Clin Exp Pharmacol Physiol, 1996 Aug, 23(8), 754 - 5 Plasma follistatin concentrations increase following lipopolysaccharide administration in sheep; Klein R et al.; 1 . The effect of inflammation induced by lipopolysaccharide (LPS) injection on plasma follistatin (FS) concentrations was investigated . 2 . Plasma FS and tumour necrosis factor-alpha concentrations increase following LPS administration in ewes . 3 . The rise in FS is similar, but more sustained, to that previously observed after surgery . 4 . These results indicate a possible functional link between FS, inflammation and the acute-phase response. Res Commun Mol Pathol Pharmacol, 1996 Aug, 93(2), 219 - 24 Suppression of enterotoxin-induced intestinal fluid secretion by wood creosote; Ataka K et al.; Wood creosote suppresses intestinal fluid secretion induced by heat-labile enterotoxin (LT) of enterotoxigenic Escherichia coli (ETEC) . When rabbit jejunum is ligated into a 5-cm segment and LT is administered locally, it actively induces intestinal fluid secretion in a dose-dependent manner . Local administration of wood creosote together with a fixed dose of LT suppressed the LT-induced fluid secretion in a dose-dependent manner . At a 50-ng/segment dose of LT, 7.4 +/- 1.1 ml (n = 5) of fluid is secreted into an intestinal segment; coadministration of wood creosote (150 micrograms/segment) significantly (p < 0.01) suppressed the fluid secretion to 2.4 +/- 2.3 ml . Based on these results, we conclude that the antidiarrheal activity of wood creosote is attributable to its antisecretory or proabsorptive effect (or both) on the intestine. J Periodontal Res, 1996 Aug, 31(6), 414 - 22 Effects of T cell adoptive transfer into nude mice on alveolar bone resorption induced by endotoxin; Ukai T et al.; Using the method of reconstitution of nude mice with T cells, we examined the effects of T cell on alveolar bone resorption induced by repeated injections of Escherichia coli endotoxin into periodontal tissue . Three mice groups (normal, nude and T cell reconstituted nude mice) were used . Endotoxin derived from E coli was repeatedly injected into the gingiva of the mice left mandibles every 48 h and the mice were killed on the day after the 1st, 4th, 7th, 10th, 13th and 20th injections of endotoxin . Alveolar bone resorption was examined histopathologically and histomorphometrically . Bone surfaces in contact with the osteoclast were defined as the site of active resorption and the ratios of active resorption were compared among the 3 mice groups . Consequently, no active resorption was found after the first injection of endotoxin in any group . After the 4th injection, active resorption was found in normal mice and T cell reconstituted nude mice and gradually rose with the increase in the injection frequency . In contrast, few osteoclasts were found even after the 10th injection in the nude mice . In addition, there were statistically significant differences between the normal mice and nude mice after the 4th and 10th injections (p < 0.05) . These findings suggested that T cell influences periodontal bone destruction induced by local administration of endotoxin during the early phases. J Clin Pathol, 1996 Aug, 49(8), 642 - 7 Antigen capture ELISA for the heat shock protein (hsp60) of Chlamydia trachomatis; Horner PJ et al.; AIMS: To develop an indirect ELISA using the heat shock protein (hsp60) of Chlamydia trachomatis as antigen . METHODS: The hsp60 gene was amplified by PCR, expressed in the vector pDEV-107 and transformed into Escherichia coli . The recombinant protein, expressed as a beta-galactosidase fusion product, was captured onto a solid phase using a monoclonal antibody directed against beta-galactosidase . Following incubation with goat anti-human antibody conjugated to peroxidase and colour development on addition of peroxidase substrate, antibody recognition of antigen was quantified by optical density at 492 nm . RESULTS: A sensitive and relatively specific ELISA to detect hsp60 has been produced, which can be exploited to determine the antibody response to C trachomatis hsp60 . CONCLUSIONS: This assay will permit the future investigation of the immunopathogenesis of persistent inflammation following C trachomatis infection. Bioorg Med Chem, 1996 Aug, 4(8), 1237 - 46 A protein radical cage slows photolysis of methylcobalamin in methionine synthase from Escherichia coli; Jarrett JT et al.; Methionine synthase from Escherichia coli is a B12-dependent enzyme that utilizes a methylcobalamin prosthetic group . In the catalytic cycle, the methyl group of methylcobalamin is transferred to homocysteine, generating methionine and cob(I)-alamin, and cob(I)alamin is then remethylated by a methyl group from methyltetrahydrofolate . Methionine synthase occasionally undergoes side reactions that produce the inactive cob(II)alamin form of the enzyme . One such reaction is photolytic homolysis of the methylcobalamin C-Co bond . Binding to the methionine synthase apoenzyme protects the methylcobalamin cofactor against photolysis, decreasing the rate of this reaction by approximately 50-fold . The X-ray structure of the cobalamin-binding region of methionine synthase suggests how the protein might protect the methylcobalamin cofactor in the resting enzyme . In particular, the upper face (methyl or beta face) of the cobalamin cofactor is in contact with several hydrophobic residues provided by an alpha-helical domain, and these residues could slow photolysis by caging the methyl radical and favoring recombination of the CH3./cob(II)alamin radical pair . We have introduced mutations at three positions in the cap domain; phenylalanine 708, phenylalanine 714, and leucine 715 have each been replaced by alanine . Calculations based on the wild-type structure predict that two of these three mutations (Phe708Ala and Leu715Ala) will increase solvent accessibility to the methylcobalamin cofactor, and in fact these mutations result in dramatic increases in the rate of photolysis . The third mutation, Phe714Ala, is not predicted to increase the accessibility of the cofactor and has only a modest effect on the photolysis rate of the enzyme . These results confirm that the alpha-helical domain covers the cofactor in the resting methylcobalamin enzyme and that residues from this domain can protect the enzyme against photolysis . Further, we show that binding the substrate methyltetrahydrofolate to the wild-type enzyme results in a saturable increase in the rate of photolysis, suggesting that substrate binding induces a conformational change in the protein that increases the accessibility of the methylcobalamin cofactor. Vaccine, 1996 Aug, 14(11), 1069 - 76 Production of recombinant SERA proteins of Plasmodium falciparum in Escherichia coli by using synthetic genes; Sugiyama T et al.; We expressed two regions of the serine repeat antigen (SERA) protein of Plasmodium falciparum in Escherichia coli by synthesizing the genes with a changed codon usage . One of the synthetic gene sequences encodes amino acid residues 17-382 (SE47') and the other encodes amino acid residues 586-802 (SE50A) . The products produced by the synthetic gene sequences in E . coli accounted for 15-30% of the total bacterial protein . Antisera against both the purified gene products prepared in rats inhibited malaria parasite growth in vitro . The anti-SE47' serum was significantly more inhibitory than the anti-SE50A serum . The described methods provide a large scale preparation of recombinant antigens for improving and producing malaria vaccine. Mol Microbiol, 1996 Aug, 21(4), 871 - 84 New components of protein folding in extracytoplasmic compartments of Escherichia coli SurA, FkpA and Skp/OmpH; Missiakas D et al.; A global search for extracytoplasmic folding catalysts in Escherichia coli was undertaken using different genetic systems that produce unstable or misfolded proteins in the periplasm . The extent of misfolding was monitored by the increased activity of the sigma E regulon that is specifically induced by misfolded proteins in the periplasm . Using multicopy libraries, we cloned two genes, surA and fkpA, that decreased the sigma E-dependent response constitutively induced by misfolded proteins . According to their sequences and their biochemical activities, SurA and FkpA belong to two different peptidyl prolyl isomerase (PPI) families . Interestingly, surA was also selected as a multicopy suppressor of a defined htrM (rfaD) null mutation . Such mutants produce a defective lipopolysaccharide that is unable to protect outer membrane proteins from degradation during folding . The SurA multicopy suppression effect in htrM (rfaD) mutant bacteria was directly associated with its ability to catalyse the folding of outer membrane proteins immediately after export . Finally, Tn10 insertions were isolated, which led to an increased activity of the sigma E regulon . Such insertions were mapped to the dsb genes encoding catalysts of the protein disulphide isomerase (PDI) family, as well as to the surA, fkpA and ompH/skp genes . We propose that these three proteins (SurA, FkpA and OmpH/Skp) play an active role either as folding catalysts or as chaperones in extracytoplasmic compartments. Mol Microbiol, 1996 Aug, 21(4), 839 - 51 The alpha subunit of RNA polymerase and transcription antitermination; Schauer AT et al.; The N gene product of coliphage gamma, with a number of host proteins (Nus factors), regulates phage gene expression by modifying RNA polymerase to a form that overrides transcription-termination signals . Mutations in host nus genes diminish this N-mediated antitermination . Here, we report the isolation and characterization of the rpoAD305E mutation, a single amino acid change in the carboxy terminal domain (CTD) of the alpha subunit of RNA polymerase, that enhances N-mediated antitermination . A deletion of the 3' terminus of rpoA, resulting in the expression of an alpha subunit missing the CTD, also enhances N-mediated antitermination and, similar to rpoAD305E, suppresses the effect of nus mutations . Thus, the N-Nus complex may be affected through contacts with the CTD of the alpha subunit of RNA polymerase, as is a group of regulatory proteins that influences initiation of transcription . What distinguishes our findings on the N-Nus complex from those of previous studies with transcription proteins is that all of the regulators characterized in those studies bind DNA and influence transcription initiation; whereas the N-Nus complex binds RNA and affects transcription elongation . A screen of some previously identified rpoA mutations that influence transcription activators revealed only one other amino acid change, L290H, in the CTD of the alpha subunit, that influences antitermination . Although our results provide evidence that interactions of the alpha subunit of RNA polymerase must be considered in forming models of transcription antitermination, they do not provide information as to whether the interactions of alpha that ultimately influence antitermination occur during initiation or during elongation of transcription. Mol Microbiol, 1996 Aug, 21(4), 787 - 97 DsbA is required for stability of the type IV pilin of enteropathogenic escherichia coli; Zhang HZ et al.; The periplasmic Escherichia coli enzyme DsbA catalyses the efficient formation of disulphide linkages in numerous extracytoplasmic proteins . Enteropathogenic E . coli, a major cause of infantile diarrhoea worldwide, expresses a type IV fimbria known as the bundle-forming pilus that promotes adherence to tissue-culture cells . In this study, we report that transposon insertions in the dsbA locus abolish adherence and dramatically reduce the level of bundlin, the major structural subunit of the pilus encoded by the bfpA locus . Adherence and bundlin levels are restored by complementation with the cloned dsbA gene . DsbA has no effect on bfpA transcription as measured with bfpA-lacZ fusions . Replacement of either cysteine codon 129 or 179 of bfpA with a serine codon results in reduced levels of bundlin, similar to the effect of the dsbA mutation . As is the case with dsbA mutants, this decreased level of bundlin is not due to decreased transcription . The half-life of bundlin as detected by pulse-chase experiments is dramatically reduced in a dsbA mutant in comparison to the wild type . The effect of DsbA on bundlin oxidation is independent of signal-peptide processing . Thus, we demonstrate that the DsbA enzyme is critical for the biogenesis of a type IV fimbria because of the essential role of a disulphide bond in the stability of the major structural subunit . These data illuminate the early steps in the biogenesis of type IV fimbriae by demonstrating that newly synthesized prepilin is a transmembrane protein accessible to periplasmic and cytoplasmic processing enzymes. Mol Microbiol, 1996 Aug, 21(4), 725 - 38 Interaction of FimB and FimE with the fim switch that controls the phase variation of type 1 fimbriae in Escherichia coli K-12; Gally DL et al.; The phase variation of type 1 fimbriae in Escherichia coli is associated with the site-specific inversion of a short DNA element . Recombination at fim requires fimB and fimE, and their products are considered to be the fim recombinases . In this study, FimB and FimE were overproduced and extracts containing the proteins were shown to (i) bind to and (ii) invert the fim switch in vitro . Phenanthroline-copper protection of DNA-protein complexes showed that both FimB and FimE bind to half-sites that flank, and overlap with, the left and right inverted repeats (IRL and IRR, respectively) of the fim switch . Alignment of the four half-sites identified a conserved 5'-CA doublet; mutation of these two bases lowers the affinity of binding of both FimB and FimE to the inverted repeats, and greatly diminishes inversion of the fim switch in vivo . The specificity of the fim recombinases observed in vivo (FimB switching in both directions; FimE switching from on-to-off only) was maintained in vitro . Furthermore, the different binding affinities of FimB and FimE for the various half-site combinations suggests that the specificity of FimE could arise, in part, from the low affinity of FimE for IRL (off). Mol Microbiol, 1996 Aug, 21(4), 695 - 702 Genetically probing the regions of ribose-binding protein involved in permease interaction; Eym Y et al.; The ribose-binding protein (RBP) of Escherichia coli, located in the periplasm, binds to ribose and mediates transport and chemotaxis . The regions on the tertiary structure of RBP that interact with the membrane permease, an ABC transporter, were genetically probed by screening a mutation using the chimeric receptor Trz . Trz is a hybrid protein between the periplasmic domain of chemoreceptor Trg and the cytoplasmic portion of osmosensor EnvZ, which provides a system for monitoring the chemotactic interaction of RBP on MacConkey agar plates when coupled with a reporter lacZ fused to an ompC gene . The expression of ompC can be increased by an interaction of ribose-bound RBP with Trz . A transport defect, either in the binding protein or in the membrane permease, causes a signalling-constitutive Lac+ phenotype of Trz even in the absence of ribose . This appears to be due to the presence of a small amount of ribose, which is normally taken up by the high-affinity transport system . By taking advantage of this, we have designed a system for genetic screening that permits a selection for mutations in the binding protein, causing specific defects in permease interaction but not in tactic interaction . Mutant RBPs that were isolated were unable to perform normal ribose uptake and to utilize ribose as a carbon source, while other functions such as taxis and sugar-binding properties were not substantially affected . The mutational changes were repeatedly found in several residues of RBP, concentrating on three surface regions and comprising two domains of the tertiary structure . We suggest that the two regions, including residues 52 and 166, are specifically involved in the permease interaction while the third region, including residues 72, 134, and others, recognizes both the permease and the chemosensory receptor. J Biomol Struct Dyn, 1996 Aug, 14(1), 25 - 30 Tryptophan intercalation in G, C containing polynucleotides: Z to B conversion of poly {d(G-5M C)} in low salt induced by a tetra peptide; Rajeswari MR; Binding of a tetra peptide, lysyl tryptophenyl glycyl lysine O-ter butyl ester (KWGK) with duplex forms of G, C containing polynucleotides, Poly {d(G-C)}, Poly {d(G-5M C)}, Poly (dG), Poly (dC) and E.coli DNA were studied under low salt conditions using UV absorption, fluorescence, and circular dichroic (C.D) spectroscopy . On addition of the peptide (upto a P/N mole ratio of 0.5), the Poly {d(G-5M C)} under low salt (1 mM Na Cl) conditions, was converted from Z to B-form as shown by the inversion of C.D spectra . The two binding constants (K1 and K2) were determined from fluorescence spectroscopy of which K2 estimates the intercalation of the tryptophenyl side chain between the base pairs of DNA and K1 estimates the electrostatic interactions between the lysyl side chains and phosphate groups . The strength of intercalation is: Z-form of Poly {d(G-5M C)} >> B form of Poly {d(G-5M C)} >> E.Coli DNA > Poly (dG).Poly (dC) . This means that peptide seems to have strong preference for Z compared to B-form and for alternating over non-alternating G, C Sequences . This suggests that tryptophan intercalation may act as a discriminating factor in recognizing Z and B-forms and may have a potential role in Protein-Nucleic acid interactions that are important for transcription. Biochem Mol Biol Int, 1996 Aug, 39(6), 1209 - 20 Organization and nucleotide sequences of ten ribosomal protein genes from the region equivalent to the S10 operon in the archaebacterium, Halobacterium halobium; Miyokawa T et al.; A determination was made of the nucleotide sequence of the 7340-bp region of a ribosomal protein gene cluster of Halobacterium halobium, which is equivalent to the S10 operon of Escherichia coli . The sequence was analyzed with the codonpreference program deduced from the halobacterial codon usage table that showed a very high GC content of the third codon position . The sequence was comprised of a string of 13 tightly linked ORFs . Most of the ORFs were homologous with ribosomal protein genes (ORF1-ORF2-rpl3-rpl4-rpl23--rpl2- rps19-rpl22-rps3-rpl29-ORF11-rps17-r pl14) . The 13-gene string was preceded by three putative AT-rich promoter sequences . The order of the genes in H . halobium essentially agreed with that of the corresponding genes of E . coli (S10-operon), except for certain deletions or insertions of additional protein genes. Biochem Mol Biol Int, 1996 Aug, 39(6), 1109 - 13 Translation initiation region plays an important role in the expression of human thrombopoietin in Escherichia coli; Jiang X et al.; A mutant human thrombopoietin (TPO) gene with a modified translation initiation region (TIR) sequence was created by site-specific mutagenesis based on the PCR technique . This mutant TPO gene encoded the same amino acid sequence as wild-type TPO gene . The wild-type TPO gene was expressed in E . coli with very low efficiency . The mutant TPO gene could reach an expression level of up to 10% of total cellular proteins in E . coli, which was much higher than the wild-type gene . The recombinant protein was mainly in the form of inclusion body which could acquire in vitro activities of human thrombopoietin after refolding. Protein Eng, 1996 Aug, 9(8), 701 - 5 Activation of E350A mutant maltodextrin phosphorylase by exogenously added acetate; Drueckes P et al.; Site-directed mutagenesis of E350 to alanine in Escherichia coli maltodextrin phosphorylase reduced both enzyme activity (100-fold) and apparent binding of the oligosaccharide substrate (10-fold), suggesting a participation of this residue in binding of the substrate in the ground and transition states . The E350A mutant enzyme was found to be activated up to 20-fold by exogenous acetate ions which substitute for the deleted side chain . In contrast, apparent binding was not affected by acetate ions, indicating a dual role for the carboxylic group of this residue in catalysis and binding . Formate also appears to activate the E350A mutant enzyme, but this effect is obscured by the strong inhibitory effect of formate on the wild-type enzyme . For propionate ions, a weak 2-fold activation was noticed, while other compounds like trifluoroacetate and acetamide had no effects on the catalytic properties of either the E350A mutant enzyme or wild-type enzyme . If E350 was substituted by a glutamine, no activation was observed upon the addition of acetate ions . However, a weak activation by formate was found, confirming that activation by acetate is caused by specific binding at the mutated site. Biol Pharm Bull, 1996 Aug, 19(8), 1026 - 31 Substrate specificity of recombinant osteoclast-specific cathepsin K from rabbits; Aibe K et al.; A cDNA clone encoding the rabbit cysteine proteinase cathepsin K, which is predominantly expressed in osteoclasts and is closely related to cathepsins L (EC 3.4.22.15) and S (EC 3.4.22.27) {Tezuka K., Tezuka Y., Maejima A., Sato T., Nemoto K., Kamioka H., Hakeda Y., Kumegawa M., J . Biol . Chem., 269, 1106 (1994)}, was expressed at high levels in Escherichia coli in a T7 expression system . The insoluble recombinant enzyme was solubilized in urea and refolded at an alkaline pH . Cathepsin K (37-kDa) was purified by gel filtration and its enzymatic characteristics were determined . The enzymatic activity of cathepsin K was strongly inhibited by cysteine proteinase inhibitors and its optimal pH was pH 5.5 . Synthetic substrate benzyloxycarbonyl-Phe-Arg-7-(4-methyl)coumaryl-amide, which is hydrolyzed by cathepsins L and S, was also cleaved by cathepsin K . On the other hand, benzyloxycarbonyl-Gly-Pro-Arg-7-(4-methyl)coumaryl-amide was the most suitable substrate for cathepsin K, but was hardly hydrolyzed by cathepsin L . The substrate specificity of cathepsin K, as determined using various chemogenic substrates, showed different characteristics from cathepsins L and S. J Mol Med, 1996 Aug, 74(8), 455 - 62 Adenoviral-mediated delivery of herpes simplex virus thymidine kinase results in tumor reduction and prolonged survival in a SCID mouse model of human ovarian carcinoma; Rosenfeld ME et al.; The herpes simplex virus thymidine kinase gene is the most widely utilized toxin for selective killing of carcinoma cells . Expression of the viral thymidine kinase gene renders cells sensitive to the toxic effects of nucleoside analogs such as ganciclovir . An advantage of this system is the "bystander effect" whereby thymidine kinase transduced tumor cells elicit a toxic effect on surrounding nontransduced tumor cells . Ovarian carcinoma appears to be an ideal candidate for gene therapy as the majority of women present with advanced stage disease, have poor prognosis for long-term survival and have the disease confined within the peritoneal cavity . Therefore the utility of an adenoviral vector to elicit an in vitro bystander effect in ovarian carcinoma cells and the therapeutic efficacy of such a system in vivo was undertaken . Immunocompetent animals were utilized to determine the maximum dose of adenovirus that could be administered without any undesirable side effects and that preimmunization had no effects on subsequent challenge . SCID mice were orthotopically transplanted with human ovarian carcinoma cells and, after establishment of tumor, given a recombinant adenovirus expressing either the herpes simplex virus thymidine kinase or the Escherichia coli beta-galactosidase gene . Half the animals from each viral group were treated with either a ganciclovir regiment (50 mg/kg daily for 14 days) or an equal volume of serum-free media . A subset of mice were killed following drug treatment and analyzed for tumor reduction . The remaining animals were followed daily for survival . The animals treated with the recombinant adenovirus expressing the herpes simplex virus thymidine kinase gene and ganciclovir had significant reduction in overall tumor burden and demonstrated statistically significant prolongation in overall survival. Inflammation, 1996 Aug, 20(4), 389 - 400 Secretion of type-1-fimbriae binding proteins from human neutrophil granulocytes; Karlsson A et al.; Granule matrix proteins secreted from human neutrophils after ionomycin stimulation were separated by SDS-PAGE, blotted onto a polyvinylidene diflouride (PVDF) membrane and overlaid with the mannose-binding lectin concanavalin A (Con A) or Escherichia coli bacteria exposing type-I-fimbriae . Four proteins of approximately 30, 40, 70 and 80 kD, respectively, derived from both the azurophil and the specific granules were shown to expose mannose-containing structures by binding of Con A . Such reactivity was also shown for a 90-kD protein from the light membrane fraction enriched in plasma membrane and secretory vesicles . When blots of granule matrix proteins were exposed to type-I-fimbriated bacteria, a total of seven proteins was recognized; four of the five Con A-binding proteins (40, 70, 80 and 90 kD) was detected also by the bacteria in addition to three proteins not detected by Con A (50, 60 and 100 kD) . The role of the secreted type-1-fimbriae binding proteins as anti-adhesin candidates is discussed. Immunopharmacol Immunotoxicol, 1996 Aug, 18(3), 457 - 63 Spirulina platensis exposure enhances macrophage phagocytic function in cats; Qureshi MA et al.; Bronchoalveolar lavage macrophages isolated from cats were cultured on glass coverslips . Macrophages were exposed to a water-soluble extract of Spirulina platensis in concentration range of 0 to 60 micrograms per mL for two hours . Spirulina-extract exposure did not cause significant macrophage cytotoxicity over untreated control cultures . Macrophage monolayers from treated and control cultures were incubated with sheep red blood cells (SRBC) as well as viable Escherichia coli . The percentages of phagocytic macrophages for both of these particulate antigens were higher (a two-fold increase in SRBC phagocytosis and over 10% increase in Escherichia coli uptake) in cultures treated with various concentrations of Spirulina-extract . However, the numbers of either types of particles internalized by phagocytic macrophage were not different between the control and treated cultures . These data which showed that Spirulina platensis extract enhances macrophage phagocytic function imply that dietary Spirulina supplementation may improve the disease resistance potential in cats. Jpn J Vet Res, 1996 Aug, 44(2), 107 - 18 Changes of serum cytokine activities and other parameters in dogs with experimentally induced endotoxic shock; Miyamoto T et al.; To study the relationship of changes of cytokines in endotoxic shock, serum tumor necrosis factor (TNF), interleukin (IL)-1 and IL-6 like activities, together with physiologic and hemodynamic responses, were examined in dogs before and after intravenous administration of lipopolysaccharide (LPS) purified from Escherichia coli in a dose of 500 micrograms/kg of body weight . The blood endotoxin concentration increased significantly at 30 min after LPS administration, and maintained high levels for 24 hr . Red blood cell counts; hemoglobin concentration and hematocrit values increased at 30 min, and these high values persisted for 24 hr . The platelet count decreased significantly at 30 min, then showed a tendency to recover, but decreased again at 24 hr . Cardiac output, cardiac index and mean arterial pressure showed transient, significant decreases at 15 min, and then returned to the baseline levels by 24 hr . TNF-like activities increased at 30 min, while IL-1-like activities did so between 30 and 60 min . The former reached the maximal levels at 2 hr and the latter at 1.5 hr . Both activities were then hardly detectable from 6 to 24 hr . IL-6-like activities elevated at 1 hr with the peak at 1.5 hr, and remained high until 24 hr. Mol Microbiol, 1996 Aug, 21(3), 613 - 20 Differential regulation of multiple overlapping promoters in flagellar class II operons in Escherichia coli; Liu X et al.; The Escherichia coli flagellar operons are divided into three categories: classes I, II and III . Expression of class II depends on expression of class I . One of the class II gene products, the FIIA protein, is an alternative sigma factor (sigma 28) required for transcription of the class III operons . In this study, we have characterized, in vitro, a role of sigma 28 in the regulation of the class II operons . Among the three class II operons examined, the fliA and fliL operons, but not the flhB operon, could be transcribed by both sigma 70 RNA polymerase holoenzyme with FihD/C (E sigma 70-FlhD/C) and sigma 28 RNA polymerase holoenzyme (E sigma 28) . The flhB operon could only be transcribed by E sigma 70-FlhD/C under the conditions used . Both the fliA and fliL operons contained two overlapping promoters oriented in tandem . The transcription of fliA directed by E sigma 28 could outcompete that by E sigma 70-FlhD/C, indicating a positive autoregulation . However, E sigma 28 could not displace E sigma 70-FlhD/C bound to the fliL promoter . The sigma 28-mediated positive regulation of the class II operons involved a mechanism in which sigma 28 competed with sigma 70 for core RNA polymerase . In addition, recruitment of core RNA polymerase from the sigma 70 -10 site to the sigma 28 -10 was facilitated by formation of E sigma 70-FlhD/C pre-initiation complex . Taken together, the three class II promoters investigated are different in terms of their regulation by sigma 28 . We propose that class II operons may be further divided into different subcategories. Mol Microbiol, 1996 Aug, 21(3), 605 - 12 Examination of AsmA and its effect on the assembly of Escherichia coli outer membrane proteins; Deng M et al.; asmA mutations were isolated as extragenic suppressors of an OmpF assembly mutant, OmpF315 . This suppressor locus produced a protein that was present in extremely low levels and could only be visualized by Western blotting in cells where AsmA expression was induced from a plasmid . Detailed fractionation analyses showed that AsmA localized with the inner membrane . Curiously, however, the mutant OmpF assembly step influenced by AsmA occurred in the outer membrane, perhaps indicating an indirect involvement of AsmA in the assembly of outer membrane proteins . Biochemical examination of the outer membrane showed that asmA null mutations reduce lipopolysaccharide (LPS) levels, thereby lowering the ratios of glycerolphospholipids to LPS and envelope proteins to LPS in the outer membrane . Despite these quantitative alterations, no apparent structural changes in LPS or major phospholipids were noted . Reduced LPS levels in asmA mutants indicate a possible role of AsmA in LPS biogenesis . Data presented in this study suggest that asmA-mediated OmpF assembly suppression may have been achieved by altering the outer membrane fluidity, thus making it more amenable for the assembly of mutant proteins. Mol Microbiol, 1996 Aug, 21(3), 557 - 65 Differential binding of BvgA to two classes of virulence genes of Bordetella pertussis directs promoter selectivity by RNA polymerase; Zu T et al.; Transcription of virulence genes of Bordetella pertussis is co-ordinately regulated by the BvgA and BvgS proteins, which are members of the two-component family of bacterial signal-transduction proteins . BvgS is the transmembrane sensor and BvgA the transcriptional regulator . By gel mobility shift assays we demonstrate that phosphorylated BvgA (BvgA approximately P) forms distinct complexes with the filamentous haemagglutinin (PFHA) promoter DNA at different BvgA approximately P: DNA ratios . DNase I protection analyses show that phosphorylation of BvgA not only enhances affinity of the protein for the binding sites of the PFHA and bvgP1 promoters, but it extends significantly the bound region towards position -35 of these promoters . Conversely, a 10-fold higher amount of BvgA approximately P is required for binding to a large DNA region, from -168 to -60, of the pertussis toxin (Ptox) promoter sequence . These findings suggest that the molecular interaction of BvgA approximately P with the Ptox promoter is different from its interaction with the PFHA and bvgP1 promoters . The sigma 70 Escherichia coli RNA polymerase (RNP) does not bind to the bvg-regulated promoters . However, following the formation of a BvgA approximately P-promoter complex, the E . coli RNP specifically recognizes and binds to the bvg-regulated promoters . Thus, BvgA approximately P exerts its action at the level of promoter recognition by directing promoter selectivity by RNP. Mol Microbiol, 1996 Aug, 21(3), 529 - 41 Assembly proteins of CS1 pili of enterotoxigenic Escherichia coli; Sakellaris H et al.; Some strains of enterotoxigenic Escherichia coli associated with human diarrhoeal disease produce a class of pili represented by those called CS1 . For the assembly of the major-pilin subunit, CooA, into pili, each of four linked genes, cooB, A, C, and D, is required . In this study, we have determined the subcellular localization of CooB, C and D, and investigated the molecular interactions of these proteins using specific antisera . CooD appears to be an integral pilus protein because it co-purifies with, and is strongly associated with, CS1 pili . In keeping with its role as an assembly protein, the CooD minor pilin (when overexpressed in CS1-piliated strains) was detected in periplasmic intermolecular complexes with the major-pilin subunit CooA . CooB is an assembly protein found exclusively in the periplasm of CS1-piliated strains . CooB also forms periplasmic intermolecular complexes with CooA, but does not constitute part of the final pilus structure . Immunoblot analysis of cell fractions showed that CooC is an outer membrane protein of CS1-piliated E . coli . Based on this information, we have proposed a model for CS1-pilus assembly which is very similar to the model for polymerization of the PapA pilin of uropathogenic E . coli . As the assembly proteins of Pap and CS1 pili are structurally unrelated, this may represent a case of convergent evolution. Cytometry, 1996 Aug 1, 24(4), 321 - 9 Enzyme-generated intracellular fluorescence for single-cell reporter gene analysis utilizing Escherichia coli beta-glucuronidase; Lorincz M et al.; We report the development of a new fluorescence-activated cell sorter (FACS)-based reporter gene system utilizing the enzymatic activity of the E . coli beta-glucuronidase (gus) gene . When loaded with the Gus substrate fluorescein-di-beta-D-glucuronide (FDGlcu), individual mammalian cells expressing and translating gus mRNA liberate sufficient levels of intracellular fluorescein for quantitative analysis by flow cytometry . This assay can be used to FACS sort viable cells based on Gus enzymatic activity, and the efficacy of the assay can be measured independently by using a fluorometric lysate assay . Furthermore, both the beta-glucuronidase and the previously described E . coli beta-galactosidase enzymes have high specificities for their cognate substrates, allowing each reporter gene to be measured by FACS independently. Biochem Mol Biol Int, 1996 Aug, 39(5), 1007 - 15 Yeast thiol-dependent protector protein expression enhances the resistance of Escherichia coli to hydrogen peroxide; Ahn SM et al.; A soluble protein from Saccharomyces cerevisiae specifically provides protection against a thiol-containing oxidation system but not against an oxidation system without thiol . This 25-kDa protein was thus named thiol-dependent protector protein (TPP) . The role of TPP in the cellular defense against oxidative stress was investigated in Escherichia coli containing an expression vector with a yeast genomic DNA fragment that encodes TPP (strain YP) and mutants in which the catalytically essential amino acid cysteine (Cys-47) has been replaced with alanine (strain YPC47A) or tryptophan (Trp-82) has been replaced with phenylalanine (strain YPW82F) by a site directed mutagenesis . There was a distinct difference between these three strains in regards to growth inhibition kinetics, viability, modulation of activities of superoxide dismutase and catalase, and the accumulation of oxidized proteins . These results suggest that TPP may play a direct role in the cellular defense against oxidative stress by functioning as an antioxidant protein. Proteins, 1996 Aug, 25(4), 506 - 9 Production and crystallization of a selenomethionyl variant of UmuD', an Escherichia coli SOS response protein; Peat TS et al.; Crystals of both native and mutant Escherichia coli UmuD' protein were obtained using the hanging drop method . Soaking the native crystals in solutions of heavy metal ions failed to produce good isomorphous derivatives, and selenomethionine substituted wild-type protein did not crystallize under conditions that gave native crystals . Site-directed mutagenesis was used to change the penultimate residue, a methionine amino acid, to either a valine or a threonine amino acid . Crystals were subsequently obtained from these mutant proteins with and without selenomethionine incorporation . Crystals of the native, the mutant, and the selenomethionine incorporated protein were all similar, crystallizing in the P4(1)2(1)2 space group. Proteins, 1996 Aug, 25(4), 501 - 5 In vivo association of protein fragments giving active AraC; Eustance RJ et al.; Frameshift mutations in a restricted portion of the arabinose operon regulatory gene araC from Escherichia coli give rise to active AraC protein, likely from the in vivo synthesis of two incomplete fragments that are active together . Synthesis of corresponding fragments, each separately inactive, from two plasmids within cells also resulted in complementation. Br J Pharmacol, 1996 Aug, 118(8), 2157 - 63 Delayed protection against ischaemia-induced ventricular arrhythmias and infarct size limitation by the prior administration of Escherichia coli endotoxin; Song W et al.; 1 . Bacterial endotoxin (lipopolysaccharide derived from Escherichia coli) was injected intraperitoneally in conscious rats in doses ranging from 0.5 to 2.5 mg kg-1 . At various times afterwards the animals were anaesthetized and subjected to a 30 min period of left coronary artery occlusion . 2 . Under these conditions the severity of ventricular arrhythmias was markedly suppressed, in comparison with saline-injected controls, but this was particularly marked with the higher doses (1.5 and 2.5 mg kg-1); the number of ventricular premature beats was reduced from 1687 +/- 227 over the 0.5 h coronary artery occlusion period to 190 +/- 46 in those rats administered 2.5 mg kg-1 endotoxin 8 h previously (P < 0.05) . The duration of ventricular tachycardia was also significantly reduced (138 +/- 26 s to 8.9 +/- 4.2 s; P < 0.01) and there was a reduction in the incidence of ventricular fibrillation (from 56% to 10%) . 3 . The time course of this protection was studied following the administration of a single dose of 2.5 mg kg-1 of endotoxin by anaesthetizing rats 4, 8 or 24 h later . Protection was apparent at each time but was particularly marked at 8 h . 4 . No rat given the highest dose of endotoxin (32 in all) died as a result of ventricular fibrillation, or from any other cause, during an occlusion, in contrast to a 26% mortality in the controls (P < 0.01) . 5 . Infarct size, measured following a 30 min period of coronary artery occlusion followed by a 3 h reperfusion period, was reduced both 8 and 24 h after the administration of 2.5 mg kg-1 endotoxin (reductions of 24.3 and 23.1% respectively; P < 0.05) . Endotoxin had no significant effect on the area at risk . 6 . The beneficial effects of endotoxin on infarct size and on ventricular arrhythmias were markedly attenuated by the prior administration of dexamethasone, 3 mg kg-1 given 1 h prior to endotoxin administration . Dexamethasone itself reduced infarct size (P < 0.05) but had no direct effect on arrhythmia severity following coronary artery occlusion . 7 . The mechanisms of this "cross-tolerance' induced by bacterial endotoxin against ischaemia-reperfusion injury remain to be elucidated but the most likely mechanisms appear to be the induction of protective enzymes or proteins (e.g . nitric oxide synthase, cyclo-oxygenase (COX) 2) probably mediated by cytokine release. Antiviral Res, 1996 Aug, 32(1), 35 - 42 A rapid assay for determination of antiviral activity against human cytomegalovirus; Hippenmeyer PJ et al.; RC256, the recombinant human cytomegalovirus (HCMV) which expresses beta-galactosidase, was used as a tool for rapid screening of compounds for antiviral activity . The effective concentration of antiviral compound needed to inhibit RC256 was identical to the concentrations needed to inhibit other strains of HCMV as measured by plaque reduction assay or virus yield reduction assay . Measurement of beta-galactosidase activity in infected cell lysates allowed determination of effective concentrations 48 h postinfection with results comparable to the longer, more laborious assays. Biotechniques, 1996 Aug, 21(2), 304 - 11 High-level inducible expression of visual pigments in transfected cells; Kazmi MA et al.; A method for high-level expression of a functionally active, recombinant human red cone opsin was developed by adding the coding sequence for the C-terminal epitope of bovine rhodopsin onto the C terminus of the cone opsin and cloning the resulting construct into the vector pMEP4 beta . The recombinant pMEP4 beta vector was transfected stably into 293-EBNA cells, and expression of the cone opsin was induced by the addition of CdCl2 into the medium . The recombinant cone opsin was reconstituted with 11-cis retinal and purified by immunoaffinity chromatography . Spectral analysis prior to and following photobleaching confirmed its identity as a red cone opsin . The protein was targeted to the cell membrane and activated bovine transducin. Shock, 1996 Aug, 6(2), 118 - 25 Differential effects of transforming growth factor-beta 1 on lipopolysaccharide induction of endothelial adhesion molecules; Kang YH et al.; In this report, we studied the effects of transforming growth factor (TGF)-beta 1 on lipopolysaccharide (LPS)-induced expression of endothelial cell (EC) adhesion molecules . Confluent human umbilical cord vein EC cultures were stimulated with Escherichia coli LPS and TGF-beta 1, alone or in combination for various times and evaluated for expression of ICAM-1, E-selectin, and VCAM-1 by immunofluorescence and radioimmunoassay . Effects of LPS and/or TGF-beta 1 on cell growth were also studied by 3H-thymidine incorporation . Both LPS and TGF-beta 1 alone stimulated EC expression of the adhesion molecules in a dose-dependent manner . The effects of TGF-beta 1 on LPS induction of the adhesion molecules varied with LPS concentration and treatment time, mode, and duration . Pretreatment with TGF-beta 1 for 24 h greatly augmented LPS induction of ICAM-1 and VCAM-1 expression, but decreased E-selectin expression . TGF-beta 1 also enhanced expression of the adhesion molecules in cells that were pretreated with 1 microgram/mL LPS for 60 min . Concomitant treatment with TGF-beta 1/LPS resulted in significant increases in ICAM-1 but decreases in VCAM-1 expression . TGF-beta 1 effects on LPS induction of the adhesion molecules were more prominent at lower LPS levels (.001, .01 microgram/mL) . Both LPS and TGF-beta 1 suppressed thymidine incorporation in a dose-related pattern . These data suggest that TGF-beta 1 has additive and antagonistic effects on LPS induction of the adhesion molecules and that the cell responsiveness to the stimuli in the expression is related to growth condition of the cells . In conclusion, our findings suggest that TGF-beta 1 exhibits pro-inflammatory and anti-inflammatory activities in human endothelial cells and may play an important role in regulating leukocyte adherence and extravasation under LPS-induced inflammatory conditions. J Pediatr Gastroenterol Nutr, 1996 Aug, 23(2), 151 - 8 Prognostic factors in hospitalized children with persistent diarrhea: implications for diet therapy; Bhatnagar S et al.; A dietary algorithm for management of persistent diarrhea in developing countries, using locally available foods, is yet to be standardized . We identified factors related to poor outcome among 75 malnourished hospitalized male patients aged 3-48 months with persistent diarrhea (> or = 14 days) treated on soy and cereal-based diet (Diet I) . The 28 patients with stool output > 60 g/k body weight on the sixth or the seventh treatment day were considered diarrhea treatment failures on Diet I . In the univariate analysis, breast feeding (p < 0.001), carbohydrate malabsorption based on low stool pH or reducing substances > 0.5% (p = 0.03), initial 24-h purge rate (p = 0.001), pneumonia (p = 0.003), or probable septicemia (p = 0.03) were associated with diarrhea treatment failures . Although 16 of these 28 patients responded to systemic antibiotics without dietary modification, all but one of the remaining recovered on a chicken puree, glucose, and oil formulation . Twenty-six children had weight loss after 7 days on Diet I as compared with the postrehydration weight . These children had lower mean age (p = 0.05), lower food intake in the first 24 h (p = 0.05) and during the initial 7 days (p < 0.01), and a higher initial excretion of enteroaggregative Escherichia coli (32 vs . 8%; p = 0.01) . In the logistic regression model, significant risk factors for diarrhea treatment failures were initial purge rates, carbohydrate malabsorption, and intercurrent systemic infection; only low food intake was associated with significant risk for weight loss . The significant association of diarrhea treatment failures with carbohydrate malabsorption suggests that in the initial diet itself, part of polysaccharide be substituted with sucrose or glucose to obtain the right balance between osmolarity and energy density . Our data suggest that prompt identification and treatment of systemic infection is critical, as its eradication achieved recovery in more than half of the treatment failures without a dietary change. Protein Sci, 1996 Aug, 5(8), 1697 - 703 Contribution of a tyrosine side chain to ribonuclease A catalysis and stability; Eberhardt ES et al.; An intricate architecture of covalent bonds and noncovalent interactions appear to position the side chain of Lys 41 properly within the active site of bovine pancreatic ribonuclease A (RNase A) . One of these interactions arises from Tyr 97, which is conserved in all 41 RNase A homologues of known sequence . Tyr 97 has a solvent-inaccessible side chain that donates a hydrogen bond to the main-chain oxygen of Lys 41 . Here, the role of Tyr 97 was examined by replacing Tyr 97 with a phenylalanine, alanine, or glycine residue . All three mutant proteins have diminished catalytic activity, with the value of Kcat being perturbed more significantly than that of Km . The free energies with which Y97F, Y97A, and Y97G RNase A bind to the rate-limiting transition state during the cleavage of poly(cytidylic acid) are diminished by 0.74, 3.3, and 3.8 kcal/mol, respectively . These results show that even though Tyr 97 is remote from the active site, its side chain contributes to catalysis . The role of Tyr 97 in the thermal stability of RNase A is large . The conformational free energies of native Y97F, Y97A, and Y97G RNase A are decreased by 3.54, 12.0, and 11.7 kcal/mol, respectively . The unusually large decrease in stability caused by the Tyr-->Phe mutation could result from a decrease in the barrier to isomerization of the Lys 41-Pro 42 peptide bond. Protein Sci, 1996 Aug, 5(8), 1625 - 32 Beta-methylthio-aspartic acid: identification of a novel posttranslational modification in ribosomal protein S12 from Escherichia coli; Kowalak JA et al.; Utilizing microscale chemical derivatization reactions and matrix-assisted laser desorption/ionization time-of-flight mass spectrometry, we have identified a novel posttranslational modification of aspartic acid, beta-methylthio-aspartic acid . The modified residue is located at position 88 in ribosomal protein S12 from Escherichia coli, a phylogenetically conserved protein that has been implicated in maintaining translational accuracy of the ribosome. Protein Sci, 1996 Aug, 5(8), 1613 - 24 Preparation and properties of pure, full-length IclR protein of Escherichia coli . Use of time-of-flight mass spectrometry to investigate the problems encountered; Donald LJ et al.; IclR protein, the repressor of the aceBAK operon of Escherichia coli, has been examined by time-of-flight mass spectrometry, with ionization by matrix assisted laser desorption or by electrospray . The purified protein was found to have a smaller mass than that predicted from the base sequence of the cloned iclR gene . Additional measurements were made on mixtures of peptides derived from IclR by treatment with trypsin and cyanogen bromide . They showed that the amino acid sequence is that predicted from the gene sequence, except that the protein has suffered truncation by removal of the N-terminal eight or, in some cases, nine amino acid residues . The peptide bond whose hydrolysis would remove eight residues is a typical target for the E . coli protease OmpT . We find that, by taking precautions to minimize Omp T proteolysis, or by eliminating it through mutation of the host strain, we can isolate full-length IclR protein (lacking only the N-terminal methionine residue) . Full-length IclR is a much better DNA-binding protein than the truncated versions: it binds the aceBAK operator sequence 44-fold more tightly, presumably because of additional contacts that the N-terminal residues make with the DNA . Our experience thus demonstrates the advantages of using mass spectrometry to characterize newly purified proteins produced from cloned genes, especially where proteolysis or other covalent modification is a concern . This technique gives mass spectra from complex peptide mixtures that can be analyzed completely, without any fractionation of the mixtures, by reference to the amino acid sequence inferred from the base sequence of the cloned gene. Protein Sci, 1996 Aug, 5(8), 1594 - 612 Ribosome-mediated translational pause and protein domain organization; Thanaraj TA et al.; Because regions on the messenger ribonucleic acid differ in the rate at which they are translated by the ribosome and because proteins can fold cotranslationally on the ribosome, a question arises as to whether the kinetics of translation influence the folding events in the growing nascent polypeptide chain . Translationally slow regions were identified on mRNAs for a set of 37 multidomain proteins from Escherichia coli with known three-dimensional structures . The frequencies of individual codons in mRNAs of highly expressed genes from E . coli were taken as a measure of codon translation speed . Analysis of codon usage in slow regions showed a consistency with the experimentally determined translation rates of codons; abundant codons that are translated with faster speeds compared with their synonymous codons were found to be avoided; rare codons that are translated at an unexpectedly higher rate were also found to be avoided in slow regions . The statistical significance of the occurrence of such slow regions on mRNA spans corresponding to the oligopeptide domain termini and linking regions on the encoded proteins was assessed . The amino acid type and the solvent accessibility of the residues coded by such slow regions were also examined . The results indicated that protein domain boundaries that mark higher-order structural organization are largely coded by translationally slow regions on the RNA and are composed of such amino acids that are stickier to the ribosome channel through which the synthesized polypeptide chain emerges into the cytoplasm . The translationally slow nucleotide regions on mRNA possess the potential to form hairpin secondary structures and such structures could further slow the movement of ribosome . The results point to an intriguing correlation between protein synthesis machinery and in vivo protein folding . Examination of available mutagenic data indicated that the effects of some of the reported mutations were consistent with our hypothesis. Protein Sci, 1996 Aug, 5(8), 1541 - 53 Crystal structures of the active site mutant (Arg-243-->Ala) in the T and R allosteric states of pig kidney fructose-1,6-bisphosphatase expressed in Escherichia coli; Stec B et al.; The active site of pig kidney fructose-1,6-bisphosphatase (EC 3.1.3.11) is shared between subunits, Arg-243 of one chain interacting with fructose-1,6-bisphosphate or fructose-2,6-bisphosphate in the active site of an adjacent chain . In this study, we present the X-ray structures of the mutant version of the enzyme with Arg-243 replaced by alanine, crystallized in both T and R allosteric states . Kinetic characteristics of the altered enzyme showed the magnesium binding and inhibition by AMP differed slightly; affinity for the substrate fructose-1,6-bisphosphate was reduced 10-fold and affinity for the inhibitor fructose-2,6-bisphosphate was reduced 1,000-fold (Giroux E, Williams MK, Kantrowitz ER, 1994, J Biol Chem 269:31404-31409) . The X-ray structures show no major changes in the organization of the active site compared with wild-type enzyme, and the structures confirm predictions of molecular dynamics simulations involving Lys-269 and Lys-274 . Comparison of two independent models of the T form structures have revealed small but significant changes in the conformation of the bound AMP molecules and small reorganization of the active site correlated with the presence of the inhibitor . The differences in kinetic properties of the mutant enzyme indicate the key importance of Arg-243 in the function of fructose-1,6-bisphosphatase . Calculations using the X-ray structures of the Arg-243-->Ala enzyme suggest that the role of Arg-243 in the wild-type enzyme is predominantly electrostatic in nature. Protein Sci, 1996 Aug, 5(8), 1453 - 65 Structure of equine infectious anemia virus proteinase complexed with an inhibitor; Gustchina A et al.; Equine infectious anemia virus (EIAV), the causative agent of infectious anemia in horses, is a member of the lentiviral family . The virus-encoded proteinase (PR) processes viral polyproteins into functional molecules during replication and it also cleaves viral nucleocapsid protein during infection . The X-ray structure of a complex of the 154G mutant of EIAV PR with the inhibitor HBY-793 was solved at 1.8 A resolution and refined to a crystallographic R-factor of 0.136 . The molecule is a dimer in which the monomers are related by a crystallographic twofold axis . Although both the enzyme and the inhibitor are symmetric, the interactions between the central part of the inhibitor and the active site aspartates are asymmetric, and the inhibitor and the two flaps are partially disordered . The overall fold of EIAV PR is very similar to that of other retroviral proteinases . However, a novel feature of the EIAV PR structure is the appearance of the second alpha-helix in the monomer in a position predicted by the structural template for the family of aspartic proteinases . The parts of the EIAV PR with the highest resemblance to human immunodeficiency virus type 1 PR include the substrate-binding sites; thus, the differences in the specificity of both enzymes have to be explained by enzyme-ligand interactions at the periphery of the active site as well. Microb Pathog, 1996 Aug, 21(2), 139 - 47 Binding of enterotoxigenic Escherichia coli expressing different colonization factors to tissue-cultured Caco-2 cells and to isolated human enterocytes; Viboud GI et al.; The binding of ETEC strains expressing different colonization factors to the human enterocyte-like cell line Caco-2 and to isolated human enterocytes were determined . Strains expressing CFA/I, CS2, CS4+CS6, CS5+CS6, CS7, CFA/III+CS6 and PCFO166 adhered well to both types of cells whereas bacteria producing CS1, CS6 only, or CS17 did not adhere to either Caco-2 cells or to jejunal human enterocytes, suggesting that similar binding structures are present in both cell types . However, in some instances, binding of bacteria to the two types of cells differed, e.g . bacteria expressing CS3 or PCFO9 bound well to human enterocytes but not to Caco-2 cells . Conversely, bacteria producing PCFO20 or PCFO159 only adhered to Caco-2 cells and not to jejunal enterocytes . With few exceptions, this inability to bind to human enterocytes was the same for cells from all parts of the small intestine . This study contradicts previous reports suggesting that the binding structures of Caco-2 cells are identical to those of human enterocytes. Genetics, 1996 Aug, 143(4), 1843 - 60 Selfish operons: horizontal transfer may drive the evolution of gene clusters; Lawrence JG et al.; A model is presented whereby the formation of gene clusters in bacteria is mediated by transfer of DNA within and among taxa . Bacterial operons are typically composed of genes whose products contribute to a single function . If this function is subject to weak selection or to long periods with no selection, the contributing genes may accumulate mutations and be lost by genetic drift . From a cell's perspective, once several genes are lost, the function can be restored only if all missing genes were acquired simultaneously by lateral transfer . The probability of transfer of multiple genes increases when genes are physically proximate . From a gene's perspective horizontal transfer provides a way to escape evolutionary loss by allowing colonization of organisms lacking the encoded functions . Since organism bearing clustered genes are more likely to act as successful donors, clustered genes would spread among bacterial genomes . The physical proximity of genes may be considered a selfish property of the operon since it affects the probability of successful horizontal transfer but may provide no physiological benefit to the host . This process predicts a mosaic structure of modern genomes in which ancestral chromosomal material is interspersed with novel, horizontally transferred operons providing peripheral metabolic functions. Genetics, 1996 Aug, 143(4), 1521 - 32 Overproduction of three genes leads to camphor resistance and chromosome condensation in Escherichia coli; Hu KH et al.; We isolated and characterized three genes, crcA, cspE and crcB, which when present in high copy confer camphor resistance on a cell and suppress mutations in the chromosomal partition gene mukB . Both phenotypes require the same genes . Unlike chromosomal camphor resistant mutants, high copy number crcA, cspE and crcB do not result in an increase in the ploidy of the cells . The cspE gene has been previously identified as a cold shock-like protein with homologues in all organisms tested . We also demonstrate that camphor causes the nucleoids to decondense in vivo and when the three genes are present in high copy, the chromosomes do not decondense . Our results implicate camphor and mukB mutations as interfering with chromosome condensation and high copy crcA, cspE and crcB as promoting or protecting chromosome folding. Plant Mol Biol, 1996 Aug, 31(5), 1009 - 20 Isolation of two differentially expressed wheat ACC synthase cDNAs and the characterization of one of their genes with root-predominant expression; Subramaniam K et al.; Two partial 1-aminocyclopropane-1-carboxylic acid (ACC) synthase cDNA clones (pWAS1, 1089 bp; and pWAS3, 779 bp) were isolated by polymerase chain reaction (PCR) using cDNA to total mRNA purified from etiolated wheat seedlings as template and degenerate oligonucleotides synthesized based on the regions of the ACC synthase amino acid sequence that are highly conserved among different plants . Northern analysis showed that the expression of the corresponding genes are differentially regulated . While the transcripts of pWAS1 were found in all the tissues of wheat that were tested with a maximum level at the early stages of spike development, pWAS3 mRNA was present almost exclusively in the root . A 5590 bp genomic clone, TA-ACS2, corresponding to pWAS3 cDNA has been isolated . The TA-ACS2 sequence consists of a 589-bp 5'-upstream region, 2743 bp of transcribed region with four exons and three introns and a 3'-downstream region of 2257 bp . Expression in Escherichia coli confirmed the ACC synthase activity of TA-ACS2 polypeptide . Sequence comparisons show that the two wheat ACC synthases are more similar to each other and to the rice ACC synthase, OS-ACS1, at the nucleotide level than at the amino acid level . The amino acid sequence of TA-ACS2 is most similar (66.1% identity) to that of broccoli . The chromosomal location of both wheat ACC synthase genes have been determined by aneuploid analysis . TA-ACS1 is located on the short arm of chromosomes 7A and 7D and on the long arm of chromosome 4A . TA-ACS2 is located on the long arm of homoeologous group 2 chromosomes. Biophys J, 1996 Aug, 71(2), 1123 - 30 High-efficiency loading, transfection, and fusion of cells by electroporation in two-phase polymer systems; Hui SW et al.; A method to concentrate drugs, DNA, or other materials with target cells in two-phase polymer systems for high-efficiency electroloading is described . The two-phase polymer system is utilized for cell and loading material selection, as well as for cell aggregation before electrofusion . The phase mixing of several water-soluble polymers is characterized, and the polyethylene glycol-Dextran (PEG m.w . 8,000 + Dextran m.w . 71,000) mixture is selected to illustrate the advantage of the two-phase systems . Fluorescently labeled Dextran or DNA is loaded into Chinese hamster ovary (CHO) and JTL cells, using electroporation in either the two-phase polymer system or the conventional single-phase suspension . The loading efficiency is 4 to 30 times higher for the two-phase system, with the best advantage at lower applied field range . Transfections of CHO, COS, Melan C, and JTL lymphoid cells using pSV-beta-galactosidase (for CHO and COS), pBK-RSV-tyrosinase, and pCP4-fucosidase plasmids, respectively, by electroporation in the two-phase polymer system and the conventional single-phase electroporation method, are compared . The former method is far superior to the latter in terms of efficiency . The threshold and optimal field strengths for the former are significantly lower than those for the latter method, so the former method is more favorable in terms of equipment requirement and safety . Electrofusion efficiency in the two-phase system is comparable to that in polyethylene glycol suspension alone and is a significant improvement from the conventional electrofusion method with dielectrophoresis . The two-phase polymer method is, therefore, a valuable technique for gene delivery to a limited cell source, as in ex vivo gene therapy. Biophys J, 1996 Aug, 71(2), 811 - 23 Hisactophilin-mediated binding of actin to lipid lamellae: a neutron reflectivity study of protein membrane coupling; Naumann C et al.; The neutron reflectivity technique is applied to determine the adsorptive interaction of the 13.5-kDa actin-binding protein hisactophilin from Dictyostelium discoideum with lipid monolayers at a lateral pressure of 21 mN/m < or = pi < or = 25 mN/m at the air-water interface . We compare binding of natural hisactophilin exhibiting a myristic acid chain membrane anchor at the N-terminus (DIC-HIS) and a fatty acid-deficient genetic product expressed in Escherichia coli (EC-HIS) . It is demonstrated that only the natural hisactophilin DIC-HIS is capable of mediating the strong binding of monomeric actin to the monolayer, where it forms a layer of about 40 A thickness corresponding to the average diameter of actin monomers . Monolayers composed of pure dimyristoyl phosphatidylcholine with fully deuterated hydrocarbon tails and headgroup (DMPC-d67) and 1:1 mixtures of this lipid with chain deuterated dimyristoyl phosphatidylglycerol (DMPG-d54) are studied on subphases consisting either of fully deuterated buffer (D2O) or of a 9:1 H2O/D2O buffer that matches the scattering length density of air (CMA buffer) . The reflectivity data are analyzed in terms of layer models, consisting of one to three layers, depending on the contrast of the buffer and the system . We show that both protein species bind tightly to negatively charged 1:1 DMPC-d67/DMPG-d54 monolayers, thereby forming a thin and most probably monomolecular protein layer of 12-15 A thickness . We find that the natural protein (DIC-HIS) partially penetrates into the lipid monolayer, in contrast to chain-deficient species (EC-HIS), which forms only an adsorbed layer . The coverage of the monolayer with DIC-HIS strongly depends on the presence of anionic DMPG in the monolayer . At a bulk protein concentration of 1.5 micrograms/ml, the molar ratio of bound protein to lipid is about 1:45 for the 1:1 lipid mixture but only 1:420 for the pure DMPC. J Neurosci Res, 1996 Aug 1, 45(3), 308 - 20 Identification and neuron specific expression of the S182/presenilin I protein in human and rodent brains; Elder GA et al.; Many individuals with familial Alzheimer disease (FAD) have mutations in a gene termed S182 or presenilin I (PS-I) . Currently, the PS-I gene product has not been identified and its function remains unknown . Here we report that affinity purified antibodies against the predicted amino acid sequence of the PS-I gene product detected in homogenates of human, mouse, and rat brains a single antigen of approximately 48 kDa . This antigen was also present in immortalized human and mouse neuronal cell cultures . Brain tissue fractionation showed that all PS-I antigen was found in the membrane fraction . In stained tissue sections of mouse central nervous system (CNS), PS-I antigen was found only in neurons throughout brain and spinal cord and was located within cell bodies, axons, and dendrites . Remarkably the relative partition among these three compartments varied dramatically . A striking feature of PS-I expression was its intense concentration in some (but not all) dendrites, at levels substantially above those in the parent perikarya . In most of the cerebrum, PS-I staining in axons was very weak or undetectable . By contrast, many axons in portions of the brainstem and in the spinal cord showed marked PS-I immunoreactivity . Similarly, staining of sections from human temporal cortex showed that PS-I was present mainly in neuronal cell bodies and dendrites . These data show that in the CNS, PS-I is expressed mainly in neurons and suggests that this protein may perform a neuron specific function . The pattern of PS-I expression in the CNS would suggest that the premature neurodegeneration associated with PS-I mutations involves a primary neuronal process rather than a secondary effect of PS-I produced in non-neuronal cells. J Neurosci Res, 1996 Aug 1, 45(3), 303 - 7 Generalized autoimmunity of the nervous system (GANS) induced by a recombinant protein composed of major pathogenic determinants of MBP, IRBP, and P2 protein: suppression of inflammation by a monoclonal antibody against activated rat T line cells; Schluesener H; We have developed a new model of generalized autoimmunity of the rat nervous system to study differential immunoregulation, barrier-function, and parenchymal inflammatory processes . We designed a multicomponent synthetic gene encoding major pathogenic determinants for Lewis rats of myelin basic protein (MBP), interphotoreceptor retinoid binding protein (IRBP), and P2 protein . Immunization with the recombinant protein induces a monophasic disease with inflammatory lesions in the eye, brain, spinal cord, and peripheral nerves . Rats recovered from GANS were tolerant against the induction of experimental autoimmune encephalomyelitis (EAE), neuritis (EAN), and uveoretinitis (EAU) by immunization with synthetic autoantigen-peptides/CFA . To demonstrate an application of GANS we have used a monoclonal antibody raised against encephalitogenic rat T lymphocytes . We show that this monoclonal antibody is suppressing not only inflammatory cell infiltration of brain and spinal cord, but as well of the eyes and the peripheral nervous system. J Neurosci Res, 1996 Aug 1, 45(3), 216 - 25 Early signaling events by endotoxin in PC12 cells: involvement of tyrosine kinase, constitutive nitric oxide synthase, cGMP-dependent protein kinase, and Ca2+ channels; Simard JM et al.; We studied the effects of endotoxin from Escherichia coli (E . coli) on Ca2+ channel activity in PC12 cells using the cell-attached patch clamp technique . Endotoxin (1-100 ng/ml) decreased channel availability (n x Po) to about one third of control values, an effect that required 3.5 +/- 1 min (mean +/- SD; n = 13) to reach steady state . The biophysical properties of the channel, including slope conductance (22 pS; 40 mM Ba2+), voltage dependence of n x Po, and open times (tau 1 = 0.78 ms, tau 2 = 8.9 ms) for the two open states at 0 mV, were not altered . The effect of endotoxin was blocked by polymyxin-B, indicating involvement of the lipid-A moiety of lipopolysaccharide, and by the tyrosine kinase (tk) inhibitor, tyrphostin . The effect of endotoxin was mimicked by 8-bromo-cGMP (100 microM), and was blocked by the inhibitor of cGMP-dependent protein kinase (PKG), H-8, suggesting involvement of the cGMP/PKG pathway . The effect of endotoxin also was blocked by the nitric oxide (NO) synthase inhibitor, NG-monomethyl-L-arginine monoacetate, suggesting involvement of nitric oxide synthase (NOS) . The rapidity of the effect of endotoxin on Ca2+ channel activity suggested that constitutive NOS (cNOS) was involved, in accordance with our finding that endotoxin-induced transcriptional induction of NOS, as measured by nitrite production, required > 6 hr . We conclude that early signaling events by endotoxin in PC12 cells involve tk, cNOS, cGMP/PKG, and Ca2+ channels. Biol Reprod, 1996 Aug, 55(2), 410 - 5 Mapping of epitopes on porcine zona pellucida-3 alpha by monoclonal antibodies inhibiting oocyte-sperm interaction; Gupta SK et al.; Zona pellucida glycoprotein 3 alpha (ZP3 alpha) has been designated as the primary sperm receptor ligand in porcine gamete interaction . In this study, epitopes were mapped on porcine ZP3 alpha (pZP3 alpha) by using monoclonal antibodies (mAbs) possessing in vitro contraceptive efficacy . Using Western blots, we tested recombinant pZP3 alpha fragments expressed as fusion proteins in Escherichia coli and corresponding to pZP3 alpha precursor protein amino acid residues 18-142 (F1), 140-243 (F2), 239-363 (F3), and 359-462 (F4), for reactivities with mAbs . MAb-403 reacted with F3, and mAbs-412 and -421 with F2 . MAb-420 showed weak reactivity with F1 . Synthesis of overlapping 12-mer peptides further resolved the epitope for mAb-420 to amino acid residues 133-144, mAb-421 to 157-168, mAb-412 to 205-216, and mAb-403 to 301-312 . MAbs-412 and -420 inhibited the binding of boar sperm to zona-encased porcine oocytes . These results, the first to define peptide epitopes of pZP3 alpha, should assist in the design of a synthetic peptide-based immunocontraceptive vaccine. Nippon Kyobu Geka Gakkai Zasshi, 1996 Aug, 44(8), 1193 - 7 {Aortic valve replacement due to Libman-Sacks endocarditis combined with infectious endocarditis}; Taniyasu N et al.; A 40-year-old female had a history of fever, arthralgia, proteinuria, and dyspnea on effort twenty years ago, and was diagnosed as SLE, renal failure, and aortic regurgitation . She also suffered from pyelonephritis and sepsis due to the infection of E . coli . Preoperative examination revealed non-active phase of SLE . Echocardiography and aortography showed massive aortic regurgitation and operation was recommended . Operative findings showed fresh vegetation on the aortic leaflets, and aortic valve replacement (Tekna-Edwards 19 mm) was performed . Histological findings of the vegetation showed Libman-Sacks endocarditis and infectious endocarditis . Predonisolone was infused intravenously to prevent the acute deterioration of SLE after the operation . She was discharged from the hospital three weeks after the operation. Microbiol Res, 1996 Aug, 151(3), 329 - 35 Study of the gelatinolytic activities Escherichia coli cells before and after starvation in seawater by substrate gel electrophoresis; Papageorgakopoulou N et al.; Previous work has shown that clinical Escherichia coli strains, starved in seawater, are able to present residual growth, with subsequent alterations to their enzymatic activities and metabolism . Gelatinolytic activity of starved cells is of importance because it appears and increases gradually with time . In this work, several forms of gelatinolytic activity were detected by SDS-polyacrylamide gel electrophoresis, differing in molecular masses and appearance, before and after starvation of E . coli cells . The enzymic forms are classified into 4 categories according to the effect of certain inhibitors on the appearance of gelatinolytic activity: a . those whose appearance is inhibited by the chelating factors EDTA, 1,10-phenanthroline and whose presence is also inhibited by N-ethylmaleimide (metalloproteinases with thiol group active); b . those affected by the presence of chelators, N-ethylmaleimide and Ca2+ (Ca2(+)-dependent metalloproteinases with thiol group active); c . those inhibited by chelators and activated in the presence of Ca2+ (Ca2(+)-dependent metalloproteinases) and d . those whose appearance is independent of the presence of inhibitors used . The forms of gelatinolytic activity of the fourth category coincide with enzyme forms that can also use casein as substrate in electrophoresis . These data suggest that there are considerable differences in the gelatinolytic pattern of clinical strains of E.coli cells before and after starvation in seawater. Microbiol Res, 1996 Aug, 151(3), 273 - 80 Expression of heat shock protein D 48.5 of Escherichia coli is subject to modulation by catabolite repression; Peruski LF Jr; The Escherichia coli heat shock regulon consists of approximately twenty polypeptides that are coordinately and transiently induced upon a temperature upshift under the control of an alternative sigma factor, designated sigma-32 or HtpR . Preliminary observations on one of the proteins of the heat shock response, protein D 48.5, suggested that its induction by sigma-32 during heat shock may be modulated by catabolite repression . In this study, a disk diffusion assay was used to screen the effect of several compounds on the expression of a lacZ fusion in the gene encoding protein D 48.5 . This assay indicated that the expression of this protein was controlled, at least in part, by the catabolite repression response . A more indepth analysis of the expression of protein D 48.5 under both steady-state and heat shock conditions was conducted in both a wild type and cya crp background . This analysis revealed that in the cya crp background, the steady-state level of protein D 48.5 was elevated relative to the wildtype, but that the heat shock induction of the protein was reduced in magnitude relative to the wild type strain, suggesting a direct link between these two global responses . The lacZ fusion in the structural gene for protein D 48.5 should prove useful as a reporter mechanism to probe the physiology and regulation of the heat shock response. Protein Expr Purif, 1996 Aug, 8(1), 75 - 84 Cloning, high-yield expression in Escherichia coli, and purification of biologically active HIV-1 Tat protein; Kirsch T et al.; We have established an expression system for full-length HIV-1 transactivator (Tat) protein in Escherichia coli . By constructing a synthetic gene for high level expression in enteric bacteria, the recombinant protein can be obtained in high yield . Fusion of the Tat sequence to an N-terminal histidine tag allows the rapid purification of the fusion protein through a single chromatographic step . After cleavage of the fusion protein with CNBr, pure Tat can be obtained through the use of a MonoS column . Reduction of the protein with Tris(2-carboxyethyl)phosphine-HCl and subsequent stepwise refolding yields biologically active Tat . Sample purity and the identity of the protein mass with the mass expected from the amino acid sequence was demonstrated by mass spectrometry . Nuclear magnetic resonance spectroscopy showed the identity of bacterially expressed and chemically synthesized Tat protein (P . Bayer et al., 1995, J . Mol . Biol . 247, 529-535) . The expression of Tat in E . coli enables isotope labeling as a prerequisite for multidimensional NMR experiments toward the elucidation of the structure of the Tat-trans-activation response element complex. Protein Expr Purif, 1996 Aug, 8(1), 41 - 7 High-level expression in Escherichia coli of the soluble, catalytic domain of rat hepatic cytochrome b5 reductase; Barber MJ et al.; A T7 expression system has been produced for the high-level production of the soluble, catalytic domain of rat hepatic cytochrome b5 reductase in Escherichia coli . The recombinant protein was purified to homogeneity using affinity chromatography on 5'-ADP agarose and gel exclusion chromatography and exhibited a molecular mass of approximately 30 kDa by polyacrylamide gel electrophoresis and a molecular mass of 30,588 by mass spectrometry . Direct sequencing of the initial 12 residues of the amino-terminus of the purified domain yielded the sequence MITLENPDIKYP, identical to that predicted from the DNA sequence . The domain incorporated a full complement of FAD with a visible absorption spectrum typical of a flavoprotein exhibiting maxima at 389 and 461 nm and a distinct shoulder at 485 nm . Addition of NADH to the protein resulted in an extensive bleaching of the visible spectrum . The recombinant domain retained both NADH:ferricyanide and NADH:cytochrome b5 reductase activities with Vmax of 48 and 26 micromol NADH consumed/min/nmol FAD, respectively, and Km of 6, 7, and 11 microM for NADH, ferricyanide, and cytochrome b5 . Comparison of the activities obtained using NADH and NADPH indicated a substantial preference for NADH as the reducing substrate . The results indicate that the recombinant protein retains the physical and catalytic properties of the native protein and represents an excellent system for probing the role of specific amino acid residues using site-directed mutagenesis. Protein Expr Purif, 1996 Aug, 8(1), 23 - 7 Functional expression of the dihydrofolate reductase domain of Leishmania major dihydrofolate reductase-thymidylate synthase bifunctional protein; Yu PL et al.; The dihydrofolate reductase (DHFR) domain of the bifunctional dihydrofolate reductase-thymidylate synthase from Leishmania major has been subcloned and expressed as a soluble protein in Escherichia coli strain PA414 harboring plasmid pLMDHFR . Homogeneous L . major DHFR was obtained by chromatography on methotrexate-Sepharose followed by DE52 . The purified enzyme migrated as a single 25-kDa protein on SDS-PAGE . The native molecular weight was determined to be 26 kDa, indicating that the isolated domain is a monomer . N-terminal sequence analysis revealed that serine, the second amino acid in the coding sequence, was the N-terminal amino acid of the protein . The enzyme showed a pH optimum similar to that of the bifunctional protein . For purified DHFR, the Km values were <1.0 microM for H2folate and <1.0 microM for NADPH . The kcat of the most active DHFR preparation was 5 s-1 . The Km and kcat values were similar to those of the bifunctional enzyme. Protein Expr Purif, 1996 Aug, 8(1), 17 - 22 Overproduction and purification of sigmaS, the Escherichia coli stationary phase specific sigma transcription factor; Nguyen LH et al.; This paper reports the overproduction and the details of a rapid method to purify active sigmaS monomers from a T7 RNA polymerase-based protein expression system . This 2-day procedure involves solubilizing inclusion bodies in sarkosyl detergent, removal of sarkosyl by dialysis, and a single gel filtration column chromatography step . The final yield of sigmaS is about 9 mg of approximately 92% purity from 0.5 g of wet weight cells . Overproduced sigmaS binds to core RNA polymerase and supports transcription from the bolAp1 promoter, a sigmaS-dependent promoter. Protein Expr Purif, 1996 Aug, 8(1), 1 - 16 Overexpression, purification, and refolding of link module from human TSG-6 in Escherichia coli: effect of temperature, media, and mutagenesis on lysine misincorporation at arginine AGA codons; Day AJ et al.; The Link module, a 98-amino-acid domain found in hyaluronan binding proteins of human tumor necrosis factor stimulated gene 6 was overexpressed in Escherichia coli . Electrospray ionization mass spectrometry revealed that only 50% of the expressed protein had the expected wild-type molecular weight, with the remaining material having between 1 and 4 arginine to lysine substitutions, arising due to misincorporation at AGA codons . The level of misincorporation was almost completely abolished by mutation of the 4 AGA codons to CGT . This mutation to high-usage arginine codons also increased the level of heterologous protein expression . Refolding of the Link module, which occurred during the purification procedure, gave two species with different disulfide bond organizations that could be separated by high-performance liquid chromatography . One of these had a disulfide bond arrangement consistent with that found in other Link modules and, by nuclear magnetic resonance spectroscopy, was shown to be folded. Anal Biochem, 1996 Aug 1, 239(2), 130 - 5 Assay of Escherichia coli dihydroorotase with enantiomeric substrate: practical preparation of carbamyl L-aspartate and high-performance liquid chromatography analysis of catalysis product; Daniel R et al.; A reasonably facile and effective procedure is described for preparing the optically active substrate of dihydroorotase (EC 3.5.2.3), N-carbamyl-L-aspartate (L-CA), which is not commercially available . Compared to the previously described methods, this procedure is not plagued by side reactions, and the L-CA is obtained in a solid form as the Mg2+/K+ mixed salt . In that salt form, the L-CA can be prepared in large quantity, and it is easier to handle and stable upon storage . L-CA can be separated from aspartate and its cyclic derivatives, dihydroorotate and hydantoin, by HPLC using a strong anion-exchange column . This HPLC method, which is used to check the purity of the synthesized carbamyl aspartate, is also effective for monitoring the enzymatic reaction. Chem Biol, 1996 Aug, 3(8), 661 - 70 Molecular basis for the binding of SH3 ligands with non-peptide elements identified by combinatorial synthesis; Feng S et al.; BACKGROUND: Protein-structure-based combinatorial chemistry has recently been used to discover several ligands containing non-peptide binding elements to the Src SH3 domain . The encoded library used has the form Cap-M1-M2-M3-PLPPLP, in which the Cap and Mi's are composed of a diverse set of organic monomers . The PLPPLP portion provided a structural bias directing the non-peptide fragment Cap-M1-M2-M3 to the SH3 specificity pocket . Fifteen ligands were selected from > 1.1 million distinct compounds . The structural basis for selection was unknown . RESULTS: The solution structures of the Src SH3 domain complexed with two ligands containing non-peptide elements selected from the library were determined by multidimensional NMR spectroscopy . The non-peptide moieties of the ligands interact with the specificity pocket of Src SH3 domain differently from peptides complexed with SH3 domains . Structural information about the ligands was used to design various homologs, whose affinities for the SH3 domain were measured . The results provide a structural basis for understanding the selection of a few optimal ligands from a large library . CONCLUSIONS: The cycle of protein-structure-based combinatorial chemistry followed by structure determination of the few highest affinity ligands provides a powerful new tool for the field of molecular recognition. Chem Biol, 1996 Aug, 3(8), 645 - 53 A Zn(II)-binding site engineered into retinol-binding protein exhibits metal-ion specificity and allows highly efficient affinity purification with a newly designed metal ligand; Schmidt AM et al.; BACKGROUND: The Zn(II)-binding site from the active center of human carbonic anhydrase II, formed by three His side chains, can be grafted onto the recombinant serum retinol-binding protein (RBP) . The artificial binding site in the resulting variant RBP/H3(A) has high affinity for Zn(II) and stabilizes the protein against denaturation . RESULTS: The metal-ion specificity of the grafted Zn(II) binding site in RBP/H3(A) was investigated . Both Cu(II) and Ni(II) bound with high affinity, although the Kd values were not as low as for Zn(II) binding . Competition experiments with the chelate ligands iminodiacetic acid (IDA) and nitrilotriacetic acid (NTA) suggested that both Ni(II) and Cu(II) bound to the protein in an octahedral manner with three vacant coordination sites, as previously observed for Zn(II) . A substituted pyrrolidine-dicarboxylic acid was designed as a structurally rigid IDA compound and coupled to a matrix . Using this support in an immobilized metal affinity chromatography (IMAC), RBP/H3(A) was purified from the bacterial cell extract in one step with unprecedented efficiency . CONCLUSIONS: Although the His3 metal-binding site used here had been removed from the substrate pocket of an enzyme and exposed to solvent on a protein surface, it showed clear selectivity for Zn(II) compared to Cu(II) and Ni(II) . Thus the properties of this structurally defined metal-binding site (which are not shared by isolated His residues or flexible oligo-His tags) can be preserved when it is added to proteins . An IMAC matrix with improved behaviour was designed, allowing highly selective purification of RBP/H3(A) and of His6-tagged RBP as well . Such rational design of supramolecular recognition may be generally useful in the fields of protein engineering and drug design. Arch Biochem Biophys, 1996 Aug 1, 332(1), 196 - 204 Cloning, characterization, and heterologous expression of cDNAs for farnesyl diphosphate synthase from the guayule rubber plant reveals that this prenyltransferase occurs in rubber particles; Pan Z et al.; Two farnesyl diphosphate synthase (FPS) cDNA's from a guayule stembark library were isolated and characterized . Both encode M(r) 39,000 proteins containing 432 amino acids that differ slightly in their deduced molecular weights and isoelectric points . They both contain the DDXXD motifs that are characteristic of prenyltransferases, and both isoforms show high homology to other plant FPS sequences but less overall homology to FPS sequences from nonplant sources . The two isoforms differ by 5% in their amino acid sequence . When expressed in Escherichia coli, each guayule isoform exhibits high specific activity that produces farnesyl diphosphate as the major isoprenoid and small amounts of geranyl diphosphate . Biochemical and immunological evidence also indicates that FPS is associated with guayule rubber particles . Antibodies to chicken FPS cross-react with both guayule isoforms expressed in E . coli and recognize a low abundance M(r) 39,000 protein in rubber particles purified from guayule stembark . Guayule FPS sequences show high homology to peptide fragments of the prenyltransferase associated with rubber particles from Hevea brasiliensis, suggesting that this enzyme may be important for rubber biosynthesis in both species. Arch Biochem Biophys, 1996 Aug 1, 332(1), 30 - 4 Human isopentenyl diphosphate: dimethylallyl diphosphate isomerase: overproduction, purification, and characterization; Hahn FM et al.; Isopentenyl diphosphate (IPP):dimethylallyl diphosphate isomerase catalyzes an essential activation step in the isoprenoid biosynthetic pathway . A human cDNA sequence {J . Xuan, J . Kowalski, A.F . Chambers, and D.T . Denhardt (1994) Genomics 20, 129-131} containing a 684-base-pair open reading frame was recently reported that encoded a protein with a significant degree of similarity to two fungal IPP isomerases {F.M . Hahn and C.D . Poulter (1995) J . Biol . Chem . 270, 11298-11303} . The human cDNA sequence was cloned into expression plasmid pFMH12 . The encoded protein was overproduced in Escherichia coli and purified to > 90% homogeneity in two steps by ion-exchange and hydrophobic interaction chromatography . The recombinant protein catalyzed the isomerization of IPP to dimethylallyl diphosphate and was maximally active at pH 7.0 in the presence of Mg2+ . The Michaelis constant for IPP was 33 microM, similar to the value of 43 microM reported for yeast IPP isomerase; Vmax = 4.1 mumol min-1 mg-1 for recombinant human IPP isomerase, approximately fivefold less than reported for the yeast enzyme {I.P . Street and C.D . Poulter (1990) Biochemistry 29, 7531-7538}. Virology, 1996 Aug 1, 222(1), 115 - 22 Analysis of a salt stable mutant of cowpea chlorotic mottle virus; Fox JM et al.; An understanding of virion assembly and disassembly requires a detailed understanding of the protein-protein and protein-nucleic acid interactions which stabilize the virion . We have characterized a mutant of cowpea chlorotic mottle virus (CCMV) that is altered in virion stability . The mutant virions resist disassembly in 1.0 M NaCl, pH 7.5, whereas the wild-type virions completely disassociate into RNA and capsid protein components . Sequence analysis of the mutant coat protein gene identified a single A to G nucleotide change at position 1484 of RNA 3 (position 134 of RNA 4), which results in a lysine to arginine change at position 42 of the coat protein . Introduction of the K42R mutation into wild-type CCMV coat protein results in a salt stable virion phenotype . Likewise, expression of the K42R mutant coat protein in Escherichia coli followed by in vitro assembly produces virions that exhibit the salt stable phenotype . Analysis of this mutation demonstrates how a single amino acid change in the primary structure of the coat protein leads to tertiary interactions which stabilize the virion. Curr Opin Struct Biol, 1996 Aug, 6(4), 491 - 8 Membrane sectors of F- and V-type H+-transporting ATPases; Fillingame RH; H+ transporting, reversible F-type ATPases (ATP synthases) and V-type ATPases share major structural features and function by similar mechanisms . Recent structural and genetic experiments provide new insights into the organization of the transmembrane and coupling sectors of the enzymes and the molecular mechanics of coupling H+ transport to ATP. Mol Carcinog, 1996 Aug, 16(4), 188 - 96 DNA base damage induced by ionizing radiation recognized by Escherichia coli UvrABC nuclease but not Nth or Fpg proteins; Roldan-Arjona T et al.; Ionizing radiation and other free radical-generating systems induce a great variety of oxidative damage to DNA bases . The major known lesions are repaired by two well-characterized DNA glycosylases of Escherichia coli, endonuclease III (Nth) and formamidopyrimidine-DNA glycosylase (Fpg), which have associated AP lyase activities . To detect and characterize potentially harmful oxidative base DNA lesions that may be repaired by alternative means, we exposed plasmid DNA to low doses of gamma-rays and removed the major base lesions by treatment with Nth and Fpg proteins . The closed circular DNA remaining after these treatments was used as a substrate of the UvrABC endonuclease complex from E . coli and as a template in a DNA polymerase arrest assay in vitro . The circular DNA contained lesions that were recognized and incised by the UvrABC nuclease and also lesions that blocked DNA polymerization in vitro . The blocking lesions were more abundant in DNA irradiated under nitrogen than under air and occurred mainly at tandem guanines; however, they were also frequent at tandem adenines and tandem cytosines. Plant Cell, 1996 Aug, 8(8), 1421 - 35 Structure and expression of a plant U1 snRNP 70K gene: alternative splicing of U1 snRNP 70K pre-mRNAs produces two different transcripts; Golovkin M et al.; The product of the U1 small nuclear ribonucleoprotein particle (U1 snRNP) 70K (U1-70K) gene, a U1 snRNP-specific protein, has been implicated in basic as well as alternative splicing of pre-mRNAs in animals . Here, we report the isolation of full-length cDNAs and the corresponding genomic clone encoding a U1-70K protein from a plant system . The Arabidopsis U1-70K protein is encoded by a single gene, which is located on chromosome 3 . Several lines of evidence indicate that two distinct transcripts (short and long) are produced from the same gene by alternative splicing of the U1-70K pre-mRNA . The alternative splicing involves inclusion or exclusion of a region (910 bp) that we named "included intron." Two transcripts were clearly detectable in all tissues tested, and the level of the transcripts varied in different organs . The deduced amino acid (427 residues) sequence from the short transcript has strong homology to the animal U1-70K protein and contains an RNA recognition motif, a glycine hinge, and an arginine-rich region characteristic of the animal U1-70K protein . The long transcript has an in-frame translational termination codon within the 910-bp included intron, resulting in a truncated protein containing only 204 amino acids . The protein encoded by the short transcript is recognized by U1 RNP-specific monoclonal antibodies and binds specifically to the Arabidopsis U1 snRNA, whereas the protein from the long transcript does not . In addition, multiple polyadenylation sites were observed in the 3' untranslated region . These results suggest a complex post-transcriptional regulation of Arabidopsis U1-70K gene expression. Eur J Biochem, 1996 Aug 1, 239(3), 887 - 96 The rabbit ileal lipid-binding protein . Gene cloning and functional expression of the recombinant protein; Stengelin S et al.; A bile-acid-binding protein of Mr 14000 has been previously identified by photoaffinity labeling in rabbit ileal brush border membrane vesicles {Kramer et al . (1993) J . Biol . Chem . 268, 18035-18046} . This peripheral membrane-associated protein was purified and identified as an ileal lipid-binding protein . It was further shown to be identical to the cytosolic 14-kDa bile-acid-binding protein from the same tissue . Starting with sequence information from tryptic fragments, we cloned and sequenced the gene and its transcript . It has four exons (123, 176, 90, 115 bp) and three introns (1372, 2291, 3137 bp) and a similar structure as the genes from other members of the fatty-acid-binding protein family . The deduced protein has 128 amino acid residues and a calculated molecular mass of 14404 Da . It exhibits high similarity to its human (83%), mouse (77%), rat (76%) and porcine (72%) counterparts . Furthermore, the recombinant protein was produced in Escherichia coli and shown to be identical to native protein from ileal tissue . Functionality of the recombinant protein was demonstrated by labeling with various photoaffinity derivatives of bile acids . Ranking of the photolabeling efficiency of these probes towards the recombinant protein was comparable to the respective ranking towards the native protein . Polyclonal antibodies that were raised in hens against the recombinant protein, specifically recognized the ileal lipid-binding protein in the brush border membrane and cytosol from rabbit ileum . In contrast, no labeling was observed with jejunal tissue . Our results suggest a specific role of the membrane-associated ileal lipid-binding protein for the process of ileal bile acid uptake. Eur J Biochem, 1996 Aug 1, 239(3), 881 - 6 A highly efficient cell-free protein synthesis system from Escherichia coli; Kim DM et al.; We modified a cell-free coupled transcription/translation system from Escherichia coli with the T7 phage RNA polymerase, and achieved a productivity as high as 0.4 mg protein/ml reaction mixture . First, we found that the optimal concentrations of phosphoenolpyruvate and poly(ethylene glycol) are interdependent; higher concentrations of the former should be used at higher concentrations of the latter . Second, the use of a condensed 30000 x g cell extract, in place of the conventional one, significantly increased the initial rate of protein synthesis . This phenomenon was demonstrated to be due to a reason other than elimination of inhibitory molecule(s) from the extract . For this system with the condensed extract, the phosphoenolpyruvate and poly(ethylene glycol) concentrations were again co-optimized, resulting in production of chloramphenicol acetyltransferase at a productivity of 0.3 mg/ml . Finally, the productivity was further increased up to 0.4 mg/ml, by supplementation of the pool of amino acids . This improved cell-free protein synthesis system is superior in productivity to any other cell-free systems reported so far, including the continuous-flow cell-free system. Eur J Biochem, 1996 Aug 1, 239(3), 842 - 9 Epitope mapping and immunoneutralization of recombinant human stem-cell factor; Mendiaz EA et al.; The epitope regions of three anti-{stem-cell factor (SCF)}g have been mapped by characterization of immunoreactivities against truncated forms of SCF in immunoblots and against synthetic peptides in solution-phase competition ELISA . Two of the antibodies, mAb 7H6 and mAb 8H7A, were raised against Escherichia coli-derived human SCF-(1-164) while the third, polyclonal antibody (pAb) 1337, was raised against a peptide corresponding to residues 3-31 of human SCF . The epitopes of mAbs 7H6 and 8H7A have been mapped to residues 61-95 and 95-110, respectively . The epitope of pAb 1337 has been mapped to residues 21-31 . The ability of the anti-SCF Ig to recognize E . coli-derived human SCF presented in various formats, i.e . partially denatured (fixed in standard ELISA or on a western blot) or native (in solution), was studied, mAb 7H6 recognized its epitope in partially denatured or native SCF with equally high affinity, while mAb 8H7A and pAb 1337 recognized their epitopes only when SCF was at least partially denatured, mAb 7H6 was found to neutralize in vitro SCF-mediated cell proliferation and SCF binding to its receptor, when present in equimolar concentrations relative to the ligand, suggesting that the epitope region is functionally significant . Evidence that the mAb 7H6 epitope is represented by discontinuous regions (residues within sequences 61-65 and 91-95 are critically involved) is presented . The observation that the mAb 7H6 epitope is discontinuous has implications for the structure of SCF. Eur J Biochem, 1996 Aug 1, 239(3), 827 - 34 Role of zinc-finger proteins Sp1 and zif268/egr-1 in transcriptional regulation of the human synaptobrevin II gene; Petersohn D et al.; Synaptobrevin II is a small integral membrane protein of synaptic vesicles that plays a key role in exocytosis . The 5'-flanking region of the human synaptobrevin II gene is very (G+C)-rich and contains a 13-bp motif that includes overlapping binding sites for the zinc finger transcription factors Sp1 and zif268/egr-1 . To test whether Sp1 and zif268/egr-1 interact with this motif, gel retardation assays were performed . These assays revealed that both transcription factors bind to the (G+C)-rich motif of the synaptobrevin II gene in vitro . The binding of Sp1 was additionally confirmed by supershift analysis with antibodies specific for Sp1 . To determine whether zif268/egr-1 plays a role in controlling synaptobrevin II gene expression, a plasmid was constructed containing the (G+C)-rich motif of the synaptobrevin II gene upstream of a minimal promoter and the Escherichia coli chloramphenicol acetyltransferase (CAT) gene as a reporter . This plasmid was transfected into CHO-K1 cells together with an expression vector encoding zif268/egr-1 . Zif268/egr-1 failed to activate transcription from this reporter gene, although it transactivated a reporter gene containing an identical (G+C)-rich motif derived from the human synapsin I promoter . Overexpression of Sp1, however, clearly activated transcription of a reporter gene under the control of the synaptobrevin II promoter (G+C)-rich sequence in Drosophila SL2 cells, which provided an Sp1-deficient background . Furthermore, a glutathione S-transferase protein containing the DNA-binding domain of Sp1 was shown to function as a dominant negative form of Sp1, reducing transcription of the synaptobrevin II promoter-CAT reporter gene in mammalian cells to basal levels . From these data, we conclude that the zif268/egr-1-binding site in the synaptobrevin II promoter is not functionally active . Instead, an overlapping Sp1-binding site in this (G+C)-rich region clearly mediates constitutive transcriptional activation. Eur J Biochem, 1996 Aug 1, 239(3), 737 - 41 The binding of nucleotides to domain I proteins of the proton-translocating transhydrogenases from Rhodospirillum rubrum and Escherichia coli as measured by equilibrium dialysis; Bizouarn T et al.; Transhydrogenase catalyses the transfer of reducing equivalents between NAD(H) and NADP(H) coupled to the translocation of protons across a membrane . The NAD(H)-binding domain of transhydrogenase (domain I protein) from Rhodospirillum rubrum and from Escherichia coli were overexpressed and purified . Nucleotide binding to the domain I proteins was determined by equilibrium dialysis . NADH and its analogue, acetylpyridine adenine dinucleotide (reduced form), bound with relatively high affinity (Kd = 32 microM and 120 microM, respectively, for the R . rubrum protein) . The binding affinity was similar at pH 8.0 and pH 9.0 in zwitterionic buffers, and at pH 7.5 in sodium phosphate buffer . NAD+ bound with lower affinity (Kd = 300 microM) . NADPH bound only very weakly (Kd > 1 mM) . Using a centrifugation procedure, Yamaguchi and Hatefi {Yamaguchi, M . & Hatefi, Y . (1993) J . Biol . Chem . 268 . 17871-17877} found that mitochondrial transhydrogenase, and a proteolytically derived domain I fragment from that enzyme, bound one NADH per dimer . They suggested that this result implied half-of-the-site reactivity for the interaction between the nucleotide ligand and the protein . However, our studies on both the E . coli and the R . rubrum recombinant transhydrogenase domain I proteins using equilibrium dialysis show that the binding stoichiometry for both NADH and the reduced form of acetylpyridine adenine dinucleotide (AcPdADH) is two nucleotides per dimer: no interaction between the monomeric units is evident . Reasons for the discrepancies between the work on bacterial and mitochondrial transhydrogenases are discussed. Eur J Biochem, 1996 Aug 1, 239(3), 732 - 6 Ser57 in the Na+/proline permease of Escherichia coli is critical for high-affinity proline uptake; Quick M et al.; Ser57 in the Na+/proline permease of Escherichia coli has been replaced with alanine, cysteine, glycine, or threonine, and properties of the corresponding putP mutants have been analyzed . Although Ser57 is not essential for activity, the amino acid side chain at this position is critical for proline uptake . Thus, alanine, cysteine, glycine, or threonine in place of Ser57 reduces the initial rate of proline transport under standard conditions to less than 10% of the wild-type value . In addition, substitution of Ser57 in the Na+/proline permease reduces the sensitivity of E . coli cells to the toxic proline analogs L-azetidine-2-carboxylate and 3.4-dehydro-D.L-proline . Replacement of Ser57 with alanine or cysteine results in apparent affinities for proline that are reduced by more than two orders of magnitude, and permeases with threonine and glycine in place of Ser57 yield apparent affinities reduced by a factor of 60 and 18 respectively, relative to wild-type . In contrast, all of the Ser57 replacements analyzed cause only small changes in Vmax values . All permease molecules containing Ser57 substitutions are inserted into the membrane in amounts comparable to the wild-type protein as shown by immunoblot analysis . These results indicate that alterations of proline transport and sensitivity to toxic proline analogs have to be attributed primarily to defects in substrate binding . It is suggested that the serine residue at position 57 of the permease is located within the substrate-binding domain of the protein. Eur J Biochem, 1996 Aug 1, 239(3), 668 - 74 Mutational studies of the amino acid residues in the combining site of Erythrina corallodendron lectin; Adar R et al.; High-resolution X-ray crystallography of the complex of the Gal/GalNAc-specific Erythrina corallodendron lectin with lactose identified the amino acid side chains that form contacts with the galactose moiety of the disaccharide . The contribution of these amino acids to the binding of different monosaccharides and oligosaccharides by the lectin was examined by site-directed mutagenesis . Replacement of Phe131, on which the galactose is stacked, by tyrosine, gave a mutant with the same hemagglutinating activity and carbohydrate specificity as the parent lectin, but replacement by alanine or valine resulted in loss of activity . Mutations of Ala88, Asp89, and Asn133 produced mutants that were also inactive whereas those of the other combining site residues, Tyr106, Ala218, and Gln219, were biologically active . None of the active mutants interacted with mannose or glucose . Thus, contrary to an earlier assumption . Ala218 is not responsible for the inability of E . corallodendron lectin to bind these sugars . Our findings also demonstrate that Gln219 is not involved in galactose binding in solution, even though this is implicated by the crystal data . Instead, our data suggest that Gln219 assists in the ligation of N-acetyllactosamine to the lectin, by interacting with the acetamide group of the disaccharide . Comparison with other legume lectins specific for mannose/glucose, galactose, N-acetylgalactosamine, L-fucose or N-acetylglucosamine, shows that only three of the combining site residues of E . corallodendron lectin occupy invariant positions both in their primary and tertiary structures . These residues are an aspartic acid and an asparagine corresponding to positions 89 and 133, respectively, in E . corallodendron lectin, and an aromatic residue, either phenylalanine (as Phe131 in this lectin), tyrosine or tryptophan . We therefore postulate that these three residues are essential for ligand binding by all such lectins, irrespective of their specificity. Eur J Biochem, 1996 Aug 1, 239(3), 597 - 601 Analysis of the mRNA cap-binding ability of human eukaryotic initiation factor-4E by use of recombinant wild-type and mutant forms; Morino S et al.; In order to identify the amino acid residues necessary for the selective recognition of the mRNA cap structure by human eukaryotic initiation factor-4E (eIF-4E), which plays a central role in the first step of mRNA translation, we prepared recombinant wild-type and fourteen mutant forms and compared their cap-binding abilities by affinity chromatography . By the direct expression of a synthetic gene encoding human eIF-4E as the soluble form in Escherichia coli and the application on a 7-methylguanosine-5'-triphosphate-Sepharose 4B cap affinity column, pure recombinant eIF-4E was prepared; the optimum pH for the binding of the mRNA cap was 7.5 . Among the amino acid residues conserved among various eIF-4E species, each of 14 functional residues was replaced with a nonpolar amino acid (alanine or leucine) . All mutant eIF-4E genes, which were constructed by site-directed mutagenesis, were expressed in the same way as the wild type, and their cap-binding abilities were compared with that of the wild type . Consequently, all eight tryptophan residues . Glu103, and two histidine residues at positions 37 and 200 in human recombinant eIF-4E were suggested to be important for the recognition of the mRNA cap structure through direct interaction and/or indirect contributions . Indirect contributions included the construction of the overall protein structure, especially the cap-binding pocket. Mil Med, 1996 Aug, 161(8), 475 - 8 Epidemiology of enterotoxigenic Escherichia coli-associated diarrheal disease occurring on board U.S . Navy ships visiting Asian ports; Orndorff GR et al.; Diarrhea represents a major health threat to U.S . military forces overseas, especially in developing countries . For military units, this illness can adversely affect combat readiness . USNAMRU-2 investigators joined several U.S . Navy ships to assess the epidemiology of diarrhea illness as a result of port visits to Asia . The primary goals were to enumerate episodes of diarrhea associated with port visits, identify epidemiologic factors leading to illness, and characterize the etiologic agents . Enterotoxigenic Escherichia coli (ETEC) was the most common organism isolated from patients presenting with diarrhea and represented 22% of diarrhea cases . Vomiting and abdominal pain differentiated ETEC from other causes of diarrhea. Plant J, 1996 Aug, 10(2), 235 - 42 Identification of a peptide methionine sulphoxide reductase gene in an oleosin promoter from Brassica napus; Sadanandom A et al.; A bidirectional promoter can be defined operationally as a short segment of DNA that regulates divergent transcription . In an attempt to investigate whether the intergenic region between the oleosin and a second open reading frame (ORFII) in Brassica napus (L.) is a divergent promoter, and also to characterize the ORFII, cDNA clones homologous to ORFII were isolated from a leaf cDNA library . A representative cDNA (clone D) of one of the two classes identified was identical, in DNA sequence, to the genomic ORFII . The second representative cDNA (clone O) was 97% identical at the nucleotide level to the genomic ORFII . The predicted amino acid sequence of the cDNA clones each exhibit homology with the peptide methionine sulphoxide reductase (PMSR) of Escherichia coli . The gene structure of ORFII was elucidated and the relative positions of the oleosin, ORFII, and the intergenic promoter region were determined . This confirms that the B . napus oleosin-ORFII intergenic region has divergent promoter activity . Consequently this is the first such plant nuclear divergent promoter identified . RFLP-mapping results showed that all four ORFII genes are linked to four of the six copies of the oleosin genes . This suggested that the bidirectional promoter locus is conserved within the B . napus genome . The ORFII gene product is targeted to the chloroplast, which is consistent with previous data indicating the presence of PMSR activity in the chloroplast . The over-expressed recombinant fusion protein (minus the transitpeptide) showed the capability to reduce peptide methionine sulphoxide residues in vitro, indicating PMSR activity . This study demonstrates that ORFII is transcribed and encodes a plant PMSR, and is the first example of the isolation of a eukaryotic PMSR gene. Plant J, 1996 Aug, 10(2), 203 - 13 Molecular cloning and characterization of a cDNA encoding the gibberellin biosynthetic enzyme ent-kaurene synthase B from pumpkin (Cucurbita maxima L.); Yamaguchi S et al.; The first committed step in the formation of diterpenoids leading to gibberellin (GA) biosynthesis is the conversion of geranylgeranyl diphosphate (GGDP) to ent-kaurene . ent-Kaurene synthase A (KSA) catalyzes the conversion of GGDP to copalyl diphosphate (CDP), which is subsequently converted to ent-kaurene by ent-kaurene synthase B (KSB) . A full-length KSB cDNA was isolated from developing cotyledons in immature seeds of pumpkin (Cucurbita maxima L.) . Degenerate oligonucleotide primers were designed from the amino acid sequences obtained from the purified protein to amplify a cDNA fragment, which was used for library screening . The isolated full-length cDNA was expressed in Escherichia coli as a fusion protein, which demonstrated the KSB activity to cyclize {3H}CDP to {3H}ent-kaurene . The KSB transcript was most abundant in growing tissues, but was detected in every organ in pumpkin seedlings . The deduced amino acid sequence shares significant homology with other terpene cyclases, including the conserved DDXXD motif, a putative divalent metal ion-diphosphate complex binding site . A putative transit peptide sequence that may target the translated product into the plastids is present in the N-terminal region. DNA Cell Biol, 1996 Aug, 15(8), 653 - 9 Identification of novel mRNA isoforms for human DNA polymerase beta; Chyan YJ et al.; Recently, we reported the organization of the thirteen exons of the human DNA polymerase beta (beta-pol) gene and the sequences of the exon-intron junctions . Splice variants of human beta-pol mRNA have been postulated to be related