliomas . Intrathecal administration of CPA prevented the development of meningeal neoplasia and led to partial regression of the parenchymal tumor mass . By contrast, C6 glioma-bearing mice receiving fibroblasts expressing the Escherichia coli lacZ gene and CPA exhibited extensive meningeal tumors and parenchymal solid brain tumors . The in situ activation of CPA by cytochrome P450 2B1 provides a novel approach not only for brain tumor gene therapy, but also for negative, drug-conditional selection of other defined cell populations. Am J Reprod Immunol, 1994 Aug, 32(1), 1 - 7 Interleukin-1 receptor antagonist (IL-1ra) production by human amnion, chorion, and decidua; Fidel PL Jr et al.; PROBLEM: This study was conducted to determine whether (1) conditioned media from unstimulated primary cultures of human amnion, chorion, or decidua contain detectable concentrations of IL-1ra in vitro, and (2) bacterial endotoxin (LPS), tumor necrosis factor-alpha (TNF-alpha), or IL-1-beta (IL-1 beta) stimulate amnion, chorion, or decidua to produce increased amounts of IL-1ra . METHOD: Placentae were obtained from women at term with intact membranes before the onset of labor . Amnion, chorion, and decidual cells were isolated by standard procedures and grown to confluence . Cells were then cultured in quadruplicate for 16 h in tissue culture medium supplemented with 10% fetal calf serum or, additionally, with various concentrations of Escherichia coli LPS, TNF-alpha, or IL-1 beta . Culture supernatants were collected, and concentrations of IL-1ra were quantitated by a sensitive and specific enzyme-linked immunosorbent assay for IL-1ra . RESULTS: Results showed that primary cultures of amnion and chorion from 4 of 9 and decidua from 10 of 12 placentae had detectable rates of production of IL-1ra (ranges: 0.08-6.5, 0.42-12.1, and 1.55-96.5 pg IL-1ra/microgram protein/16 h, respectively) . In addition, LPS (10-1,000 ng/ml) and IL-1 beta (0.1-10 ng/ml), but not TNF-alpha (0.01-100 ng/ml), stimulated decidual cells to release/secrete increased amounts of IL-1ra compared with media alone (range: 2.5-400 pg IL-1ra/microgram protein/16 h, P < 0.0001) . In contrast, neither LPS, TNF-alpha, or IL-1 beta could stimulate amnion or chorion to release/secrete IL-1ra . CONCLUSIONS: These results indicate (1) that amnion, chorion, and predominantly decidua, can release or secrete IL-1ra in vitro, and (2) that LPS and IL-1 beta can stimulate decidual cells to produce increased amounts of IL-1ra. Photochem Photobiol, 1994 Aug, 60(2), 125 - 33 DNA photolyase from the fungus Neurospora crassa . Purification, characterization and comparison with other photolyases; Eker AP et al.; A phr-gene from the filamentous fungus Neurospora crassa was overexpressed in Escherichia coli cells, yielding a biologically active photolyase . After purification till apparent homogeneity, the 66 kDa protein was found to contain equimolar amounts of 5,10-methenyltetrahydrofolic acid (MTHF) and FAD, classifying it as an MTHF-type photolyase . Compared to other MTHF photolyases the absorption maximum of Neurospora photolyase is shifted from ca 380 nm to 391 nm (epsilon = 34,800), while an additional shoulder is present at 465 nm . In dark-adapted enzyme the FAD chromophore is predominantly present in the oxidized form, in contrast with E . coli and Saccharomyces cerevisiae photolyase, which contain mainly semiquinone or fully reduced FAD, respectively . Preillumination or dithionite treatment converted oxidized FAD in Neurospora photolyase into the fully reduced form, with a concomitant shift of the absorption maximum from 391 to 396 nm and disappearance of the 465 nm shoulder . The action spectrum of photoreactivation coincides with the absorption spectrum of preilluminated (reduced) photolyase, extending the spectral region of MTHF-type photolyases from 380 till 396 nm . A quantum yield of 0.57 was obtained for the overall repair reaction . Comparison of spectral properties of FAD in Neurospora photolyase and the model compound lumiflavin points to an apolar microenvironment of photolyase-bound FAD . Neurospora photolyase has distinct advantages over E . coli photolyase as it is more stable and contains a full complement of chromophores. J Immunoassay, 1994 Aug, 15(3), 239 - 49 A monoclonal inhibition enzyme immunoassay for detection of antibodies against hepatitis B core antigen: confirmation of an immunodominant epitope; Pujol FH et al.; Monoclonal antibodies (mAbs) were raised against hepatitis B virus core produced by a recombinant clone of Escherichia coli (rHBc) . The three mAbs recognized rHBc by Western blot, suggesting that they reacted with non-conformational epitopes . Competition experiments between mAbs and human anti-HBc sera confirmed the existence of an immunodominant HBc epitope within the viral antigen . A monoclonal competition enzyme immunoassay using an IgM mAb conjugated to biotin and streptavidin-peroxidase as the detection system yielded 99% sensitivity and 100% specificity, when compared to other commercial assays. Microbiology, 1994 Aug, 140 ( Pt 8), 2135 - 42 Molecular cloning of a gene encoding the immunogenic 21 kDa protein of Cowdria ruminantium; Mahan SM et al.; Major immunogenic polypeptides (21, 32, 40, 46, 58, 85 and 160 kDa) of Cowdria ruminantium were identified by immunoprecipitation and immunoblotting . A pUC13 library of C . ruminantium genomic DNA was screened with hyperimmune sheep serum to identify Escherichia coli colonies which expressed genes encoding these immunogenic proteins . A recombinant E . coli colony, F5.2, was identified containing plasmid insert DNA of 2773 bp . The cloned DNA insert contained two long open reading frames (ORFs) of 627 bp (complete) and 831 bp (incomplete), both potentially encoding proteins containing an N-terminal signal peptide . Deletion experiments suggested that the hyperimmune sheep serum recognized a protein that was encoded by the 627 bp ORF . The 627 bp ORF was amplified by polymerase chain reaction (PCR), subcloned and expressed to a high level in E . coli . A sheep antiserum made to the expressed recombinant fusion protein recognized a 21 kDa protein of all strains of C . ruminantium tested, confirming that the 627 bp ORF encodes a native 21 kDa protein in C . ruminantium . Similarly, the recombinant protein was recognized by all sera tested from heartwater-infected animals . The antigenic conservation of the 21 kDa protein and its immunogenic nature are reasons for further testing of this recombinant protein in subunit diagnostic tests. Microbiology, 1994 Aug, 140 ( Pt 8), 2109 - 14 Cloning and DNA sequence analysis of the region containing attP of the temperate phage phi AR29 of Prevotella ruminicola AR29; Gregg K et al.; Phage phi AR29 was shown to exist as a prophage integrated into the chromosome of Prevotella ruminicola AR29 . By DNA hybridization studies, the point of integrative recombination on the phage genome (attP) was located on a 4.5 kb EcoRV fragment . After preliminary mapping with restriction endonucleases, a 2.8 kb EcoRV/HindIII fragment was isolated, cloned in Escherichia coli and sequenced . DNA hybridization localized the attP site to the vicinity of an internal DraI site . Sequence analysis showed the presence of several direct and inverted repeats around the attP site, with consensus core sequences similar to the integrase binding sites of phage lambda . Two open reading frames are present adjacent to attP (ORF1 and ORF2) . The predicted polypeptide product of ORF1 has a region of structural similarity to known integrases . Although the predicted product of ORF2 shows at best weak homology with known excisionases, no other ORFs occur in the sequence upstream from ORF1, leaving ORF2 as the most likely candidate for this role . However, if ORF2 does represent an xis gene, then this putative integration module would possess a notable difference from that of other temperate phages in the inversion of the positions of int and xis relative to attP . The proposed phi AR29 integration module is being used to develop phage-based integrative vector systems for the genetic manipulation of rumen bacteria. Microbiology, 1994 Aug, 140 ( Pt 8), 1937 - 44 Inhibition of lipid biosynthesis induces the expression of the pspA gene; Bergler H et al.; Treatment of Escherichia coli with diazaborine strongly induces the synthesis of a 28 kDa protein which is associated with the cytoplasmic membrane . The partial amino acid sequence proved that this protein is identical to the phage shock protein PspA . The kinetics of the expression of the pspA gene were determined in an E . coli strain which carried a pspA-lacZ fusion in the chromosome . PspA synthesis is independent of the growth phase . It is, however, strongly induced when fatty acid biosynthesis is inhibited by diazaborine or cerulenin . Treatment with either compound also causes dose-dependent inhibition of phospholipid biosynthesis whose degree correlates with the induction of PspA . Another cause of induction of PspA synthesis is treatment of E . coli with globomycin, which is an inhibitor of the processing of lipoproteins. Free Radic Res, 1994 Aug, 21(2), 75 - 83 Singlet oxygen induces predominantly G to T transversions on a single-stranded shuttle vector replicated in monkey cells; Ribeiro DT et al.; To elucidate the mechanisms of mutagenesis by singlet oxygen DNA damage in mammalian cells, a SV40-derived single-stranded shuttle vector was exposed to the water soluble endoperoxide 3,3'-(1,4-naphthylidene) dipropionate (NDPO2) . The damaged vector was transfected into monkey COS7 cells and the plasmid progeny exhibited up to 10 fold increase on the mutation frequency in the supF target gene, when compared to untreated vector . The sequence in the supF locus of such mutants revealed that singlet oxygen-induced mutagenesis in single-stranded vector is significantly different from spontaneous mutagenesis . Among the base substitutions, most of the mutations involved deoxyguanosines, being G to T transversions the predominant type of change . The data indicate that mutagenesis by singlet oxygen in mammalian cells may be generated by an error prone bypass of damaged deoxyguanosines at the template DNA. Plant J, 1994 Aug, 6(2), 259 - 69 Characterization of an Arabidopsis cDNA for a soluble epoxide hydrolase gene that is inducible by auxin and water stress; Kiyosue T et al.; A cDNA (1122 bp) was isolated from a cDNA library prepared from Arabidopsis thaliana L . that had been subjected to drought stress for 1 h . The sequencing of a genomic clone corresponding to the cDNA and S1 mapping analysis revealed that the cDNA lacked the first 6 bp from its translational start (ATG) . The resulting open reading frame encodes a polypeptide of 321 amino acids, and the calculated molecular weight of this polypeptide is 36,423 Da . The deduced amino acid sequence shows a high degree of similarity to C terminal halves of those of soluble epoxide hydrolases (sEHs) of human, mouse and rat, 35.5%, 34.1% and 33.1%, respectively . The cDNA was expressed in Escherichia coli cells, and the expressed protein migrates at 40 kDa when analyzed by SDS-PAGE . The recombinant protein at 40 kDa is much smaller than the mammalian sEH (58 kDa) but has characteristics of activity and inhibition similar to the mammalian sEHs when assayed with the substrate trans-stilbene oxide and the inhibitors 4-fluorochalcone oxide (4FCO), (2R,3R)-3-(4-nitrophenyl) glycidol (RRNPG), and (2S,3S)-3-(4-nitrophenyl)glycidol (SSNPG), which indicates that the cDNA did encode a soluble epoxide hydrolase of A . thaliana (AtsEH) . Drought stress, but not heat or cold stress, slightly increased the accumulation of the mRNAs for AtsEH . The level of AtsEH transcripts increased strongly after treatment with a plant hormone, auxin (2,4-dichlorophenoxyacetic acid, 2,4-D; naphthalene-acetic acid, NAA; and indole-3-acetic acid, IAA) in young, pre-bolting plants . Treatment with cytokinin (6-benzylaminopurine, BA), abscisic acid (ABA) or gibberellin (GA3) had no detectable effect on AtsEH transcript levels . The transcripts for AtsEH gene were detected in the aerial vegetative organs of bolting plants (i.e . stems and leaves), but not in roots, flowers and seeds . The possible function of AtsEH is discussed . A similar sEH cDNA has recently been characterized in potato (Stapleton et al., 1994). Plant Cell, 1994 Aug, 6(8), 1145 - 55 Identification of the 100-kD victorin binding protein from oats; Wolpert TJ et al.; The fungus Cochliobolus victoriae, the causal agent of victoria blight of oats, produces the host-specific toxin victorin . Sensitivity of oats to victorin, and thus susceptibility to the fungus, is controlled by a single dominant gene . This gene is believed to also confer resistance to the crown rust pathogen Puccinia coronata . In the case of victoria blight, the gene has been hypothesized to condition susceptibility by encoding a toxin receptor . A 100-kD victorin binding protein (VBP) has been identified; it binds radiolabeled victorin derivatives in a ligand-specific manner and in a genotype-specific manner in vivo . The VBP may function as a toxin receptor . In vitro translation coupled with indirect immunoprecipitation was used to identify the mRNA for the 100-kD VBP, and fractionated mRNAs were used to prepare cDNA libraries enriched in the relative abundance of cDNA for the 100-kD VBP . A 3.4-kb cDNA clone was isolated that, when subjected to a 400-bp 5' deletion, was capable of directing the synthesis of a protein in Escherichia coli, which reacted to an antibody specific for the 100-kD VBP . Peptide mapping, by limited proteolysis, indicated that the protein directed by the cDNA is the 100-kD VBP . Nucleotide sequence analysis of the cDNA revealed extensive homology to a previously cloned cDNA for the P protein component of the multienzyme complex glycine decarboxylase . Glycine decarboxylase is a nuclear-encoded, mitochondrial enzyme complex . Protein gel blot analysis indicated that the 100-kD VBP copurifies with mitochondria . Based on analysis of in vitro translation products, nucleotide sequence homology, mitochondrial localization, and the widespread species distribution of the 100-kD VBP, we concluded that the 100-kD VBP is the P protein component of glycine decarboxylase. Plant Cell, 1994 Aug, 6(8), 1107 - 21 Molecular structure and enzymatic function of lycopene cyclase from the cyanobacterium Synechococcus sp strain PCC7942; Cunningham FX Jr et al.; A gene encoding the enzyme lycopene cyclase in the cyanobacterium Synechococcus sp strain PCC7942 was mapped by genetic complementation, cloned, and sequenced . This gene, which we have named crtL, was expressed in strains of Escherichia coli that were genetically engineered to accumulate the carotenoid precursors lycopene, neurosporene, and zeta-carotene . The crtL gene product converts the acyclic hydrocarbon lycopene into the bicyclic beta-carotene, an essential component of the photosynthetic apparatus in oxygen-evolving organisms and a source of vitamin A in human and animal nutrition . The enzyme also converts neurosporene to the monocyclic beta-zeacarotene but does not cyclize zeta-carotene, indicating that desaturation of the 7-8 or 7'-8' carbon-carbon bond is required for cyclization . The bleaching herbicide 2-(4-methylphenoxy)triethylamine hydrochloride (MPTA) effectively inhibits both cyclization reactions . A mutation that confers resistance to MPTA in Synechococcus sp PCC7942 was identified as a point mutation in the promoter region of crtL . The deduced amino acid sequence of lycopene cyclase specifies a polypeptide of 411 amino acids with a molecular weight of 46,125 and a pI of 6.0 . An amino acid sequence motif indicative of FAD utilization is located at the N terminus of the polypeptide . DNA gel blot hybridization analysis indicated a single copy of crtL in Synechococcus sp PCC7942 . Other than the FAD binding motif, the predicted amino acid sequence of the cyanobacterial lycopene cyclase bears little resemblance to the two known lycopene cyclase enzymes from nonphotosynthetic bacteria . Preliminary results from DNA gel blot hybridization experiments suggest that, like two earlier genes in the pathway, the Synechococcus gene encoding lycopene cyclase is homologous to plant and algal genes encoding this enzyme. FEMS Microbiol Rev, 1994 Aug, 14(4), 315 - 23 Roles of manganese and iron in the regulation of the biosynthesis of manganese-superoxide dismutase in Escherichia coli; Hassan HM et al.; Aerobic life-style offers both benefits and risks to living cells . The major risk comes from the formation of reactive oxygen intermediates (i.e . superoxide radical, O2-; hydrogen peroxide, H2O2; and hydroxyl radical, OH.) during normal oxygen metabolism . However, living cells are able to cope with oxygen toxicity by virtue of a unique set of antioxidant enzymes that scavenge O2- and H2O2, and prevent the formation OH. . Superoxide dismutases (SODs; EC 1.15.1.1) are metalloenzymes essential for aerobic survival . Escherichia coli contains two forms of this enzyme: an iron-containing enzyme (FeSOD) and a manganese-containing enzyme (MnSOD) . In E . coli, MnSOD biosynthesis is under rigorous control . The enzyme is induced in response to a variety of environmental stress conditions including exposure to oxygen, redox cycling compounds such as paraquat which exacerbate the level of intracellular superoxide radicals, iron chelation (i.e . iron deprivation), and oxidants . A model for the regulation of the MnSOD has been proposed in which the MnSOD gene (sodA) is negatively regulated at the level of transcription by an iron-containing redox-sensitive repressor protein . The effect of iron-chelation most probably results in removal of the iron necessary for repressor activity . Recent studies have shown that sodA expression is regulated by three iron-dependent regulatory proteins, Fur (ferric uptake regulation), Fnr (fumarate nitrate regulation) and SoxR (superoxide regulon), and by the ArcA/ArcB (aerobic respiration control) system . The potential Fur-, Fnr- and ArcA-binding sites in the sodA promoter region have been identified by using different cis-acting regulatory mutations that caused anaerobic derepression of the gene.(ABSTRACT TRUNCATED AT 250 WORDS) J Pharmacol Exp Ther, 1994 Aug, 270(2), 665 - 74 Biochemical and pharmacological characterization of high-affinity trimetoquinol analogs on guinea pig and human beta adrenergic receptor subtypes: evidence for partial agonism; Fraundorfer PF et al.; Radioligand binding assays were used to characterize the interaction of a series of trimetoquinol {1-(3',4',5'-trimethoxybenzyl)-6,7-dihydroxy-1,2,3,4-tetrahydroisoqui nol ine; TMQ} analogs with beta adrenergic receptors (beta-AR) . The results indicated that TMQ analogs bound with similar affinities to guinea pig (heart, lung and skeletal muscle) and human (beta-AR in Escherichia coli) beta-1- and beta-2-AR subtypes . However, the isomers of TMQ and 8-fluoro-TMQ bound stereoselectively to beta-AR with the S-isomers having affinities at least 112- and 8-fold greater, respectively, than their corresponding R-isomers . In general, a direct relationship existed between TMQ analog binding to guinea pig beta-AR and functional activity on guinea pig right atria (beta-1) and trachea (beta-2) . For selected halogenated TMQ analogs (3',5'-diiodo-TMQ, 3'-iodo-TMQ, 5,8-difluoro-TMQ and 5-iodo-TMQ) which had higher beta-AR affinities than TMQ, but were less potent beta-AR agonists than TMQ, this relationship was not seen . To explain this, the function of the TMQ analogs was analyzed at the level of the beta-AR-associated effector mechanism (i.e., G-protein and adenylyl cyclase) . In Chinese hamster ovary cells expressing human beta-2-AR, TMQ and halogenated analogs bound to the receptor with high affinity (nanomolar range); however, they failed to effectively couple with beta-AR-associated G-protein and only partially activated receptor-associated adenylyl cyclase . Receptor occupancies of 0.14, 2 and 23% were required for (-)-isoproterenol, S-(-)-TMQ and 3'5'-diiodo-TMQ to produce equivalent cyclic AMP accumulations in human beta-2-AR Chinese hamster ovary cells . Thus, TMQ and halogenated TMQ derivatives bind stereoselectively to beta-AR with high affinity, and may be classified as partial beta-AR agonists. J Bacteriol, 1994 Aug, 176(16), 4949 - 57 Identification and characterization of a gene cluster mediating enteroaggregative Escherichia coli aggregative adherence fimbria I biogenesis; Savarino SJ et al.; The aggregative pattern of adherence (AA) exhibited by enteroaggregative Escherichia coli upon HEp-2 cells is a plasmid-associated property which correlates with aggregative adherence fimbria I (AAF/I) expression and human erythrocyte hemagglutination . By using cloning and mutagenesis strategies, two noncontiguous plasmid segments (designated regions 1 and 2) required for AA expression have previously been identified in enteroaggregative E . coli 17-2 . TnphoA mutagenesis was performed on clones containing region 1, and 16 TnphoA mutants which were negative for the AA phenotype were analyzed . The TnphoA insertion site for each mutant was determined by junctional DNA sequencing . All 16 mutations occurred within a 4.6-kb span in region 1 . Nucleotide sequence analysis of the region revealed four contiguous open reading frames, designated aggDCBA, in the same span . AA-negative TnphoA insertions into all open reading frames except aggB were obtained . On the basis of mutational analysis and protein homology data, it is inferred that aggA, aggC, and aggD are involved in biogenesis of AAF/I, encoding a major fimbrial subunit, outer membrane usher, and periplasmic fimbrial chaperone, respectively . By immunogold electron microscopy, polyclonal antiserum raised against the aggA gene product decorated AAF/I fimbriae, affirming that AggA encodes an AAF/I subunit. J Bacteriol, 1994 Aug, 176(15), 4691 - 9 AggR, a transcriptional activator of aggregative adherence fimbria I expression in enteroaggregative Escherichia coli; Nataro JP et al.; Enteroaggregative Escherichia coli (EAggEC) has been associated with persistent pediatric diarrhea in the developing world, yet the pathogenetic mechanisms of EAggEC infection are unknown . Our previous data have suggested that aggregative adherence of some EAggEC strains to HEp-2 cells is mediated by flexible, bundle-forming fimbriae, which we have termed aggregative adherence fimbriae I (AAF/I) . Genes sufficient to confer expression of AAF/I are located on the 60-MDa plasmid of EAggEC 17-2; AAF/I genes are present as two unlinked plasmid regions (regions 1 and 2), separated by 9 kb of DNA . Here we report the complete DNA sequencing of region 2 and the identification of an open reading frame which is involved in the expression of AAF/I . One open reading frame of 794 bp encodes a protein (designated AggR) with a predicted molecular size of 29.4 kDa, which shows a high degree of amino acid sequence identity to CfaR and other members of the AraC class of gene regulators . The cloned aggR gene (or, alternatively, a cloned cfaR gene) was sufficient to complement a region 1 clone to confer AAF/I expression . To further substantiate the role of aggR in the regulation of AAF/I, we constructed a 289-bp in-frame aggR deletion and replaced the native gene in 17-2 by allelic exchange, using the temperature-sensitive vector pIB307 . The resulting aggR deletions were negative for AAF/I expression, but expression was restored when the aggR gene (cloned into pBluescript II SK) was reintroduced into the aggR mutant . RNA slot blot experiments using a probe for the putative AAF/I pilin subunit (aggA) revealed that aggR operates as a transcriptional activator of aggA expression . aggA::phoA fusions were constructed in 17-2 and in 17-2 delta aggR . AggR was found to promote expression of the aggA gene under a variety of conditions of temperature, osmolarity, oxygen tension, and medium . At acid pH, aggA expression was maximal and was regulated by both AggR-dependent and AggR-independent mechanisms. J Bacteriol, 1994 Aug, 176(15), 4664 - 8 Why does Escherichia coli have two primary pathways for synthesis of glutamate? Helling RB. Escherichia coli has two primary pathways for glutamate synthetase-glutamate synthase pathway is known to be essential for synthesis at low ammonium concentrations and for regulation of the glutamine pool, but the necessity for glutamate dehydrogenase (GDH) has been uncertain . The results of competition experiments between the wild type and a GDH-deficient mutant during nutrient-limited growth and of direct enzyme measurements suggest that GDH is used in glutamate synthesis when the cell is limited for energy (and carbon) but ammonium and phosphate are present in excess, while the glutamine synthetase-glutamate synthase pathway is used when the cell is not under energy limitation . The use of alternative routes for glutamate synthesis implies that the energy cost of biosynthesis may be less when energy is limited than when energy is unlimited. Infect Immun, 1994 Aug, 62(8), 3305 - 10 Hemagglutinating and chemotactic properties of synthetic peptide segments of fimbrial protein from Porphyromonas gingivalis; Ogawa T et al.; Porphyromonas gingivalis 381 fimbriae, their synthetic peptide segments, and lipopolysaccharide (LPS) were examined for hemagglutinating and migration-stimulating activities . P . gingivalis 381 fimbriae clearly caused hemagglutination, and several oligopeptide segments such as FP381(61-80), FP381(171-185), and FP381(302-321), corresponding to the amino acid residue numbers based on the amino acid sequence of fimbrillin proposed by Dickinson et al . (D . P . Dickinson, M . A . Kubiniec, F . Yoshimura, and R . J . Genco, J . Bacteriol . 170:1658-1665, 1988), were also demonstrated to agglutinate erythrocytes although less effectively than the native fimbriae . Furthermore, P . gingivalis 381 LPS but not Escherichia coli O55:B5 LPS definitely exhibited hemagglutination . P . gingivalis fimbriae as well as their synthetic peptides possessing hemagglutinating activity enhanced the chemotaxically induced migration of human peripheral blood monocytes . The results of the analyses using synthetic peptide FP381(61-80), its related compounds, and an analog suggested that the amino acid sequence XLTXXLTXXNXX within fimbrial protein molecules may play an important role structurally in the attachment of the protein to host cells such as erythrocytes and monocytes. Arch Biochem Biophys, 1994 Aug 1, 312(2), 317 - 25 Reversion mutations in the beta subunit mutants of Escherichia coli F1-ATPase with defective subunit assembly: implications for structure and function of the amino-terminal region; Miki J et al.; We analyzed reversion mutations of the mutants with a substitution of Leu-40 by Pro or Glu-41 by Lys in the beta subunit of Escherichia coli F1-ATPase . These mutants had an altered molecular assembly of the alpha and beta subunit on the membranes and lost the binding activity of the beta subunit to the monoclonal antibody beta 31 . For the mutation of Leu-40 to Pro, we found that all reversion mutations Gln, or Ser besides the wild-type Leu . These pseudo reversion mutations restored the cell growth on minimal agar supplemented with succinate as the sole carbon source, the assembly of the alpha and beta subunits, and also the ATPase activities in the membranes . These results suggested that Leu-40 itself is not an essential residue for the function of the beta subunit but that the residue contributes to a conformation around residue 40, which is important for the assembly of the alpha and beta subunits onto the membranes . For the mutation of Glu-41 to Lys, pseudo reversion mutations were found at the original residue 41 as Lys to Gln or Asn and also at Arg-218 to Cys or His in addition to the original Glu-41 to Lys mutation . These suppression mutations recovered the cell growth, indicating the recovery of ATP synthesis . The ATPase activity was high in cells with the Lys-41 to Glu mutation but those were relatively low in those with other reversion mutations . The assembly of the alpha and beta subunits recovered in the revertants to the wild-type level, except for the Lys-41 to Asn mutation, which resulted in a decreased amount of the alpha subunit on the membranes . These results suggested that though Glu-41 is not an essential residue, it plays an important role in the assembly of the alpha and beta subunit on the membranes . The results also suggested that residues Arg-218 and Glu-41 are located close together and interact with each other, either directly or indirectly.(ABSTRACT TRUNCATED AT 400 WORDS) J Neurochem, 1994 Aug, 63(2), 495 - 500 Secreted form of amyloid beta/A4 protein precursor (APP) binds to two distinct APP binding sites on rat B103 neuron-like cells through two different domains, but only one site is involved in neuritotropic activity; Ninomiya H et al.; Recent studies have identified the Alzheimer's disease amyloid beta/A4 protein precursor (APP) as a trophic and/or tropic protein on several types of cells, including fibroblasts, primary culture neurons, PC12 cells, and B103 neuron-like cells . Many trophic proteins bind heparin, and it is believed that the heparin-binding domain is crucial for the trophic activity of these proteins . APP also binds heparin . The current studies were undertaken to examine the hypothesis that the neuritotropic activity of APP requires heparin binding . It was found that APP produced in E . coli bound B103 cells through detergent-extractable molecules . Approximately 50% of the binding sites were heparinase-sensitive, and heparin and heparan sulfate competed for APP binding to these sites . The heparinase-insensitive sites were recognized by a stretch of 17 amino acids of APP (residues 319-335) that contains the neuritotropic activity of APP . A mutant APP with a deletion at this site was capable of binding to the heparinase-sensitive sites, although this molecule was not neuritotropic to B103 neuron-like cells . Therefore, the neuritotropic site and the heparin-binding site are distinct in APP, and the neuritotropic effect of APP is produced through its binding to detergent-extractable and heparinase-insensitive sites. Circ Shock, 1994 Aug, 43(4), 155 - 60 Mediators of endotoxin-induced leukocyte adhesion in mesenteric postcapillary venules; Harris NR et al.; Intravital video microscopy was used to monitor and quantify leukocyte-endothelial cell adherence, leukocyte emigration, venular shear rate, and leukocyte rolling velocity in cat mesenteric venules exposed to E . coli endotoxin lipopolysaccharides (LPS) . LPS induced a steady decrease in venular shear rate and leukocyte rolling velocity while increasing leukocyte adherence and emigration . The molecular determinants and chemical mediators of LPS-induced leukocyte-endothelial cell adhesion were investigated using monoclonal antibodies (MAbs) against the adhesion glycoproteins CD11/CD18 on leukocytes (MAb IB4) and ICAM-1 (MAb RR1/1) on endothelial cells as well as superoxide dismutase (SOD) and a platelet-activating factor receptor antagonist (WEB 2086) . Leukocyte adherence was significantly (P < .05) reduced by administration of either the CD11/CD18 MAb or SOD, while the PAF receptor antagonist and ICAM-1 MAb had no effect . None of the four agents studied significantly altered LPS-induced leukocyte emigration, venular shear rate, or leukocyte rolling velocity . These results indicate that the leukocyte adherence induced by LPS is dependent on the adhesion glycoprotein CD11/CD18 and superoxide, and that an endothelial cell ligand other than ICAM-1 participates in this adhesion process. Glycoconj J, 1994 Aug, 11(4), 274 - 81 Ricin B chain fragments expressed in Escherichia coli are able to bind free galactose in contrast to the full length polypeptide; Wales R et al.; Deleted forms of ricin B chain (RTB) containing only one of the two galactose binding sites were produced in E . coli and targeted to the periplasm by fusion to the ompA or ompF signal sequences . The proteins were then isolated from the periplasm and their sugar binding properties assessed . Previous studies investigating the properties of such proteins produced in Xenopus laevis oocytes suggested that deleted forms of RTB, when not glycosylated, retain their ability to bind simple sugars, unlike the full-length unglycosylated proteins . When produced in E . coli however we found that only one, EB733, of a number of deleted forms of RTB closely related to those previously produced in Xenopus laevis oocytes, bound to simple sugars . All of the deletion forms of RTB were found to bind in the asialofetuin binding assay; an assay which has been previously utilized to measure binding of lectins to the terminal galactose residues of glycoprotein oligosaccharides . However, in contrast to glycosylated RTB, binding of the deletion mutants could be competed to only a small degree or not at all with galactose . The only deletion mutant observed to bind to free galactose when produced in E . coli corresponded closely to the complete domain 2 of RTB . It is assumed that this mutant forms a stable structure similar to that of the C-terminal domain in the full-length protein . The structural integrity of EB733 was not only suggested by its sugar binding properties and solubility but also by its consistently higher level of expression and the absence of any apparent susceptibility to E . coli proteases. Zhongguo Yi Xue Ke Xue Yuan Xue Bao, 1994 Aug, 16(4), 290 - 5 {Expression of HPV16E6 gene and preparation of monoclonal antibody against the expression product}; Gui C et al.; The recombinant plasmid PAS1-HPV16E6 containing the HPV16E6 gene was expressed in E . coli AR120 under nalidixic acid induction . A 19KD expression protein was isolated, purified and identified . Mice were immunized with the purified E6 expression protein . A murine hybridoma, RAC6, was obtained by fusing splenic cells from an immunized BABL/c mouse with mouse myeloma cell line SP2/0-Ag14 cells, followed by screening in HAT medium, cloning and recloning in methyl cellulose . The hybridoma RAC6 stably produces specific monoclonal antibody against the E6 expression protein. Zentralbl Bakteriol, 1994 Aug, 281(2), 201 - 13 Prevalence of attaching and effacing Escherichia coli in stool samples from patients and controls; Schmidt H et al.; Enteropathogenic E . coli (EPEC) and enterohemorrhagic E . coli (EHEC) have the ability to cause 'attaching and effacing' (AE) lesions; the genes necessary to cause AE in both of these pathogroups have been identified and termed eae . Using colony hybridization, we screened 237 stool samples from patients with diarrhea, and 237 stool samples from age-matched controls for the presence of E . coli carrying eae . Individual colonies harbouring eae could be recovered from 7 (2.9%) of the patient stools, as well as from 6 (2.5%) of the control stools . All these E . coli isolates were positive in the fluorescence actin staining (FAS) test . In addition, all the samples were also probed for Shiga-like toxin (slt) genes and the EPEC adherence factor (EAF) to evaluate whether testing for eae identified all EHEC and class I EPEC . Of the 7 patient samples harbouring E . coli with eae, 4 had E . coli with eae and slt genes, and 2 had E . coli with eae and EAF sequences . In 2 of the 237 patient stools, E . coli which were eae and EAF negative but slt probe positive could be recovered . These 2 E . coli strains were non-reactive in the FAS test . Of the control samples, none of the E . coli strains, including the 6 samples containing eae positive strains, possessed EAF or slt-sequences . In concrete terms, the similar eae incidence found in both E . coli isolates from patients and controls is currently of limited clinical diagnostic value and more importantly, the eae probe could not identify all slt-harbouring E . coli . On the basis of these results, the use of the eae-probe cannot be recommended in preference to the slt probes for the detection of EHEC. Zentralbl Bakteriol, 1994 Aug, 281(2), 130 - 9 Expression and plasmid transfer of genes coding for the fimbrial antigen F107 in porcine Escherichia coli strains; Wittig W et al.; Expression of the fimbrial antigen F107 of porcine E . coli strains on agar plates was achieved by microaerobic cultivation . For part of the strains of some types, addition of alizarin yellow and eosin to the agar medium proved to be necessary . Some of these strains reacted distinctly positive only when the small colonies growing between the larger ones on alizarin-yellow agar were tested . The fimbrial antigen of the Swiss strain 107/86 was provisionally designated the F107ab variant and that of the Hungarian strain 2134P and the Czech strain 8813, the F107ac variant . The F107 genetic determinants were found to be often linked with those encoding haemolysin production and are frequently carried by plasmids. Biochem Mol Biol Int, 1994 Aug, 34(1), 209 - 16 Tryptophanase from Escherichia coli: catalytic and spectral properties in water-organic solvents; Faleev NG et al.; In water-methanol and water-dimethylformamide (DMF) (1:1 v/v) solutions tryptophanase from E.coli retains its abilities to form a quinonoid complex with quasisubstrates and to catalyze the decomposition of S-o-nitrophenyl-L-cysteine (SOPC) . Both the KM and Vmax values decrease in water-organic media . The affinities of tryptophanase for L-alanine, L-tryptophan, oxindolyl-L-alanine and indole in aqueous methanol are decreased, the effect being stronger for the more hydrophobic substances . In a water solution tryptophanase catalizes the reaction of SOPC with indole to form L-tryptophan while in water-organic solvents only decomposition of SOPC is observed. Biochem Mol Biol Int, 1994 Aug, 34(1), 109 - 17 Expression of human acylphosphatase in Escherichia coli affects intracellular calcium levels; Liguri G et al.; In vitro experiments demonstrated the ability of acylphosphatase to hydrolyze the phosphorylated intermediate that is formed during the activity of Na+, K(+)- and Ca(2+)-ATPases of mammalian cells membranes . In order to investigate the effect of this enzyme on intracellular cation levels, a synthetic gene for human muscle acylphosphatase has been expressed in E . coli strains BL21 and JM101 . Intracellular total steady-state calcium concentration, as measured by isotopic exchange, was significantly higher in transformed cells as compared to controls and the rising was dependent on the level of acylphosphatase expression . Accordingly also free intracellular calcium concentration, as measured by Fura-2 fluorescence, increased in transformed cells . On the other hand, phosphate levels were not affected by the expression of acylphosphatase, while sodium and rubidium levels increase in transformed cells . Intracellular pH resulted to be slightly affected by the expression of acylphosphatase, cytoplasm of transformed JM101 bacteria being more alkaline (pH 7.45) as compared to control cells (pH 7.40) . On the basis of these results, it can be suggested that acylphosphatase acts in vivo by regulating the cation transport in E . coli. J Bioenerg Biomembr, 1994 Aug, 26(4), 435 - 45 Energy-transducing nicotinamide nucleotide transhydrogenase: nucleotide sequences of the genes and predicted amino acid sequences of the subunits of the enzyme from Rhodospirillum rubrum; Yamaguchi M et al.; Based on the amino acid sequence of the N-terminus of the soluble subunit of the Rhodospirillum rubrum nicotinamide nucleotide transhydrogenase, two oligonucleotide primers were synthesized and used to amplify the corresponding DNA segment (110 base pairs) by the polymerase chain reaction . Using this PCR product as a probe, one clone with the insert of 6.4 kbp was isolated from a genomic library of R . rubrum and sequenced . This sequence contained three open reading frames, constituting the genes nntA1, nntA2, and nntB of the R . rubrum transhydrogenase operon . The polypeptides encoded by these genes were designated alpha 1, alpha 2, and beta, respectively, and are considered to be the subunits of the R . rubrum transhydrogenase . The predicted amino acid sequence of the alpha 1 subunit (384 residues; molecular weight 40276) has considerable sequence similarity to the alpha subunit of the Escherichia coli and the N-terminal 43-kDa segment of the bovine transhydrogenases . Like the latter, it has a beta alpha beta fold in the corresponding region, and the purified, soluble alpha 1 subunit cross-reacts with antibody to the bovine N-terminal 43-kDa fragment . The predicted amino acid sequence of the beta subunit of the R . rubrum transhydrogenase (464 residues; molecular weight 47808) has extensive sequence identity with the beta subunit of the E . coli and the corresponding C-terminal sequence of the bovine transhydrogenases . The chromatophores of R . rubrum contain a 48-kDa polypeptide, which cross-reacts with antibody to the C-terminal 20-kDa fragment of the bovine transhydrogenase . The predicted amino acid sequence of the alpha 2 subunit of the R . rubrum enzyme (139 residues; molecular weight 14888) has considerable sequence identity in its C-terminal half to the corresponding segments of the bovine and the alpha subunit of the E . coli transhydrogenases. Eur J Epidemiol, 1994 Aug, 10(4), 413 - 20 Analysis of the entire nucleotide sequence of the cryptic plasmid QpH1 from Coxiella burnetti; Thiele D et al.; The complete plasmid QpH1 from Coxiella burnetti, isolate 'Nine Mile', phase I, was cloned as NotI fragment with a size of 37329 bp . The entire plasmid was sequenced by the chain termination method after EcoRI subcloning . 37 open reading frames coding for polypeptides larger than 100 amino acid residues were determined . The predicted polypeptide products of the open reading frames were compared by computer analysis with reported protein sequences . Homologies of predicted polypeptide products to analogous proteins are described. Exp Eye Res, 1994 Aug, 59(2), 151 - 9 A group of novel glutathione S-transferase isozymes showing high activity towards 4-hydroxy-2-nonenal are present in bovine ocular tissues; Srivastava SK et al.; Recently, a mouse glutathione S-transferase (GST) isozyme, mGSTA4-4, which belongs to a distinct group of GSTs has been characterized in our laboratory . During the present studies, Western blot analyses of bovine ocular tissues using the antibodies raised against the recombinant mGSTA4-4 obtained by expression in Escherichia coli revealed that the orthologs of mGSTA4-4 were present in cornea, retina, iris-ciliary body and sclera, but absent in lens . These novel GST isozymes of bovine ocular tissues were purified by immunoaffinity chromatography using the antibodies against rec-mGSTA4-4 and were designated as bGST 5.8 (their pI value being 5.8) . Amino acid sequences of CNBr fragments of bGST 5.8 from cornea, sclera, retina and iris-ciliary body showed high degree of primary structure homologies with the corresponding regions of mGSTA4-4 indicating these bovine GST isozymes were distinct from the alpha . mu and pi group GSTs and were the newest members of the group of GSTs to which mGSTA4-4 belongs . There were significant differences among the amino acid sequences of bGST 5.8 of cornea and iris-ciliary body and retina suggesting presence of at least two closely related genes at bGST 5.8 locus . bGST 5.8 isozymes showed high activity toward 4-HNE (four-to-five-fold higher than that towards 1-chloro-2,4-dinitrobenzene), expressed GSH-peroxidase activity towards fatty acid hydroperoxides and phospholipid hydroperoxides, and showed GSH-conjugating activity towards fatty acid epoxides suggesting that these isozymes may play an important role in protection mechanism against the endogenous toxicants formed during lipid peroxidation. J Biochem (Tokyo), 1994 Aug, 116(2), 416 - 25 cDNA cloning and characterization of mitochondrial import stimulation factor (MSF) purified from rat liver cytosol; Alam R et al.; We identified a liver cytosolic protein factor that stimulated the import of wheat germ lysate-synthesized precursor proteins into mitochondria . It was termed mitochondrial import stimulation factor or MSF {Hachiya, N . et al . (1993) EMBO J . 12, 1579-1586} . It consisted of 32-kDa (MSFL) and 30-kDa (MSFS) polypeptides as assessed by SDS-PAGE . MSF recognized the presequence portion of mitochondrial precursor proteins and catalyzed the depolymerization and unfolding of in vitro synthesized mitochondrial precursor proteins in an ATP-dependent manner . We report here the cDNA cloning and characterization of MSF . Microsequencing of MSFL and MSFS showed that they belonged to a highly conserved, widely distributed eukaryotic protein family, collectively designated as 14-3-3 proteins . We cloned the cDNA of MSFL and that of one component of MSFS (MSFS1) from a rat liver cDNA library . The cloned cDNAs were separately expressed in Escherichia coli and the expressed proteins were purified to homogeneity . The purified recombinant MSFL and MSFS1 stimulated mitochondrial import of adrenodoxin precursor (pAd) synthesized in vitro with wheat germ lysate translation system . Recombinant MSFL or MSFS1 had the ability to bind with denatured pAd and they kept the precursor in an import-competent state . Rabbit polyclonal antibodies raised against the recombinant proteins inhibited the import-stimulation activity of rat liver cytosol as well as that of authentic purified MSF . Identification of MSF as 14-3-3 proteins establishes a novel function for this family of proteins and indicates their role as cytosolic chaperones to aid many important cellular events. J Biochem (Tokyo), 1994 Aug, 116(2), 399 - 405 Production of human salivary type cysteine proteinase inhibitors (cystatins) by an Escherichia coli system and partial characterization of recombinant cystatin S and its mutant (117 arginine-->tryptophan); Saitoh E et al.; The cDNAs encoding the precursors of cystatin SN, cystatin S, and two mutants of cystatin S (-18R-->W; 117R-->W) were expressed in Escherichia coli JM109 with isopropyl-beta-D-thio-galactoside (IPTG) induction . Premature cystatin S with the original signal {-20MARPLCTLLLLMATLAGALA} was processed and a large amount of the mature form was produced . A mutation (-18R-->W) in the signal reduced its accumulation in periplasmic space remarkably . The amount of cystatin SN accumulated in the periplasm was slightly smaller than that of cystatin S . The periplasmic fraction was prepared by cold osmotic-shock treatment and the expressed cystatins were detected using anti-cystatin S antibody . Recombinant cystatin S and its mutant (117R-->W) were purified from the periplasmic fractions with an ion exchange column of DEAE-cellulose . The amino (N-) terminal 10 residues of recombinant cystatin S was sequenced to be SSSKEENRII-, which is exactly identical to that of the authentic mature cystatin S . Recombinant cystatin S and the mutant showed virtually the same inhibitory properties for ficin, papain and cathepsin B as the native cystatin S and its monophosphorylated form . The inhibitory activity of the both recombinant cystatins for cathepsin C was weaker than those of the native cystatin S and phosphorylated cystatin S. J Biochem (Tokyo), 1994 Aug, 116(2), 393 - 8 Recombinant human tyrosine hydroxylase types 1-4 show regulatory kinetic properties for the natural (6R)-tetrahydrobiopterin cofactor; Nasrin S et al.; Human tyrosine hydroxylase (hTH) exists in four isoforms . The four recombinant hTH isoenzymes types 1-4 (hTH1-4) produced in and purified from Escherichia coli showed regulatory kinetic properties for the natural (6R)-L-erythro-tetrahydrobiopterin (RBPH4) as a cofactor . In contrast, the unnatural cofactor (6S)-L-erythro-tetrahydrobiopterin (SBPH4) and a synthetic cofactor (6RS)-methyl-tetrahydropterin (6MPH4) showed usual kinetic characteristics with each of hTH1-4 . Substrate inhibition by tyrosine was observed for each of hTH1-4 with natural RBPH4 . Two different Km values for pterin cofactor were observed at a high concentration (200 microM) of L-tyrosine only with natural RBPH4, in contrast to a single Km value for unnatural SBPH4 or synthetic 6MPH4 . The present results suggest that in the presence of relatively high concentrations (approximately 100 microM) of tyrosine in vivo, RBPH4 cofactor may have a regulatory role for the activity of all four human isoenzymes in vivo . We also found that recombinant hTH1 and 3 were more unstable than hTH2 and 4, suggesting that the 4-amino-acid insertion in hTH2 and 4 may be responsible for the relative stability of hTH2 and 4 isoenzymes. J Biochem (Tokyo), 1994 Aug, 116(2), 368 - 73 Accumulation of platelet-activating factor acetylhydrolase in the peritoneal cavity of guinea pig after endotoxin shock; Karasawa K et al.; We examined the production of PAF, a mediator of shock, and LysoPAF, an inactive metabolite of PAF, in the guinea pig peritoneal cavity after i.p . administration of Escherichia coli LPS . Within 1 h of LPS administration, the level of PAF in the peritoneal fluid increased from 4.9 to 37.2 pmol/animal and decreased to the control value thereafter . In contrast, the level of lysoPAF gradually rose from 63.5 to 268 pmol/animal for up to 6 h . The activity of acetylhydrolase, which converts PAF to lysoPAF, in the peritoneal cavity increased in parallel with the increase in the lysoPAF level . The enzyme was distinguishable from phospholipase A2, because p-bromophenacylbromide (p-BPB), Ca2+, and ethylenediaminetetraacetic acid (EDTA) did not affect its enzymatic activity . In addition, this acetylhydrolase revealed similar biochemical properties to that detected in plasma . Both acetylhydrolases were resistant to trypsin treatment and had the same apparent molecular weight, as shown by gel-filtration column chromatography . These results suggest that the acetylhydrolase, which accumulates in the peritoneal cavity, infiltrates from the plasma in response to LPS, and then participates in the exclusion of PAF during endotoxin shock. J Biochem (Tokyo), 1994 Aug, 116(2), 285 - 90 The NhaB Na+/H+ antiporter is essential for intracellular pH regulation under alkaline conditions in Escherichia coli; Shimamoto T et al.; We isolated a mutant of Escherichia coli which was defective in an Na+/H+ antiporter and grew poorly under alkaline conditions {Ishikawa, T., Hama, H., Tsuda, T., and Tsuchiya, T . (1987) J . Biol . Chem . 262, 7443-7446} . Later, it was concluded that the defective Na+/H+ antiporter in the mutant was the NhaB system, and the nhaB gene was mapped to 25.6 min on the E . coli chromosome {Thelen, P., Tsuchiya, T., and Goldberg, E.B . (1991) J . Bacteriol . 173, 6553-6557} . We found that the NhaB-defective cells cannot grow in a high pH medium . Furthermore, intracellular pH in the mutant cells was almost the same as extracellular pH between 7.9 and 9.1, that is, intracellular pH was not regulated at this pH range . On the other hand, intracellular pH of the wild-type cells was maintained at about 7.6 when the extracellular pH was between 7.6 and 8.5 . Thus, the NhaB Na+/H+ antiporter is essential for the regulation of intracellular pH under alkaline conditions in E . coli . Introduction of nhaA gene into the mutant cells increased Na+/H+ antiporter activity, but did not restore the defective growth and defective intracellular pH regulation under alkaline conditions. Mol Cell Endocrinol, 1994 Aug, 104(1), 81 - 93 Overexpression of rainbow trout estrogen receptor domains in Escherichia coli: characterization and utilization in the production of antibodies for immunoblotting and immunocytochemistry; Pakdel F et al.; Complementary DNA fragments that encode central and C-terminal domains of rainbow trout estrogen receptor (rtER) were expressed in Escherichia coli as fusion proteins with glutathione-S-transferase (GST) . Both fusion proteins were induced by IPTG and could readily be detected as a 53-55 kDa band in crude extracts or in insoluble fraction after polyacrylamide gel electrophoresis and Coomassie blue staining . These recombinant proteins were solubilized and partially purified (ca . 60-75%) using centrifugation and different concentrations of urea . Gel mobility shift assays revealed that the hybrid protein containing ER central domain forms a specific complex with a synthetic estrogen-response-element . Similarly, we showed by steroid-binding assays that the hybrid protein containing the ER C-terminal domain binds specifically estrogen and not other steroids . These hybrid receptors were further isolated by electroelution after electrophoresis and used to immunize rabbits . Polyclonal antibodies from each antiserum were purified using GST-rtER fusion proteins . The specificity of these purified antibodies was confirmed by Western blot analysis using extracts from yeast and COS-1 cells transfected with rtER cDNA expression vectors . In these cells, rtER level was about 300-500 fmol/mg of protein, and the receptor was found as a single band migrating as a 65 kDa polypeptide . Interestingly, Western blot analysis with both purified antibodies directed against central or C-terminal regions of rtER revealed two receptor forms in trout liver nuclear extracts: a major form migrating as 65 kDa protein also observed in transfected cells, and a minor band at 71 kDa specific to the liver . Both receptor form levels were strongly induced by estradiol whereas they were virtually undetectable in untreated male trout livers . Immunocytochemistry performed on brain and pituitary of female trout revealed the presence of rtER in neurons located in the ventral telencephalon, preoptic area and mediobasal hypothalamus, as well as cells in the proximal pars distalis of the pituitary. Biochem Soc Trans, 1994 Aug, 22(3), 801 - 5 High-resolution, non-crystallographic structural studies of large integral membrane proteins; Watts A; The work described here clearly demonstrates that n.m.r . is a viable method with which to resolve molecular details about membrane proteins and can give information at a resolution comparable with that gained from crystallographic studies, where, in the limited number of cases studied so far, such information is available . Sensitivity is not a major problem although other difficulties may prevent particular kinds of information being resolved . However, because bond orientational details are obtained ab initio, the method is quite model independent and interpretationally unique . The requirements for solid-state n.m.r . methods to be applied to a large membrane protein are that: a sufficient amount of functionally active protein is available; the protein should be located in bilayers; and suitable specific isotopic enrichment is achieved, in ideal cases, either by chemical or biosynthetic means . Achieving these requirements will need to draw on a range of skills, possibly genetic or chemical or both, and a full understanding of the biochemistry is essential to ensure that any structural information gained is functionally relevant . The structural information potentially available includes: (a) conformation details about specific parts of a protein or its binding sites; (b) distance measurements between specific labels introduced into the protein; (c) dynamics of particular parts of the protein; (d) chain folding and residue orientation; and (e) the mechanistic changes that can occur during a functional cycle . Much developmental work still needs to be done both on the sample handling instrumental side and with the theoretical aspect of the method.(ABSTRACT TRUNCATED AT 250 WORDS) Biokhimiia, 1994 Aug, 59(8), 1238 - 44 {Determination of homoserine kinase activity by chromatographic separation and measurement of reaction products}; Gening LV et al.; A highly specific procedure for quantitative assay of the homoserine kinase activity in an optimized enzymatic reaction using 14C-labelled homoserine or {gamma 33P}-ATP as substrates, and paper or thin-layer chromatography for separation of the formed o-phosphohomoserine, is described . The procedure is simple, sensitive and allows the assay for the activity of both purified and non-purified homoserine kinases. J Comput Aided Mol Des, 1994 Aug, 8(4), 405 - 20 Quantitative structure-activity relationships by neural networks and inductive logic programming . I . The inhibition of dihydrofolate reductase by pyrimidines; Hirst JD et al.; Neural networks and inductive logic programming (ILP) have been compared to linear regression for modelling the QSAR of the inhibition of E . coli dihydrofolate reductase (DHFR) by 2,4-diamino-5-(substituted benzyl)pyrimidines, and, in the subsequent paper {Hirst, J.D., King, R.D . and Sternberg, M.J.E . J . Comput.-Aided Mol . Design, 8 (1994) 421}, the inhibition of rodent DHFR by 2,4-diamino-6,6-dimethyl-5-phenyl-dihydrotriazines . Cross-validation trials provide a statistically rigorous assessment of the predictive capabilities of the methods, with training and testing data selected randomly and all the methods developed using identical training data . For the ILP analysis, molecules are represented by attributes other than Hansch parameters . Neural networks and ILP perform better than linear regression using the attribute representation, but the difference is not statistically significant . The major benefit from the ILP analysis is the formulation of understandable rules relating the activity of the inhibitors to their chemical structure. J Comput Aided Mol Des, 1994 Aug, 8(4), 367 - 88 Nucleotide-binding properties of adenylate kinase from Escherichia coli: a molecular dynamics study in aqueous and vacuum environments; Kern P et al.; The complex of adenylate kinase with its transition-state inhibitor has been studied by molecular dynamics simulations in water and in vacuum environments with the GROMOS force field over a period of 300 ps . The adenylate kinase, a member of the nucleotide-binding protein family, was exemplarily chosen for the inspection of the nucleotide-binding properties in the active site . The ligand binding and the domain movements have been studied in detail over the simulation period and compared with the crystal structure . Secondary structure transitions and domain closures defined those parts of the structure which are involved in an induced-fit movement of the enzyme . The presence of more stable hydrogen bonds on the substrate side leads to the assumption that substrate binding is more specific than cosubstrate binding . Reliable results were achieved only if water was explicitly included in the stimulation. Eur J Surg, 1994 Aug, 160(8), 437 - 42 Macrophage phagocytic dysfunction and reduced metabolic response in experimental obstructive jaundice; Ding JW et al.; OBJECTIVE: To investigate the metabolic response as measured by microcalorimetry and the phagocytic activity or peritoneal macrophages that had been harvested from jaundiced and normal rats and incubated with Escherichia coli in vitro . DESIGN: Open laboratory study . SETTING: University departments of surgery and immunology . MATERIAL: 12 Male Sprague-Dawley rats . INTERVENTIONS: Ligation and transsection of the common bile duct (n = 6) or sham operation (n = 6) . MAIN OUTCOME MEASURES: Metabolic response (pW/cell) measured by microcalorimetry and phagocytic function assessed by light microscopy after Giemsa stain two weeks after operation . RESULTS: The mean (SEM) maximal metabolic response of macrophages and the metabolic rate one hour after inoculation with E coli were significantly reduced in jaundiced rats compared with controls (6.95 (1.95) compared with 27.39 (7.24), p = 0.005, and 5.50 (1.05) compared with 20.10 (3.35) p = 0.016, respectively) as were the number of E coli phagocytosed by macrophages harvested from jaundiced animals (p = 0.0002) . CONCLUSION: The reduced metabolic response and phagocytosis of E coli by peritoneal macrophages in rats by biliary obstruction is a sign of depressed reticuloendothelial function . This mechanism may explain the high incidence of infective complications inpatients with obstructive jaundice. Pharmacol Ther, 1994 Aug, 63(2), 199 - 207 Metabolic suicide genes in gene therapy; Mullen CA; This article reviews uses of metabolic suicide genes in gene therapy . Suicide genes encode novel nonmammalian enzymes that can convert a relatively nontoxic prodrug into a highly toxic agent . Cells genetically transduced to express such genes essentially commit metabolic suicide in the presence of the appropriate prodrug . Three metabolic suicide genes are described: herpes simplex thymidine kinase, Escherichia coli cytosine deaminase and varicella zoster thymidine kinase . Transfer and expression of these genes into mammalian cells is described . Preclinical models of suicide gene therapy of cancer and human immunodeficiency virus are discussed, and several clinical trials employing suicide genes are described. Protein Eng, 1994 Aug, 7(8), 997 - 1004 Novel substrate specificity engineered in the arabinose binding protein; Declerck N et al.; The L-arabinose binding protein (ABP) of Escherichia coli naturally binds L-arabinose and D-galactose with very high affinity and, with reduced affinity, a variety of other sugars that differ only at the C5 position of the pyranose ring . However, there are stringent specificity requirements at the 1, 2, 3 and 4 positions . Based on the high resolution crystallographic structure of the ligand-protein complex, remodelling of the binding pocket was attempted to shift the specificity towards C1-substituted galactosides . To create space in the vicinity of the reducing end of bound galactose, four residues, Lys10, Asp90, Thr147 and Leu145, have been mutated for residues with smaller side chains . Forty-seven mutants containing different combinations of these mutations were tested by fluorometry for their ability to bind methyl-beta-D-galactoside (met-beta-Gal) or iso-propyl-beta-D-thio-galactoside (IPTG) . Two double-residue mutants carrying Ser at position 147 and Ala or Gly at position 90 appeared of particular interest for being able to bind met-beta-Gal or IPTG, respectively, and no longer galactose . Fluorescence experiments and molecular modelling indicate that the mode of binding of the new substrates to the mutant proteins might be similar to that of the natural ligands to wild-type ABP. Protein Eng, 1994 Aug, 7(8), 977 - 84 Structural basis for the difference in thermodynamic properties between the two cysteine proteinase inhibitors human stefins A and B; Jerala R et al.; Homology modelling has been used to model stefin A based on the X-ray structure of stefin B . Several models have been produced by interactive modelling or positioning of the side chains by Monte Carlo procedure with simulated annealing . The quality of models was evaluated by calculation of the free energy of hydration, 3D-1D potential or buried area of surface accessibility . Stefin A is a thermostable protein, exhibiting a two-state denaturation, while stefin B denatures at a 40 degrees C lower temperature and forms a stable molten globule intermediate under mild denaturing conditions . From the tertiary structures, thermodynamic functions were predicted, conforming closely to the experimental calorimetric results . Polar and apolar buried areas of surface accessibility were obtained by structural deconvolution of the thermograms . It is suggested that the basic difference between the stefins is the domination of hydrophobic interaction in the stabilization of stefin B, which is due to its non-specific nature leading to the formation of a molten globule intermediate . Modelling of stefin A predicts increased numbers of hydrogen bonds which stabilize it and increase the cooperativity of its denaturation. Protein Eng, 1994 Aug, 7(8), 969 - 76 Cooperative deformation of a de novo designed protein; Tanaka T et al.; A de novo protein design has been made to understand the unique packing of natural proteins that have a beta/alpha-barrel fold . A carefully designed 207 amino acid sequence was synthesized using an Escherichia coli expression system and the structural and thermodynamic characteristics of the purified protein were studied . At neutral pH the protein is soluble and monomeric, with large amounts of secondary structure and a hydrophobic core, although the broad resonance peaks of its NMR spectrum suggest that the designed protein does not have a unique structure with tightly packed side chains . In an H-D exchange experiment, no amido protons of the designed protein exchanged slowly with deuterons . At acidic pH, thermal unfolding was observed with a remarkable change in the excess heat capacity measured directly by a differential scanning microcalorimeter . The enthalpy and entropy differences at 110 degrees C, extrapolated from analyzed thermodynamic parameters, are approximately 1/3 of the common values for natural proteins . These measurements indicate that the folding is significantly cooperative as expected, but that the protein is still loosely packed. Protein Eng, 1994 Aug, 7(8), 1017 - 26 Multivalent Fvs: characterization of single-chain Fv oligomers and preparation of a bispecific Fv; Whitlow M et al.; Single-chain Fv proteins are known to aggregate and form multimeric species . We report here that these molecules represent a new class of molecular assembly, which we have termed multivalent Fvs . Each binding site in a multivalent Fv comprises the variable light-chain (VL) domain from a single-chain Fv, and the variable heavy-chain (VH) domain from a second single-chain Fv . Each single-chain Fv in a multivalent Fv is part of two binding sites . We have characterized the multivalent forms of the 4-4-20, CC49 and B6.2 sFvs . The degree of multivalent Fv formation is linker-dependent . Multivalent Fvs cannot form in the absence of an intact linker . Multivalent Fvs can be stabilized by their antigen . The conversion between different forms of the multivalent Fvs can be catalyzed by disassociating agents such as 0.5 M guanidine hydrochloride with 20% ethanol . Multivalent Fvs have significantly different stabilities depending on the specific variable domains from which they are constructed . Two models have been proposed for the structure of a multivalent Fv . We have tested each model by attempting to produce a heterodimer from the anti-fluorescein 4-4-20 and anti-tumor CC49 variable regions . We successfully produced a 4-4-20/CC49 heterodimer that comprises two mixed sFvs . The first mixed sFv is composed of the 4-4-20 VL domain, a 12 residue linker and the CC49 Vh domain . The second mixed sFv is composed of a CC49 VL domain, a 12 residue linker and the 4-4-20 VH domain . The 4-4-20/CC49 heterodimer bound both fluorescein and the tumor-associated glycoprotein-72 antigen . These results support a VH/VL 'rearrangement' model in which each variable domain of a multivalent Fv binding site comes from a different polypeptide chain. Mol Biochem Parasitol, 1994 Aug, 66(2), 309 - 18 Molecular characterization of the alpha-subunit of Trichomonas vaginalis hydrogenosomal succinyl CoA synthetase; Lahti CJ et al.; The anaerobic, parasitic protist, Trichomonas vaginalis, is characterized by the absence of mitochondria and the presence of double membrane bound organelles called hydrogenosomes . Succinyl-coenzyme A synthetase is a hydrogenosomal enzyme which catalyzes the formation of ATP via substrate-level phosphorylation . We have characterized genes encoding the alpha subunit of the hydrogenosomal protein succinyl-coenzyme A synthetase (SCS) . The alpha-SCS of T . vaginalis is encoded by a multigene family composed of 3 similar genes that do not appear allelic . These 3 alpha-SCS genes encode a protein with a calculated molecular mass of approximately 32.5 kDa that has > 50% identity (> 70% similarity with alpha-SCSs from Escherichia coli, Thermus flavus, and rat liver mitochondria . Antibodies raised against recombinant T vaginalis alpha-SCS expressed in bacteria were used to isolate alpha-SCS proteins from purified hydrogenosomes . These proteins partition into the soluble fraction of hydrogenosomes treated with sodium carbonate at high pH, consistent with a matrix localization in the organelle . Amino-terminal sequencing of purified alpha-SCS proteins shows that mature proteins lack a short, leader sequence of 9 amino acids . These amino terminal sequences which are cleaved from T . vaginalis alpha-SCSs are similar to each other and to all other leader sequences identifed on hydrogenosomal proteins. FEMS Immunol Med Microbiol, 1994 Aug, 9(2), 143 - 50 Expression of antigenically distinct fimbriae with hemagglutination and HeLa cell adherence properties by an enteroaggregative Escherichia coli strain belonging to the enteropathogenic serogroup; Nandy RK et al.; An enteroaggregative Escherichia coli (EAggEC) strain (DS92), isolated from a case of infantile diarrhea, was shown to express mannose-resistant hemagglutination and HeLa cell adhering properties when grown at 37 degrees C but not at 28 degrees C . Cellular adherence properties of DS92, which belonged to enteropathogenic serogroup 0125, were shown to correlate well with the expression of fimbriae that were encoded by a 112 kb plasmid . The fimbriae of the EAggEC strain DS92 were composed of 20 kDa subunit proteins and were serologically distinct from fimbrial or non-fimbrial cell surface antigen(s) of other diarrheagenic E . coli strains including the reference EAggEC strain 17-2 . Interestingly, the 20-kDa fimbrial protein was found to be antigenically related to 18- and 14.5-kDa cell surface proteins of two other locally isolated EAggEC strains belonging to the enteropathogenic serogroup 086. Biochem Mol Biol Int, 1994 Aug, 33(6), 1117 - 26 The prolactin of European sea bass (Dicentrarchus labrax L.): cloning of cDNA and efficient expression in Escherichia coli; Doliana R et al.; The cDNA encoding sea bass (Dicentrarchus labrax) prolactin (sbPRL) was obtained by reverse transcription-polymerase chain reaction (RT/PCR) from pituitary RNA with degenerate primers designed on the basis of the cDNAs of the two PRLs (tPRL188 and tPRL177) from the tilapia, Oreochromis niloticus . The sbPRL cDNA encodes a preprotein of 212 amino acids composed of a putative signal peptide of 24 residues and a mature protein of 188 amino acids that is the homologue of tiPRL188 . The cDNA coding for the mature protein was cloned into the pAX4a+ expression vector and expressed efficiently in Escherichia coli as a beta-galactosidase-fusion protein . To split the fusion protein, a sequence encoding the hexapeptide, (Asn-Gly)3, that contains three Asn-Gly hydroxylamine-cleavable bonds, had been previously introduced by PCR upstream of the sbPRL cDNA . N-terminal sequencing confirmed that the cleaved product corresponded to sbPRL . An antiserum raised against the recombinant hormone detected by immunoblotting a single band in sea bass pituitaries and two bands in tilapia pituitaries, suggesting the occurrence of a single PRL form in sea bass. Biosci Biotechnol Biochem, 1994 Aug, 58(8), 1490 - 5 Carboxypeptidase Taq, a thermostable zinc enzyme, from Thermus aquaticus YT-1: molecular cloning, sequencing, and expression of the encoding gene in Escherichia coli; Lee SH et al.; The gene for carboxypeptidase Taq, a thermostable metallo-carboxypeptidase from Thermus aquaticus YT-1, was cloned and sequenced . The gene comprised an open reading frame of 1,536 base pairs with a GTG initiation codon and a TGA termination codon, which encodes a protein of 56,210 Da consisting of 511 amino acid residues . The GTG initiation codon of the gene was replaced with ATG by site-directed mutagenesis, and then the gene was expressed in Escherichia coli . The enzyme purified from E . coli cells showed the same properties as those of carboxypeptidase Taq prepared from T . aquaticus cells